U.S. patent application number 16/532212 was filed with the patent office on 2020-01-16 for nucleophilic catalysts for oxime linkage.
The applicant listed for this patent is Baxalta GmbH, Baxalta Incorporated. Invention is credited to Stefan Haider, Andreas Ivens, Hanspeter Rottensteiner, Jurgen Siekmann, Peter Turecek, Oliver Zoechling.
Application Number | 20200017543 16/532212 |
Document ID | / |
Family ID | 45556599 |
Filed Date | 2020-01-16 |
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United States Patent
Application |
20200017543 |
Kind Code |
A1 |
Haider; Stefan ; et
al. |
January 16, 2020 |
NUCLEOPHILIC CATALYSTS FOR OXIME LINKAGE
Abstract
The invention relates to materials and methods of conjugating a
water soluble polymer to an oxidized carbohydrate moiety of a
therapeutic protein comprising contacting the oxidized carbohydrate
moiety with an activated water soluble polymer under conditions
that allow conjugation. More specifically, the present invention
relates to the aforementioned materials and methods wherein the
water soluble polymer contains an active aminooxy group and wherein
an oxime or hydrazone linkage is formed between the oxidized
carbohydrate moiety and the active aminooxy group on the water
soluble polymer, and wherein the conjugation is carried out in the
presence of a nucleophilic catalyst.
Inventors: |
Haider; Stefan;
(Prinzersdorf, AT) ; Ivens; Andreas; (Zurich,
CH) ; Rottensteiner; Hanspeter; (Vienna, AT) ;
Siekmann; Jurgen; (Vienna, AT) ; Turecek; Peter;
(Klosterneuburg, AT) ; Zoechling; Oliver; (Vienna,
AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baxalta Incorporated
Baxalta GmbH |
Bannockburn
Zug |
IL |
US
CH |
|
|
Family ID: |
45556599 |
Appl. No.: |
16/532212 |
Filed: |
August 5, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15281616 |
Sep 30, 2016 |
10414793 |
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16532212 |
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14136233 |
Dec 20, 2013 |
9492555 |
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15281616 |
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13194038 |
Jul 29, 2011 |
8642737 |
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14136233 |
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12843542 |
Jul 26, 2010 |
8637640 |
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13194038 |
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61369186 |
Jul 30, 2010 |
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61347136 |
May 21, 2010 |
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61228828 |
Jul 27, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/60 20170801;
C07K 1/1077 20130101; C07K 1/34 20130101; A61K 47/61 20170801; C07K
14/755 20130101; C07K 1/20 20130101 |
International
Class: |
C07K 1/107 20060101
C07K001/107; C07K 1/20 20060101 C07K001/20; C07K 1/34 20060101
C07K001/34; C07K 14/755 20060101 C07K014/755; A61K 47/61 20060101
A61K047/61; A61K 47/60 20060101 A61K047/60 |
Claims
1.-72. (canceled)
73. A modified therapeutic protein comprising an activated water
soluble polymer conjugated to an oxidized carbohydrate moiety on a
therapeutic protein through forming a hydrazone linkage, wherein
said activated water soluble polymer contains an active hydrazide
group and is selected from the group consisting of polyethylene
glycol (PEG), branched PEG, PolyPEG.RTM. (Warwick Effect Polymers;
Coventry, UK), polysialic acid (PSA), starch, hydroxyalkyl starch
(HAS), hydroxylethyl starch (HES), carbohydrate, polysaccharides,
pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan
sulfate, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO),
polyalkylene glycol (PAG), polypropylene glycol (PPG),
polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),
polycarboxylate, polyvinylpyrrolidone, polyphosphazene,
polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid
anhydride, and poly(l-hydroxymethylethylene hydroxymethylformal)
(PHF); wherein the modified therapeutic protein is prepared by a
method comprising the steps of: a) oxidizing a carbohydrate moiety
on a therapeutic protein by incubating said protein with an
oxidizing agent selected from the group consisting of sodium
periodate (NaIO.sub.4), lead tetraacetate (Pb(OAc).sub.4) and
potassium perruthenate (KRuO.sub.4); and b) forming a hydrazone
linkage between the oxidized carbohydrate moiety of the therapeutic
protein and the activated water soluble polymer containing an
active hydrazide group in the presence of a nucleophilic catalyst
under conditions allowing formation of said hydrazone linkage; and
wherein the nucleophilic catalyst is m-toluidine.
74. The modified therapeutic protein of claim 73, wherein the
therapeutic protein is selected from the group consisting of Factor
IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), Von Willebrand
Factor (VWF), Factor V (FV), Factor X (FX), Factor XI (FXI), Factor
XII (FXII), thrombin (FII), protein C, protein S, tPA, PAI-1,
tissue factor (TF), ADAMTS 13 protease, IL-1 alpha, IL-1 beta,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, human growth hormone (HGH),
tumor necrosis factor-alpha (TNF-alpha), colony stimulating
factor-1 (CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony
stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha),
consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL-32 alpha,
IL-33, thrombopoietin (TPO), Ang-1, Ang-2, Ang-4, Ang-Y,
angiopoietin-like polypeptide 1 (ANGPTL1), angiopoietin-like
polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide 3 (ANGPTL3),
angiopoietin-like polypeptide 4 (ANGPTL4), angiopoietin-like
polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide 6 (ANGPTL6),
angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin, vascular
endothelial growth factor (VEGF), angiogenin, activin A, activin B,
activin C, bone morphogenic protein-1, bone morphogenic protein-2,
bone morphogenic protein-3, bone morphogenic protein-4, bone
morphogenic protein-5, bone morphogenic protein-6, bone morphogenic
protein-7, bone morphogenic protein-8, bone morphogenic protein-9,
bone morphogenic protein-10, bone morphogenic protein-11, bone
morphogenic protein-12, bone morphogenic protein-13, bone
morphogenic protein-14, bone morphogenic protein-15, bone
morphogenic protein receptor IA, bone morphogenic protein receptor
IB, bone morphogenic protein receptor II, brain derived
neurotrophic factor, cardiotrophin-1, ciliary neurotrophic factor,
ciliary neurotrophic factor receptor, cripto, cryptic,
cytokine-induced neutrophil chemotactic factor 1, cytokine-induced
neutrophil chemotactic factor 2.alpha., cytokine-induced neutrophil
chemotactic factor 2.beta., .beta. endothelial cell growth factor,
endothelin 1, epidermal growth factor, epigen, epiregulin,
epithelial-derived neutrophil attractant, fibroblast growth factor
4, fibroblast growth factor 5, fibroblast growth factor 6,
fibroblast growth factor 7, fibroblast growth factor 8, fibroblast
growth factor 8b, fibroblast growth factor 8c, fibroblast growth
factor 9, fibroblast growth factor 10, fibroblast growth factor 11,
fibroblast growth factor 12, fibroblast growth factor 13,
fibroblast growth factor 16, fibroblast growth factor 17,
fibroblast growth factor 19, fibroblast growth factor 20,
fibroblast growth factor 21, fibroblast growth factor acidic,
fibroblast growth factor basic, glial cell line-derived
neurotrophic factor receptor .alpha.1, glial cell line-derived
neurotrophic factor receptor .alpha.2, growth related protein,
growth related protein .alpha., growth related protein .alpha.,
growth related protein .gamma., heparin binding epidermal growth
factor, hepatocyte growth factor, hepatocyte growth factor
receptor, hepatoma-derived growth factor, insulin-like growth
factor I, insulin-like growth factor receptor, insulin-like growth
factor II, insulin-like growth factor binding protein, keratinocyte
growth factor, leukemia inhibitory factor, leukemia inhibitory
factor receptor .alpha., nerve growth factor, nerve growth factor
receptor, neuropoietin, neurotrophin-3, neurotrophin-4, oncostatin
M (OSM), placenta growth factor, placenta growth factor 2,
platelet-derived endothelial cell growth factor, platelet derived
growth factor, platelet derived growth factor A chain, platelet
derived growth factor AA, platelet derived growth factor AB,
platelet derived growth factor B chain, platelet derived growth
factor BB, platelet derived growth factor receptor .alpha.,
platelet derived growth factor receptor 13, pre-B cell growth
stimulating factor, stem cell factor (SCF), stem cell factor
receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor
.alpha., transforming growth factor .beta., transforming growth
factor .beta.1, transforming growth factor .beta.1.2, transforming
growth factor .beta.2, transforming growth factor .beta.3,
transforming growth factor .beta.5, latent transforming growth
factor .beta.1, transforming growth factor .beta. binding protein
I, transforming growth factor .beta. binding protein II,
transforming growth factor .beta. binding protein III, thymic
stromal lymphopoietin (TSLP), tumor necrosis factor receptor type
I, tumor necrosis factor receptor type II, urokinase-type
plasminogen activator receptor, phospholipase-activating protein
(PUP), insulin, lectin, ricin, prolactin, chorionic gonadotropin,
follicle-stimulating hormone, thyroid-stimulating hormone, tissue
plasminogen activator, IgG, IgE, IgM, IgA, and IgD,
.alpha.-galactosidase, .beta.-galactosidase, DNAse, fetuin,
luteinizing hormone, estrogen, albumin, lipoproteins, fetoprotein,
transferrin, thrombopoietin, urokinase, integrin, thrombin, leptin,
Humira (adalimumab), Prolia (denosumab), Enbrel (etanercept), a
protein in Table 1, or a biologically active fragment, derivative
or variant thereof.
75. The modified therapeutic protein of claim 74, wherein the
therapeutic protein has biological activity of a blood coagulation
protein.
76. The modified therapeutic protein of claim 75, wherein the blood
cogulation protein is selected from the group consisting of FVIIa,
FVIII and FIX.
77. The modified therapeutic protein of claim 73, wherein the water
soluble polymer is PEG or PSA.
78. The modified therapeutic protein of claim 73, wherein the
oxidizing agent is sodium periodate (NaIO.sub.4) and is added in an
amount to result in a final concentration between about 50 .mu.M
and about 1000 .mu.M under conditions comprising a time period
between about 0.1 minutes and 120 minutes; a temperature between
about 2.degree. C. and about 37.degree. C.; in the presence or
absence of light; and with or without stirring.
79. The modified therapeutic protein of claim 73, wherein the
m-toluidine is provided at a concentration between 1 mM and about
50 mM.
80. The modified therapeutic protein of claim 74, wherein the
therapeutic protein has biological activity of FVIIa, FVIII or FIX;
wherein the water soluble polymer is PEG or PSA; wherein the
oxidizing agent is sodium periodate (NaIO4) and is added in an
amount to result in a final concentration between about 50 .mu.M
and about 1000 .mu.M; and wherein the m-toluidine is provided at a
concentration between 1 mM and about 50 mM.
81. The modified therapeutic protein of claim 80, wherein the
oxidizing agent is sodium periodate (NaIO.sub.4) and is added in an
amount to result in a final concentration of 400 .mu.M; and wherein
the m-toluidine is provided at a concentration of 10 mM.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 15/281,616, filed Sep. 30, 2016, which
is a continuation application of U.S. patent application Ser. No.
14/136,233, filed Dec. 20, 2013, now U.S. Pat. No. 9,492,555, which
is a continuation application of U.S. patent application Ser. No.
13/194,038, filed Jul. 29, 2011, now U.S. Pat. No. 8,642,737, which
claims benefit to U.S. Provisional No. 61/369,186, filed Jul. 30,
2010, and is a Continuation-In-Part of U.S. patent application Ser.
No. 12/843,542, filed Jul. 26, 2010, now U.S. Pat. No. 8,637,640,
which claims benefit of to U.S. Provisional No. 61/347,136, filed
May 21, 2010 and U.S. Provisional No. 61/228,828, filed Jul. 27,
2009, all of which are incorporated herein by reference in its
entirety.
REFERENCE TO A SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, is
named "SEQUENCE LISTING" and is 4,096 kilobytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to materials and methods for
conjugating a water soluble polymer to a protein.
BACKGROUND OF THE INVENTION
[0004] The preparation of conjugates by forming a covalent linkage
between the water soluble polymer and the therapeutic protein can
be carried out by a variety of chemical methods. PEGylation of
polypeptide drugs protects them in circulation and improves their
pharmacodynamic and pharmacokinetic profiles (Harris and Chess, Nat
Rev Drug Discov. 2003; 2:214-21). The PEGylation process attaches
repeating units of ethylene glycol (polyethylene glycol (PEG)) to a
polypeptide drug. PEG molecules have a large hydrodynamic volume
(5-10 times the size of globular proteins), are highly water
soluble and hydrated, non-toxic, non-immunogenic and rapidly
cleared from the body. PEGylation of molecules can lead to
increased resistance of drugs to enzymatic degradation, increased
half-life in vivo, reduced dosing frequency, decreased
immunogenicity, increased physical and thermal stability, increased
solubility, increased liquid stability, and reduced aggregation.
The first PEGylated drugs were approved by the FDA in the early
1990s. Since then, the FDA has approved several PEGylated drugs for
oral, injectable, and topical administration.
[0005] Polysialic acid (PSA), also referred to as colominic acid
(CA), is a naturally occurring polysaccharide. It is a homopolymer
of N-acetylneuraminic acid with .alpha.(2.fwdarw.8) ketosidic
linkage and contains vicinal diol groups at its non-reducing end.
It is negatively charged and a natural constituent of the human
body. It can easily be produced from bacteria in large quantities
and with pre-determined physical characteristics (U.S. Pat. No.
5,846,951). Because the bacterially-produced PSA is chemically and
immunologically identical to PSA produced in the human body,
bacterial PSA is non-immunogenic, even when coupled to proteins.
Unlike some polymers, PSA acid is biodegradable. Covalent coupling
of colominic acid to catalase and asparaginase has been shown to
increase enzyme stability in the presence of proteolytic enzymes or
blood plasma. Comparative studies in vivo with polysialylated and
unmodified asparaginase revealed that polysialylation increased the
half-life of the enzyme (Fernandes and Gregoriadis, Int J Pharm.
2001; 217:215-24).
[0006] Coupling of PEG-derivatives to peptides or proteins is
reviewed by Roberts et al. (Adv Drug Deliv Rev 2002; 54:459-76).
One approach for coupling water soluble polymers to therapeutic
proteins is the conjugation of the polymers via the carbohydrate
moieties of the protein. Vicinal hydroxyl (OH) groups of
carbohydrates in proteins can be easily oxidized with sodium
periodate (NaIO4) to form active aldehyde groups (Rothfus et Smith,
J Biol Chem 1963; 238:1402-10; van Lenten et Ashwell, J Biol Chem
1971; 246:1889-94). Subsequently the polymer can be coupled to the
aldehyde groups of the carbohydrate by use of reagents containing,
for example, an active hydrazide group (Wilchek M and Bayer E A,
Methods Enzymol 1987; 138:429-42). A more recent technology is the
use of reagents containing aminooxy groups which react with
aldehydes to form oxime linkages (WO 96/40662, WO2008/025856).
[0007] Additional examples describing conjugation of a water
soluble polymer to a therapeutic protein are described in WO
06/071801 which teaches the oxidation of carbohydrate moieties in
Von Willebrand factor and subsequent coupling to PEG using
hydrazide chemistry; US Publication No. 2009/0076237 which teaches
the oxidation of rFVIII and subsequent coupling to PEG and other
water soluble polymers (e.g. PSA, HES, dextran) using hydrazide
chemistry; WO 2008/025856 which teaches oxidation of different
coagulation factors, e.g. rFIX, FVIII and FVIIa and subsequent
coupling to e.g., PEG, using aminooxy chemistry by forming an oxime
linkage; and U.S. Pat. No. 5,621,039 which teaches the oxidation of
FIX and subsequent coupling to PEG using hydrazide chemistry.
[0008] Recently, an improved method was described comprising mild
periodate oxidation of sialic acids to generate aldehydes followed
by reaction with an aminooxy group containing reagent in the
presence of catalytic amounts of aniline (Dirksen A., and Dawson P
E, Bioconjugate Chem. 2008; 19, 2543-8; and Zeng Y et al., Nature
Methods 2009; 6:207-9). The aniline catalysis dramatically
accelerates the oxime ligation, allowing the use of very low
concentrations of the reagent. The use of nucelophilic catalysts
are also described in Dirksen, A., et al., J Am Chem Soc.,
128:15602-3 (2006); Dirksen, A., et al., Angew chem. Int Ed.,
45:7581-4 (2006); Kohler, J. J., ChemBioChem., 10:2147-50 (2009);
Giuseppone, N., et al., J Am Chem Soc., 127:5528-39 (2005); and
Thygesen, M. B., et al., J Org Chem., 75:1752-5 (2010).
[0009] Although aniline catalysis can accelerate the oxime ligation
allowing short reaction times and the use of low concentrations of
the aminooxy reagent, aniline has toxic properties that must be
considered when, for example, the conjugated therapeutic protein to
form the basis of a pharmaceutical. For example, aniline has been
shown to induce methemoglobinemia (Harrison, J. H., and Jollow, D.
J., Molecular Pharmacology, 32(3) 423-431, 1987). Long-term dietary
treatment of rats has been shown to induce tumors in the spleen
(Goodman, D G., et al., J Natl Cancer Inst., 73(1):265-73, 1984).
In vitro studies have also shown that aniline has the potential to
induce chromosome mutations and has the potentially genotoxic
activity (Bombhard E. M. et Herbold B, Critical Reviews in
Toxicology 35, 783-835, 2005).
[0010] Considering the potentially dangerous properties of aniline
and notwithstanding the methods available of conjugating water
soluble polymers to therapeutic proteins, there remains a need to
develop materials and methods for conjugating water soluble
polymers to proteins that improves the protein's pharmacodynamic
and/or pharmacokinetic properties while minimizing the costs
associated with the various reagents and minimizing the health
risks to the patient recipient.
SUMMARY OF THE INVENTION
[0011] The present invention provides materials and methods for
conjugating polymers to proteins that improves the protein's
pharmacodynamic and/or pharmacokinetic properties while minimizing
the costs associated with the various reagents and the health risks
to the patient recipients when the conjugation reaction is
catalyzed by a nucleophilic catalyst. In various embodiments of the
invention, alternative catalysts to substitute for aniline are
provided.
[0012] In one embodiment, a method of conjugating a water soluble
polymer to an oxidized carbohydrate moiety of a therapeutic protein
is provided comprising contacting the oxidized carbohydrate moiety
with an activated water soluble polymer under conditions that allow
conjugation; said water soluble polymer containing an active
aminooxy group and is selected from the group consisting of
polyethylene glycol (PEG), branched PEG, PolyPEG.RTM. (Warwick
Effect Polymers; Coventry, UK), polysialic acid (PSA), starch,
hydroxyalkyl starch (HAS), hydroxylethyl starch (HES),
carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic
acid, chondroitin sulfate, dermatan sulfate, starch, dextran,
carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene
glycol (PAG), polypropylene glycol (PPG), polyoxazoline,
polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate,
polyvinylpyrrolidone, polyphosphazene, polyoxazoline,
polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid
anhydride, poly(l-hydroxymethylethylene hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC); and
said carbohydrate moiety oxidized by incubation with a buffer
comprising an oxidizing agent selected from the group consisting of
sodium periodate (NaIO4), lead tetraacetate (Pb(OAc)4) and
potassium perruthenate (KRuO4); wherein an oxime linkage is formed
between the oxidized carbohydrate moiety and the active aminooxy
group on the water soluble polymer; and wherein said oxime linkage
formation is catalyzed by a nucleophilic catalyst selected from the
group consisting of o-amino benzoic acid, m-amino benzoic acid,
p-amino benzoic acid, sulfanilic acid, o-aminobenzamide,
o-toluidine, m-toluidine, p-toluidine, o-anisidine, m-anisidine,
and p-anisidine.
[0013] In another embodiment, a method of conjugating a water
soluble polymer to an oxidized carbohydrate moiety of a therapeutic
protein is provided comprising contacting the oxidized carbohydrate
moiety with an activated water soluble polymer under conditions
that allow conjugation; said therapeutic protein selected from the
group consisting of Factor IX (FIX), Factor VIII (FVIII), Factor
VIIa (FVIIa), Von Willebrand Factor (VWF), Factor FV (FV), Factor X
(FX), Factor XI (FXI), Factor XII (FXII), thrombin (FII), protein
C, protein S, tPA, PAI-1, tissue factor (TF), ADAMTS 13 protease,
IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony
stimulating factor-1 (CSF-1), M-CSF, SCF, GM-CSF, granulocyte
colony stimulating factor (G-CSF), EPO, interferon-alpha
(IFN-alpha), consensus interferon, IFN-beta, IFN-gamma, IFN-omega,
IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,
IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL-32
alpha, IL-33, thrombopoietin (TPO), Ang-1, Ang-2, Ang-4, Ang-Y,
angiopoietin-like polypeptide 1 (ANGPTL1), angiopoietin-like
polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide 3 (ANGPTL3),
angiopoietin-like polypeptide 4 (ANGPTL4), angiopoietin-like
polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide 6 (ANGPTL6),
angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin, vascular
endothelial growth factor (VEGF), angiogenin, activin A, activin B,
activin C, bone morphogenic protein-1, bone morphogenic protein-2,
bone morphogenic protein-3, bone morphogenic protein-4, bone
morphogenic protein-5, bone morphogenic protein-6, bone morphogenic
protein-7, bone morphogenic protein-8, bone morphogenic protein-9,
bone morphogenic protein-10, bone morphogenic protein-11, bone
morphogenic protein-12, bone morphogenic protein-13, bone
morphogenic protein-14, bone morphogenic protein-15, bone
morphogenic protein receptor IA, bone morphogenic protein receptor
IB, bone morphogenic protein receptor II, brain derived
neurotrophic factor, cardiotrophin-1, ciliary neutrophic factor,
ciliary neutrophic factor receptor, cripto, cryptic,
cytokine-induced neutrophil chemotactic factor 1, cytokine-induced
neutrophil, chemotactic factor 2.alpha., cytokine-induced
neutrophil chemotactic factor 2.beta.,.beta. endothelial cell
growth factor, endothelin 1, epidermal growth factor, epigen,
epiregulin, epithelial-derived neutrophil attractant, fibroblast
growth factor 4, fibroblast growth factor 5, fibroblast growth
factor 6, fibroblast growth factor 7, fibroblast growth factor 8,
fibroblast growth factor 8b, fibroblast growth factor 8c,
fibroblast growth factor 9, fibroblast growth factor 10, fibroblast
growth factor 11, fibroblast growth factor 12, fibroblast growth
factor 13, fibroblast growth factor 16, fibroblast growth factor
17, fibroblast growth factor 19, fibroblast growth factor 20,
fibroblast growth factor 21, fibroblast growth factor acidic,
fibroblast growth factor basic, glial cell line-derived neutrophic
factor receptor .alpha.1, glial cell line-derived neutrophic factor
receptor .alpha.2, growth related protein, growth related protein
.alpha., growth related protein .beta., growth related protein
.gamma., heparin binding epidermal growth factor, hepatocyte growth
factor, hepatocyte growth factor receptor, hepatoma-derived growth
factor, insulin-like growth factor I, insulin-like growth factor
receptor, insulin-like growth factor II, insulin-like growth factor
binding protein, keratinocyte growth factor, leukemia inhibitory
factor, leukemia inhibitory factor receptor .alpha., nerve growth
factor nerve growth factor receptor, neuropoietin, neurotrophin-3,
neurotrophin-4, oncostatin M (OSM), placenta growth factor,
placenta growth factor 2, platelet-derived endothelial cell growth
factor, platelet derived growth factor, platelet derived growth
factor A chain, platelet derived growth factor AA, platelet derived
growth factor AB, platelet derived growth factor B chain, platelet
derived growth factor BB, platelet derived growth factor receptor
.alpha., platelet derived growth factor receptor .beta., pre-B cell
growth stimulating factor, stem cell factor (SCF), stem cell factor
receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor
.alpha., transforming growth factor (3, transforming growth factor
.beta.1, transforming growth factor .beta.1.2, transforming growth
factor .beta.2, transforming growth factor .beta.3, transforming
growth factor .beta.5, latent transforming growth factor .beta.1,
transforming growth factor .beta. binding protein I, transforming
growth factor .beta. binding protein II, transforming growth factor
.beta. binding protein III, thymic stromal lymphopoietin (TSLP),
tumor necrosis factor receptor type I, tumor necrosis factor
receptor type II, urokinase-type plasminogen activator receptor,
phospholipase-activating protein (PUP), insulin, lectin ricin,
prolactin, chorionic gonadotropin, follicle-stimulating hormone,
thyroid-stimulating hormone, tissue plasminogen activator, IgG,
IgE, IgM, IgA, and IgD, .alpha.-galactosidase,
.beta.-galactosidase, DNAse, fetuin, leutinizing hormone, estrogen,
insulin, albumin, lipoproteins, fetoprotein, transferrin,
thrombopoietin, urokinase, integrin, thrombin, leptin, Humira
(adalimumab), Prolia (denosumab), Enbrel (etanercept), a protein in
Table 1, or a biologically active fragment, derivative or variant
thereof; said water soluble polymer containing an active aminooxy
group and is selected from the group consisting of polyethylene
glycol (PEG), branched PEG, PolyPEG.RTM. (Warwick Effect Polymers;
Coventry, UK), polysialic acid (PSA), starch, hydroxyalkyl starch
(HAS), hydroxylethyl starch (HES), carbohydrate, polysaccharides,
pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan
sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide
(PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG),
polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),
polycarboxylate, polyvinylpyrrolidone, polyphosphazene,
polyoxazoline, polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, poly(l-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC); and
said carbohydrate moiety oxidized by incubation with a buffer
comprising an oxidizing agent selected from the group consisting of
sodium periodate (NaIO4), lead tetraacetate (Pb(OAc)4) and
potassium perruthenate (KRuO4); wherein an oxime linkage is formed
between the oxidized carbohydrate moiety and the active aminooxy
group on the water soluble polymer; and wherein in said oxime
linkage formation is catalyzed by a nucleophilic catalyst selected
from the group consisting of o-amino benzoic acid, m-amino benzoic
acid, p-amino benzoic acid, sulfanilic acid, o-aminobenzamide,
o-toluidine, m-toluidine, p-toluidine, o-anisidine, m-anisidine,
and p-anisidine.
[0014] In still another embodiment, an aforementioned method is
provided wherein a solution comprising an initial concentration of
the therapeutic protein between about 0.3 mg/ml and about 3.0 mg/ml
is adjusted to a pH value between about 5.0 and about 8.0 prior to
contacting with the activated water soluble polymer.
[0015] As used herein, the term "about" means a value above or
below a stated value. In various embodiments, the term "about"
includes the stated value plus or minus 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% of the stated
value.
[0016] In yet another embodiment, an aforementioned method is
provided wherein the initial concentration of the therapeutic
protein is about 1.0 mg/ml and the pH is about 6.0. In a related
embodiment, the initial concentration of the therapeutic protein is
about 0.75 mg/ml and the pH is about 6.0. In still another related
embodiment, the initial concentration of the therapeutic protein is
about 1.25 mg/ml and the pH is about 6.0.
[0017] In another embodiment, an aforementioned method is provided
wherein the therapeutic protein is contacted by a desired excess
concentration of activated water soluble polymer, wherein the
excess concentration is between about 1-molar and about 300-molar
excess. In another embodiment, the excess concentration is about
50-fold molar excess.
[0018] In still another embodiment, an aforementioned method is
provided wherein the therapeutic protein is incubated with the
activated water soluble polymer under conditions comprising a time
period between about 0.5 hours and about 24 hours; a temperature
between about 2.degree. C. and about 37.degree. C.; in the presence
or absence of light; and with or without stirring. In another
embodiment, the conditions comprise a time period of about 120
minutes, a temperature of about 22.degree. C., the absence of
light; and with stirring. As used herein, the term "stirring" is
meant to include stirring at various speeds and intensities (e.g.,
gentle stirring) by commonly used laboratory or manufacturing
equipment and products.
[0019] In another embodiment, an aforementioned method is provided
wherein the nucleophilic catalyst is added in an amount to result
in a final concentration between about 1.0 mM and about 50 mM
nucleophilic catalyst, under conditions comprising a time period
between about 0.1 minutes and about 30 minutes; a temperature
between about 2.degree. C. and about 37.degree. C.; in the presence
or absence of light; and with or without stirring. In another
embodiment, the final concentration of the nucleophilic catalyst is
about 10 mM, and the conditions comprise a time period of up to
about 15 minutes, a temperature of about 22.degree. C., the absence
of light; and with stirring.
[0020] In still another embodiment, an aforementioned method is
provided wherein the oxidizing agent is added in an amount to
result in a final concentration between about 50 .mu.M and about
1000 .mu.M oxidizing agent, under conditions comprising a time
period between about 0.1 minutes and 120 minutes; a temperature
between about 2.degree. C. and about 37.degree. C.; in the presence
or absence of light; and with or without stirring. In another
embodiment, the final concentration of oxidizing agent is about 400
.mu.M, and the conditions comprise a time period of about 10
minutes, a temperature of about 22.degree. C., the absence of light
and with stirring.
[0021] In yet another embodiment, an aforementioned method is
provided wherein the conjugating the water soluble polymer to the
oxidized carbohydrate moiety of the therapeutic protein is stopped
by the addition of a quenching agent selected from the group
consisting of L-cysteine, methionine, glutathione, glycerol, sodium
meta bisulfate (Na2S2O5), tryptophane, tyrosine, histidine or
derivatives thereof, kresol, imidazol, and combinations thereof;
wherein the quenching agent is added in an amount to result in a
final concentration between about 1 mM and about 100 mM quenching
agent, under conditions comprising a time period between about 5
minutes and about 120 minutes; a temperature between about
2.degree. C. and about 37.degree. C.; in the presence or absence of
light; and with or without stirring. In another embodiment, the
quenching agent is L-cysteine. In still another embodiment, the
L-cysteine is added to result in a final concentration of about 10
mM and the conditions comprise a time period of about 60 minutes, a
temperature of about 22.degree. C., the absence of light and with
stirring.
[0022] In another embodiment, an aforementioned method is provided
comprising: a) a first step comprising adjusting the pH value of a
solution comprising the therapeutic protein to a pH value between
about 5.0 and about 8.0, wherein the therapeutic protein
concentration is between about 0.3 mg/ml and about 3.0 mg/ml; b) a
second step comprising oxidizing one or more carbohydrates on the
therapeutic protein, wherein the oxidizing agent is added to the
solution in the first step to result in a final concentration
between about 50 .mu.M and about 1000 .mu.M, under conditions
comprising a time period between about 0.1 minutes and about 120
minutes; a temperature between about 2.degree. C. and about
37.degree. C.; in the presence or absence of light, and with or
without stirring; c) a third step comprising contacting the
therapeutic protein with a desired excess concentration of
activated water soluble polymer, wherein the excess concentration
is between about 1-molar excess and about 300-molar excess, under
conditions comprising a time period between about 0.5 hours and
about 24 hours, a temperature between about 2.degree. C. and about
37.degree. C.; in the presence or absence of light; and with or
without stirring; d) a fourth step comprising adding a nucleophilic
catalyst to the solution of the third step, wherein the
nucleophilic catalyst is added to result in a final concentration
between about 1 mM and about 50 mM, under conditions comprising a
time period between about 0.1 minutes and about 30 minutes; a
temperature between about 2.degree. C. and about 37.degree. C.; in
the presence or absence of light, and with or without stirring; e)
a fifth step wherein the therapeutic protein is incubated with the
activated water soluble polymer and nucleophilic catalyst under
conditions that allow conjugation of the activated water-soluble
polymer to one or more oxidized carbohydrates on the therapeutic
protein, said conditions comprising a time period between about 0.5
hours and about 24 hours, a temperature between about 2.degree. C.
and about 37.degree. C.; in the presence or absence of light, and
with or without stirring; and f) a sixth step wherein the
conjugating the water soluble polymer to the one or more oxidized
carbohydrates of the therapeutic protein in the fifth step is
stopped by the addition of a quenching agent selected from the
group consisting of L-cysteine, methionine, glutathione, glycerol,
Na2S205 (sodium meta bisulfite), tryptophane, tyrosine, histidine
or derivatives thereof, kresol, imidazol, and combinations thereof;
wherein the quenching agent is added to result in a final
concentration of about 1 mM and about 100 mM, under conditions
comprising a time period between about 5 minutes and about 120
minutes; a temperature between about 2.degree. C. and about
37.degree. C.; in the presence or absence of light, and with or
without stirring. In another embodiment, the initial concentration
of the therapeutic protein in the first step is about 1 mg/ml and
the pH is about 6.0; wherein the final concentration of oxidizing
agent in the second step is about 400 .mu.M, and the conditions in
the fifth step comprise a time period of about 10 minutes, a
temperature of about 22.degree. C., the absence of light and with
stirring; wherein the excess concentration in the third step is
about 50 molar excess; wherein the conditions in the third step
comprise a time period of about 15 minutes, a temperature of about
22.degree. C., the absence of light and with stirring; wherein the
final concentration of the nucleophilic catalyst in the fourth step
is about 10 mM, and the conditions in the fourth step comprise a
time period of about 15 minutes, a temperature of about 22.degree.
C., the absence of light and with stirring; wherein the conditions
of incubating the therapeutic protein with the activated water
soluble polymer and nucleophilic catalyst in the fifth step
comprise a time period of about 2 hours; a temperature of about
22.degree. C.; the absence of light; and with stirring; and wherein
the quenching agent in the sixth step is L-cysteine; and wherein
the L-cysteine is added to result in a final concentration of about
10 mM and the conditions in the sixth step comprise a time period
of about 60 minutes, a temperature of about 22.degree. C., the
absence of light and with stirring.
[0023] In another embodiment, an aforementioned method is provided
wherein the water soluble polymer is PSA. In another embodiment the
PSA is comprised of about 10-300 sialic acid units. In another
embodiment, the water soluble polymer is PEG. In another
embodiment, the water soluble polymer is HES. In still another
embodiment, the water soluble polymer is HAS.
[0024] In still another embodiment, an aforementioned method is
provided wherein the therapeutic protein is FIX. In another
embodiment, the therapeutic protein is FVIIa. In another
embodiment, the therapeutic protein is FVIII.
[0025] In yet another embodiment, an aforementioned method is
provided wherein the oxidizing agent is sodium periodate
(NaIO4).
[0026] In another embodiment, an aforementioned method is provided
wherein the oxidized carbohydrate moiety of the therapeutic protein
is located in the activation peptide of the blood coagulation
protein.
[0027] In one embodiment, an aforementioned method is provided
wherein PSA is prepared by reacting an activated aminooxy linker
with oxidized PSA; wherein the aminooxy linker is selected from the
group consisting of: [0028] a) a 3-oxa-pentane-1,5-dioxyamine
linker of the formula:
[0028] ##STR00001## [0029] b) a
3,6,9-trioxa-undecane-1,11-dioxyamine linker of the formula:
##STR00002##
[0030] and [0031] c) a
3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine linker of the
formula:
##STR00003##
[0032] wherein the PSA is oxidized by incubation with a oxidizing
agent to form a terminal aldehyde group at the non-reducing end of
the PSA. In a related embodiment, the aminooxy linker is
3-oxa-pentane-1,5-dioxyamine.
[0033] In still another embodiment, an aforementioned method is
provided wherein the oxidizing agent is NaIO4.
[0034] In another embodiment, an aforementioned method is provided
wherein the nucleophilic catalyst is provided at a concentration
between about 1 mM and about 50 mM. In one embodiment, the
nucleophilic catalyst is m-toluidine. In still another embodiment,
the m-toluidine is present in the conjugation reaction at a
concentration of about 10 mM.
[0035] In yet another embodiment, an aforementioned method is
provided further comprising the step of reducing an oxime linkage
in the conjugated therapeutic protein by incubating the conjugated
therapeutic protein in a buffer comprising a reducing compound
selected from the group consisting of sodium cyanoborohydride
(NaCNBH3), ascorbic acid (vitamin C) and NaBH3. In one embodiment,
the reducing compound is sodium cyanoborohydride (NaCNBH3).
[0036] In still another embodiment, an aforementioned method is
provided further comprising the step of purifying the conjugated
therapeutic protein. In another embodiment, the conjugated
therapeutic protein is purified by a method selected from the group
consisting of chromatography, filtration and precipitation. In
another embodiment, the chromatography is selected from the group
consisting of Hydrophobic Interaction Chromatography (HIC), Ion
Exchange chromatography (IEC), Size exclusion chromatography (SEC),
Affinity chromatography, and Reversed-phase chromatography. In
still another embodiment, an anti-chaotropic salt is used in a
chromotagraphy loading step and in a chromatography washing step.
In yet another embodiment, the chromatography takes place in a
column. In another embodiment, the column comprises a
chromatography resin selected from the group consisting of
Phenyl-Sepharose FF and Butyl-Sepharose FF. In another embodiment,
the resin is present in the column at a bed height of between about
5 cm and about 20 cm. In one embodiment, the bed height is about 10
cm.
[0037] In another embodiment, an aforementioned method is provided
comprising one or more washing steps wherein flow direction is set
to up-flow and wherein the flow rate is between about 0.2 cm/min
and about 6.7 cm/min. As used herein, the term "down-flow" refers
to a flow direction from the top of the chromatographic column to
the bottom of the chromatographic column (normal flow
direction/standard mode). As used herein, the term "up-flow" refers
to a flow direction from the bottom to the top of the column
(reversed flow direction). In one embodiment, the flow rate is
about 2 cm/min.
[0038] In another embodiment, an aforementioned method is provided
comprising one or more elution steps wherein flow direction is set
to down-flow and wherein the flow rate is between about 0.1 cm/min
and about 6.7 cm/min. In a related embodiment, the flow rate is
about 1 cm/min.
[0039] In still another embodiment, an aforementioned method is
provided comprising concentrating the conjugated therapeutic
protein by ultra-/diafiltration (UF/DF). In another embodiment, the
final concentration of therapeutic protein is between about 0.5 and
about 3 mg/ml.
[0040] In another embodiment, an aforementioned method is provided
wherein the therapeutic protein comprises between about 5 and about
11 water-soluble polymer moieties. In another embodiment, the
therapeutic protein comprises between about 1 and about 3
water-soluble polymers.
[0041] In still another embodiment, an aforementioned method is
provided wherein the conjugated therapeutic protein is purified
using chromatography; wherein an anti-chaotropic salt is used for a
loading step and for a washing step; the method comprising one or
more washing steps wherein flow direction is set to up-flow and
wherein the flow rate is between about 0.2 cm/min and about 6.7
cm/min and one or more elution steps wherein flow direction is set
to down-flow and wherein the flow rate is between about 0.2 cm/min
andabout 6.7 cm/min; further comprising concentrating the
conjugated therapeutic protein by ultra-/diafiltration (UF/DF). In
another embodiment, the chromatography is hydrophobic interaction
chromatography (HIC); wherein the one or more washing steps flow
rate is about 2 cm/min; and wherein the one or more elution steps
flow rate is about 1 cm/min.
[0042] In another embodiment, a modified therapeutic protein
produced by any of the aforementioned methods is provided.
[0043] In still another embodiment, a method of forming an oxime
linkage between an oxidized carbohydrate moiety on a therapeutic
protein and an activated water soluble polymer containing an active
aminooxy group is provided comprising the steps of: a) oxidizing a
carbohydrate moiety on a therapeutic protein by incubating said
protein with an oxidizing agent selected from the group consisting
of sodium periodate (NaIO4), lead tetraacetate (Pb(OAc)4) and
potassium perruthenate (KRuO4); and b) forming an oxime linkage
between the oxidized carbohydrate moiety of the therapeutic protein
and the activated water soluble polymer containing an active
aminooxy group in the presence of a nuclephilic catalyst under
conditions allowing formation of said oxime linkage; wherein said
water soluble polymer containing an active aminooxy group is
selected from the group consisting polyethylene glycol (PEG),
branched PEG, PolyPEG.RTM. (Warwick Effect Polymers; Coventry, UK),
polysialic acid (PSA), starch, hydroxyalkyl starch (HAS),
hydroxylethyl starch (HES), carbohydrate, polysaccharides,
pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan
sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide
(PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG),
polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),
polycarboxylate, polyvinylpyrrolidone, polyphosphazene,
polyoxazoline, polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, poly(l-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC); wherein
the nucleophilic catalyst is selected from the group consisting of
o-amino benzoic acid, m-amino benzoic acid, p-amino benzoic acid,
sulfanilic acid, o-aminobenzamide, o-toluidine, m-toluidine,
p-toluidine, o-anisidine, m-anisidine, and p-anisidine.
[0044] In yet another embodiment, a method of forming an oxime
linkage between an oxidized carbohydrate moiety on a therapeutic
protein and an activated water soluble polymer containing an active
aminooxy group is provided comprising the steps of: a) oxidizing a
carbohydrate moiety on a therapeutic protein by incubating said
protein with an oxidinzing agent selected from the group consisting
of sodium periodate (NaIO4), lead tetraacetate (Pb(OAc)4) and
potassium perruthenate (KRuO4); and b) forming an oxime linkage
between the oxidized carbohydrate moiety of the therapeutic protein
and the activated water soluble polymer containing an an active
aminooxy group in the presence of a nuclephilic catalyst under
conditions allowing formation of said oxime linkage; wherein the
therapeutic protein is selected from the group consisting of Factor
IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), Von Willebrand
Factor (VWF), Factor FV (FV), Factor X (FX), Factor XI (FXI),
Factor XII (FXII), thrombin (FII), protein C, protein S, tPA,
PAI-1, tissue factor (TF), ADAMTS 13 protease, IL-1 alpha, IL-1
beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating
factor-1 (CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony
stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha),
consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL-32 alpha,
IL-33, thrombopoietin (TPO), Ang-1, Ang-2, Ang-4, Ang-Y,
angiopoietin-like polypeptide 1 (ANGPTL1), angiopoietin-like
polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide 3 (ANGPTL3),
angiopoietin-like polypeptide 4 (ANGPTL4), angiopoietin-like
polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide 6 (ANGPTL6),
angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin, vascular
endothelial growth factor (VEGF), angiogenin, activin A, activin B,
activin C, bone morphogenic protein-1, bone morphogenic protein-2,
bone morphogenic protein-3, bone morphogenic protein-4, bone
morphogenic protein-5, bone morphogenic protein-6, bone morphogenic
protein-7, bone morphogenic protein-8, bone morphogenic protein-9,
bone morphogenic protein-10, bone morphogenic protein-11, bone
morphogenic protein-12, bone morphogenic protein-13, bone
morphogenic protein-14, bone morphogenic protein-15, bone
morphogenic protein receptor IA, bone morphogenic protein receptor
IB, bone morphogenic protein receptor II, brain derived
neurotrophic factor, cardiotrophin-1, ciliary neutrophic factor,
ciliary neutrophic factor receptor, cripto, cryptic,
cytokine-induced neutrophil chemotactic factor 1, cytokine-induced
neutrophil, chemotactic factor 2a, cytokine-induced neutrophil
chemotactic factor 2.beta.,.beta. endothelial cell growth factor,
endothelin 1, epidermal growth factor, epigen, epiregulin,
epithelial-derived neutrophil attractant, fibroblast growth factor
4, fibroblast growth factor 5, fibroblast growth factor 6,
fibroblast growth factor 7, fibroblast growth factor 8, fibroblast
growth factor 8b, fibroblast growth factor 8c, fibroblast growth
factor 9, fibroblast growth factor 10, fibroblast growth factor 11,
fibroblast growth factor 12, fibroblast growth factor 13,
fibroblast growth factor 16, fibroblast growth factor 17,
fibroblast growth factor 19, fibroblast growth factor 20,
fibroblast growth factor 21, fibroblast growth factor acidic,
fibroblast growth factor basic, glial cell line-derived neutrophic
factor receptor .alpha.1, glial cell line-derived neutrophic factor
receptor .alpha.2, growth related protein, growth related protein
.alpha., growth related protein .beta., growth related protein
.gamma., heparin binding epidermal growth factor, hepatocyte growth
factor, hepatocyte growth factor receptor, hepatoma-derived growth
factor, insulin-like growth factor I, insulin-like growth factor
receptor, insulin-like growth factor II, insulin-like growth factor
binding protein, keratinocyte growth factor, leukemia inhibitory
factor, leukemia inhibitory factor receptor .alpha., nerve growth
factor nerve growth factor receptor, neuropoietin, neurotrophin-3,
neurotrophin-4, oncostatin M (OSM), placenta growth factor,
placenta growth factor 2, platelet-derived endothelial cell growth
factor, platelet derived growth factor, platelet derived growth
factor A chain, platelet derived growth factor AA, platelet derived
growth factor AB, platelet derived growth factor B chain, platelet
derived growth factor BB, platelet derived growth factor receptor
.alpha., platelet derived growth factor receptor .beta., pre-B cell
growth stimulating factor, stem cell factor (SCF), stem cell factor
receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor
.alpha., transforming growth factor .beta., transforming growth
factor .beta.1, transforming growth factor .beta.1.2, transforming
growth factor .beta.2, transforming growth factor .beta.3,
transforming growth factor .beta.5, latent transforming growth
factor .beta.1, transforming growth factor .beta. binding protein
I, transforming growth factor .beta. binding protein II,
transforming growth factor .beta. binding protein III, thymic
stromal lymphopoietin (TSLP), tumor necrosis factor receptor type
I, tumor necrosis factor receptor type II, urokinase-type
plasminogen activator receptor, phospholipase-activating protein
(PUP), insulin, lectin ricin, prolactin, chorionic gonadotropin,
follicle-stimulating hormone, thyroid-stimulating hormone, tissue
plasminogen activator, IgG, IgE, IgM, IgA, and IgD,
.alpha.-galactosidase, .beta.-galactosidase, DNAse, fetuin,
leutinizing hormone, estrogen, insulin, albumin, lipoproteins,
fetoprotein, transferrin, thrombopoietin, urokinase, integrin,
thrombin, leptin, Humira (adalimumab), Prolia (denosumab), Enbrel
(etanercept), a protein from Table 1, or a biologically active
fragment, derivative or variant thereof; wherein said water soluble
polymer containing an active aminooxy group is selected from the
group consisting of polyethylene glycol (PEG), branched PEG,
PolyPEG.RTM. (Warwick Effect Polymers; Coventry, UK), polysialic
acid (PSA), starch, hydroxyalkyl starch (HAS), hydroxylethyl starch
(HES), carbohydrate, polysaccharides, pullulane, chitosan,
hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch,
dextran, carboxymethyl-dextran, polyalkylene oxide (PAO),
polyalkylene glycol (PAG), polypropylene glycol (PPG),
polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),
polycarboxylate, polyvinylpyrrolidone, polyphosphazene,
polyoxazoline, polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, poly(l-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC); wherein
the nucleophilic catalyst is selected from the group consisting of
o-amino benzoic acid, m-amino benzoic acid, p-amino benzoic acid,
sulfanilic acid, o-aminobenzamide, o-toluidine, m-toluidine,
p-toluidine, o-anisidine, m-anisidine, and p-anisidine.
[0045] In yet another embodiment, a method of forming a hydrazone
linkage between an oxidized carbohydrate moiety on a therapeutic
protein and an activated water soluble polymer containing an active
hydrazide group is provided comprising the steps of: a) oxidizing a
carbohydrate moiety on a therapeutic protein by incubating said
protein with an oxidinzing agent selected from the group consisting
of sodium periodate (NaIO4), lead tetraacetate (Pb(OAc)4) and
potassium perruthenate (KRuO4); and b) forming a hydrazone linkage
between the oxidized carbohydrate moiety of the therapeutic protein
and the activated water soluble polymer containing an an active
hydrazide group in the presence of a nuclephilic catalyst under
conditions allowing formation of said hydrazone linkage; wherein
said water soluble polymer containing an active hydrazide group is
selected from the group consisting of polyethylene glycol (PEG),
branched PEG, PolyPEG.RTM. (Warwick Effect Polymers; Coventry, UK),
polysialic acid (PSA), starch, hydroxyalkyl starch (HAS),
hydroxylethyl starch (HES), carbohydrate, polysaccharides,
pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan
sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide
(PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG),
polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),
polycarboxylate, polyvinylpyrrolidone, polyphosphazene,
polyoxazoline, polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, poly(l-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC); wherein
the nucleophilic catalyst is selected from the group consisting of
o-amino benzoic acid, m-amino benzoic acid, p-amino benzoic acid,
sulfanilic acid, o-aminobenzamide, o-toluidine, m-toluidine,
p-toluidine, o-anisidine, m-anisidine, and p-anisidine.
[0046] In another embodiment, a method of forming a hydrazone
linkage between an oxidized carbohydrate moiety on a therapeutic
protein and an activated water soluble polymer containing an active
hydrazide group comprising the steps of: a) oxidizing a
carbohydrate moiety on a therapeutic protein by incubating said
protein with an oxidinzing agent selected from the group consisting
of sodium periodate (NaIO4), lead tetraacetate (Pb(OAc)4) and
potassium perruthenate (KRuO4); and b) forming a hydrazone linkage
between the oxidized carbohydrate moiety of the therapeutic protein
and the activated water soluble polymer containing an an active
hydrazide group in the presence of a nuclephilic catalyst under
conditions allowing formation of said hydrazone linkage; wherein
the therapeutic protein is selected from the group consisting of
Factor IX (FIX), Factor VIII (FVIII), Factor VIIa (FVIIa), Von
Willebrand Factor (VWF), Factor FV (FV), Factor X (FX), Factor XI
(FXI), Factor XII (FXII), thrombin (FII), protein C, protein S,
tPA, PAI-1, tissue factor (TF), ADAMTS 13 protease, IL-1 alpha,
IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating
factor-1 (CSF-1), M-CSF, SCF, GM-CSF, granulocyte colony
stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha),
consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL-32 alpha,
IL-33, thrombopoietin (TPO), Ang-1, Ang-2, Ang-4, Ang-Y,
angiopoietin-like polypeptide 1 (ANGPTL1), angiopoietin-like
polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide 3 (ANGPTL3),
angiopoietin-like polypeptide 4 (ANGPTL4), angiopoietin-like
polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide 6 (ANGPTL6),
angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin, vascular
endothelial growth factor (VEGF), angiogenin, activin A, activin B,
activin C, bone morphogenic protein-1, bone morphogenic protein-2,
bone morphogenic protein-3, bone morphogenic protein-4, bone
morphogenic protein-5, bone morphogenic protein-6, bone morphogenic
protein-7, bone morphogenic protein-8, bone morphogenic protein-9,
bone morphogenic protein-10, bone morphogenic protein-11, bone
morphogenic protein-12, bone morphogenic protein-13, bone
morphogenic protein-14, bone morphogenic protein-15, bone
morphogenic protein receptor IA, bone morphogenic protein receptor
IB, bone morphogenic protein receptor II, brain derived
neurotrophic factor, cardiotrophin-1, ciliary neutrophic factor,
ciliary neutrophic factor receptor, cripto, cryptic,
cytokine-induced neutrophil chemotactic factor 1, cytokine-induced
neutrophil, chemotactic factor 2a, cytokine-induced neutrophil
chemotactic factor 2.beta.,.beta. endothelial cell growth factor,
endothelin 1, epidermal growth factor, epigen, epiregulin,
epithelial-derived neutrophil attractant, fibroblast growth factor
4, fibroblast growth factor 5, fibroblast growth factor 6,
fibroblast growth factor 7, fibroblast growth factor 8, fibroblast
growth factor 8b, fibroblast growth factor 8c, fibroblast growth
factor 9, fibroblast growth factor 10, fibroblast growth factor 11,
fibroblast growth factor 12, fibroblast growth factor 13,
fibroblast growth factor 16, fibroblast growth factor 17,
fibroblast growth factor 19, fibroblast growth factor 20,
fibroblast growth factor 21, fibroblast growth factor acidic,
fibroblast growth factor basic, glial cell line-derived neutrophic
factor receptor .alpha.1, glial cell line-derived neutrophic factor
receptor .alpha.2, growth related protein, growth related protein
.alpha., growth related protein .beta., growth related protein
.gamma., heparin binding epidermal growth factor, hepatocyte growth
factor, hepatocyte growth factor receptor, hepatoma-derived growth
factor, insulin-like growth factor I, insulin-like growth factor
receptor, insulin-like growth factor II, insulin-like growth factor
binding protein, keratinocyte growth factor, leukemia inhibitory
factor, leukemia inhibitory factor receptor .alpha., nerve growth
factor nerve growth factor receptor, neuropoietin, neurotrophin-3,
neurotrophin-4, oncostatin M (OSM), placenta growth factor,
placenta growth factor 2, platelet-derived endothelial cell growth
factor, platelet derived growth factor, platelet derived growth
factor A chain, platelet derived growth factor AA, platelet derived
growth factor AB, platelet derived growth factor B chain, platelet
derived growth factor BB, platelet derived growth factor receptor
.alpha., platelet derived growth factor receptor .beta., pre-B cell
growth stimulating factor, stem cell factor (SCF), stem cell factor
receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor
.alpha., transforming growth factor .beta., transforming growth
factor .beta.1, transforming growth factor .beta.1.2, transforming
growth factor .beta.2, transforming growth factor .beta.3,
transforming growth factor .beta.5, latent transforming growth
factor .beta.1, transforming growth factor .beta. binding protein
I, transforming growth factor .beta. binding protein II,
transforming growth factor .beta. binding protein III, thymic
stromal lymphopoietin (TSLP), tumor necrosis factor receptor type
I, tumor necrosis factor receptor type II, urokinase-type
plasminogen activator receptor, phospholipase-activating protein
(PUP), insulin, lectin ricin, prolactin, chorionic gonadotropin,
follicle-stimulating hormone, thyroid-stimulating hormone, tissue
plasminogen activator, IgG, IgE, IgM, IgA, and IgD,
.alpha.-galactosidase, .beta.-galactosidase, DNAse, fetuin,
leutinizing hormone, estrogen, insulin, albumin, lipoproteins,
fetoprotein, transferrin, thrombopoietin, urokinase, integrin,
thrombin, leptin, Humira (adalimumab), Prolia (denosumab), Enbrel
(etanercept), a protein from Table 1, or a biologically active
fragment, derivative or variant thereof; wherein said water soluble
polymer containing an active hydrazide group is selected from the
group consisting of polyethylene glycol (PEG), branched PEG,
PolyPEG.RTM. (Warwick Effect Polymers; Coventry, UK), polysialic
acid (PSA), starch, hydroxyalkyl starch (HAS), hydroxylethyl starch
(HES), carbohydrate, polysaccharides, pullulane, chitosan,
hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch,
dextran, carboxymethyl-dextran, polyalkylene oxide (PAO),
polyalkylene glycol (PAG), polypropylene glycol (PPG),
polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),
polycarboxylate, polyvinylpyrrolidone, polyphosphazene,
polyoxazoline, polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, poly(l-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC); wherein
the nucleophilic catalyst is selected from the group consisting of
o-amino benzoic acid, m-amino benzoic acid, p-amino benzoic acid,
sulfanilic acid, o-aminobenzamide, o-toluidine, m-toluidine,
p-toluidine, o-anisidine, m-anisidine, and p-anisidine.
[0047] In another embodiment, an aforementioned method is provided
wherein the water soluble polymer containing an active aminooxy
group is prepared by a method comprising: incubating a solution
comprising an oxidized water-soluble polymer with an activated
aminooxy linker comprising an active aminooxy group under
conditions that allow the formation of a stable oxime linkage
between the oxidized water-soluble polymer and the activated
aminooxy linker, said conditions comprising a time period between
about 1 minute and about 24 hours; a temperature between about
2.degree. C. and about 37.degree. C.; in the presence or absence of
light, and with or without stirring; thereby forming a water
soluble polymer containing an active aminooxy group; and b)
purifying the water soluble polymer containing an active aminooxy
group by a method selected from the group consisting of
chromatography, filtration and precipitation. The term "activated
water-soluble polymer" refers, in one embodiment, to a
water-soluble polymer containing an aldehyde group.
[0048] In yet another embodiment, an aforementioned method is
provided wherein the water soluble polymer containing an active
aminooxy group is prepared by a method comprising: a) incubating a
solution comprising an oxidized water-soluble polymer with an
activated aminooxy linker comprising an active aminooxy group under
conditions that allow the formation of a stable oxime linkage
between the oxidized water-soluble polymer and the activated
aminooxy linker, said conditions comprising a time period between
about 1 minute and about 24 hours; a temperature between about
2.degree. C. and about 37.degree. C.; in the presence or absence of
light, and with or without stirring; thereby forming a water
soluble polymer containing an active aminooxy group; b) incubating
a solution comprising the water soluble polymer containing an
active aminooxy group of step a) with a reducing agent under
conditions that allow the formation of a stable alkoxamine linkage
between the oxidized water-soluble polymer and the activated
aminooxy linker, said conditions comprising a time period between
about 1 minute and about 24 hours; a temperature between about
2.degree. C. and about 37.degree. C.; in the presence or absence of
light; and with or without stirring; and c) purifying the water
soluble polymer containing an active aminooxy group by a method
selected from the group consisting of chromatography, filtration
and precipitation.
[0049] In still another embodiment, an aforementioned method is
provided wherein the water soluble polymer containing an active
aminooxy group is prepared by a method comprising: a) incubating a
solution comprising an oxidized water-soluble polymer with an
activated aminooxy linker comprising an active aminooxy group under
conditions that allow the formation of a stable oxime linkage
between the oxidized water-soluble polymer and the activated
aminooxy linker, said conditions comprising a time period between
about 1 minute and about 24 hours; a temperature between about
2.degree. C. and about 37.degree. C.; in the presence or absence of
light, and with or without stirring; thereby forming a water
soluble polymer containing an active aminooxy group; b) incubating
a solution comprising the water soluble polymer containing an
active aminooxy group of step a) with a nucleophilic catalyst under
conditions comprising a time period between 1 minute and 24 hours;
a temperature between 2.degree. C. and 37.degree. C.; in the
presence or absence of light; and with or without stirring; and c)
purifying the water soluble polymer containing an active aminooxy
group by a method selected from the group consisting of
chromatography, filtration and precipitation.
[0050] In yet another embodiment, an aforementioned method is
provided wherein the water soluble polymer containing an active
aminooxy group is prepared by a method comprising: a) incubating a
solution comprising an oxidized water-soluble polymer with an
activated aminooxy linker comprising an active aminooxy group under
conditions that allow the formation of a stable oxime linkage
between the oxidized water-soluble polymer and the activated
aminooxy linker, said conditions comprising a time period between
about 1 minute and about 24 hours; a temperature between about
2.degree. C. and about 37.degree. C.; in the presence or absence of
light, and with or without stirring; thereby forming a water
soluble polymer containing an active aminooxy group; b) incubating
a solution comprising the water soluble polymer containing an
active aminooxy group of step a) with a nucleophilic catalyst under
conditions comprising a time period between 1 minute and 24 hours;
a temperature between 2.degree. C. and 37.degree. C.; in the
presence or absence of light; and with or without stirring; c)
incubating a solution comprising the water soluble polymer
containing an active aminooxy group of step b) with a reducing
agent under conditions that allow the formation of a stable
alkoxamine linkage between the oxidized water-soluble polymer and
the activated aminooxy linker, said conditions comprising a time
period between about 1 minute and about 24 hours; a temperature
between about 2.degree. C. and about 37.degree. C.; in the presence
or absence of light; and with or without stirring; and d) purifying
the water soluble polymer containing an active aminooxy group by a
method selected from the group consisting of chromatography,
filtration and precipitation.
[0051] In another embodiment, an aforementioned method is provided
wherein the oxidized water soluble polymer is selected from the
group consisting of polyethylene glycol (PEG), branched PEG,
PolyPEG.RTM. (Warwick Effect Polymers; Coventry, UK), polysialic
acid (PSA), starch, hydroxyalkyl starch (HAS), hydroxylethyl starch
(HES), carbohydrate, polysaccharides, pullulane, chitosan,
hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch,
dextran, carboxymethyl-dextran, polyalkylene oxide (PAO),
polyalkylene glycol (PAG), polypropylene glycol (PPG),
polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),
polycarboxylate, polyvinylpyrrolidone, polyphosphazene,
polyoxazoline, polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, poly(l-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC), and
wherein said water-soluble polymer is oxidized by incubation with a
oxidizing agent to form a terminal aldehyde group at the
non-reducing end of the water-soluble polymer. In one embodiment,
the water-soluble polymer is PSA.
[0052] In another embodiment, an aforementioned method is provided
wherein the oxidizing agent is NaIO4.
[0053] In still another embodiment, an aforementioned method is
provided wherein the aminooxy linker is selected from the group
consisting of: [0054] a) a 3-oxa-pentane-1,5-dioxyamine linker of
the formula:
[0054] ##STR00004## [0055] b) a
3,6,9-trioxa-undecane-1,11-dioxyamine linker of the formula:
##STR00005##
[0055] and [0056] c) a
3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine linker of the
formula:
##STR00006##
[0057] In yet another embodiment, an aforementioned method is
provided wherein the reducing agent is selected from the group
consisting of sodium cyanoborohydride (NaCNBH3), ascorbic acid
(vitamin C) and NaBH3. In one embodiment, the reducing agent is
sodium cyanoborohydride (NaCNBH3).
[0058] In another embodiment, an aforementioned method is provided
wherein the nucleophilic catalyst is selected from the group
consisting of o-amino benzoic acid, m-amino benzoic acid, p-amino
benzoic acid, sulfanilic acid, o-aminobenzamide, o-toluidine,
m-toluidine, p-toluidine, o-anisidine, m-anisidine, and
p-anisidine. In one embodiment, the nucleophilic catalyst is
m-toluidine. In another embodiment, the nucleophilic catalyst is
added in an amount to result in a final concentration between about
1.0 mM and about 50 mM nucleophilic catalyst.
[0059] In another embodiment, an aforementioned method is provided
further comprising concentrating the conjugated therapeutic protein
by ultra-/diafiltration (UF/DF).
[0060] In another embodiment, a method of conjugating a water
soluble polymer to an oxidized carbohydrate moiety of a blood
coagulation protein is provided comprising contacting the oxidized
carbohydrate moiety with an activated water soluble polymer under
conditions that allow conjugation;
[0061] said blood coagulation protein selected from the group
consisting of Factor IX (FIX), Factor VIII (FVIII), Factor VIIa
(FVIIa), Von Willebrand Factor (VWF), Factor FV (FV), Factor X
(FX), Factor XI (FXI), Factor XII (FXII), thrombin (FII), protein
C, protein S, tPA, PAI-1, tissue factor (TF) and ADAMTS 13 protease
or a biologically active fragment, derivative or variant
thereof;
[0062] said water soluble polymer containing an active aminooxy
group and is selected from the group consisting of polyethylene
glycol (PEG), branched PEG, polysialic acid (PSA), carbohydrate,
polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitin
sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran,
polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene
glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl
alcohol (PVA), polycarboxylate, polyvinylpyrrolidone,
polyphosphazene, polyoxazoline, polyethylene-co-maleic acid
anhydride, polystyrene-co-maleic acid anhydride,
poly(l-hydroxymethylethylene hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC); and
[0063] said carbohydrate moiety oxidized by incubation with a
buffer comprising an oxidizing agent selected from the group
consisting of sodium periodate (NaIO4), lead tetraacetate
(Pb(OAc)4) and potassium perruthenate (KRuO4); wherein an oxime
linkage is formed between the oxidized carbohydrate moiety and the
active aminooxy group on the water soluble polymer.
FIGURES
[0064] FIG. 1 shows the primary structure of coagulation Factor IX
(SEQ ID NO: 1).
[0065] FIG. 2 shows the coupling of oxidized rFIX to
aminooxy-PSA.
[0066] FIG. 3 shows the synthesis of the water soluble di-aminoxy
linkers 3-oxa-pentane-1,5-dioxyamine and
3,6,9-trioxa-undecane-1,11-dioxyamine.
[0067] FIG. 4 shows the preparation of aminooxy-PSA.
[0068] FIG. 5 shows the visualization of PSA-FIX conjugates
prepared in the presence of different catalysts by SDS PAGE. a)
Comparison of aniline with m-toluidine using different
concentrations; b) Comparison of aniline with o-aminobenzoic acid,
m-aminobenzoic acid, p-aminobenzoic acid, p-aminobenzamide and
sulfanilic acid; c) Comparison of aniline and m-toluidine with
o-anisidine and m-anisidine.
[0069] FIG. 6 shows percent of polysialylation with various
nucleophilic catalysts.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The pharmacological and immunological properties of
therapeutic proteins can be improved by chemical modification and
conjugation with polymeric compounds such as polyethylene glycol
(PEG), branched PEG, polysialic acid (PSA), hydroxyalkyl starch
(HAS), hydroxylethyl starch (HES), carbohydrate, polysaccharides,
pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan
sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide
(PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG),
polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA),
polycarboxylate, polyvinylpyrrolidone, polyphosphazene,
polyoxazoline, polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, poly(l-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC). The
properties of the resulting conjugates generally strongly depend on
the structure and the size of the polymer. Thus, polymers with a
defined and narrow size distribution are usually preferred in the
art. Synthetic polymers like PEG can be manufactured easily with a
narrow size distribution, while PSA can be purified in such a
manner that results in a final PSA preparation with a narrow size
distribution. In addition PEGylation reagents with defined polymer
chains and narrow size distribution are on the market and
commercially available for a reasonable price.
[0071] The addition of a soluble polymer, such as through
polysialylation, is one approach to improve the properties of
therapeutic proteins such as the blood coagulation protein FIX, as
well as other coagulation proteins (e.g., VWF, FVIIa (see, e.g., US
2008/0221032A1, incorporated herein by reference) and FVIII).
Therapeutic Proteins
[0072] In certain embodiments of the invention, the aforementioned
polypeptides and polynucleotides are exemplified by the following
therapeutic proteins: enzymes, antigens, antibodies, receptors,
blood coagulation proteins, growth factors, hormones, and ligands.
In certain embodiments, the therapeutic protein is a blood
coagulation protein such as Factor IX (FIX), Factor VIII (FVIII),
Factor VIIa (FVIIa), Von Willebrand Factor (VWF), Factor FV (FV),
Factor X (FX), Factor XI (FXI), Factor XII (FXII), thrombin (FII),
protein C, protein S, tPA, PAI-1, tissue factor (TF) or ADAMTS 13
protease. In one embodiment, a therapeutic protein according to the
invention is a glycoprotein or, in various embodiments, a protein
that is not naturally glycosylated in vivo (i.e., a protein that
does not contain a natural glycosylation site or a protein that is
not glycosylated in a host cell prior to purification).
[0073] In certain embodiments, the therapeutic protein is
immunoglobulins, cytokines such IL-1 alpha, IL-1 beta, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1),
M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF),
EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta,
IFN-gamma, IFN-omega, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14,
IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23,
IL-24, IL-31, IL-32 alpha, IL-33, thrombopoietin (TPO),
angiopoietins, for example Ang-1, Ang-2, Ang-4, Ang-Y, the human
angiopoietin-like polypeptides ANGPTL1 through 7, vitronectin,
vascular endothelial growth factor (VEGF), angiogenin, activin A,
activin B, activin C, bone morphogenic protein-1, bone morphogenic
protein-2, bone morphogenic protein-3, bone morphogenic protein-4,
bone morphogenic protein-5, bone morphogenic protein-6, bone
morphogenic protein-7, bone morphogenic protein-8, bone morphogenic
protein-9, bone morphogenic protein-10, bone morphogenic
protein-11, bone morphogenic protein-12, bone morphogenic
protein-13, bone morphogenic protein-14, bone morphogenic
protein-15, bone morphogenic protein receptor IA, bone morphogenic
protein receptor IB, bone morphogenic protein receptor II, brain
derived neurotrophic factor, cardiotrophin-1, ciliary neutrophic
factor, ciliary neutrophic factor receptor, cripto, cryptic,
cytokine-induced neutrophil chemotactic factor 1, cytokine-induced
neutrophil, chemotactic factor 2a, cytokine-induced neutrophil
chemotactic factor 2.beta.,.beta. endothelial cell growth factor,
endothelin 1, epidermal growth factor, epigen, epiregulin,
epithelial-derived neutrophil attractant, fibroblast growth factor
4, fibroblast growth factor 5, fibroblast growth factor 6,
fibroblast growth factor 7, fibroblast growth factor 8, fibroblast
growth factor 8b, fibroblast growth factor 8c, fibroblast growth
factor 9, fibroblast growth factor 10, fibroblast growth factor 11,
fibroblast growth factor 12, fibroblast growth factor 13,
fibroblast growth factor 16, fibroblast growth factor 17,
fibroblast growth factor 19, fibroblast growth factor 20,
fibroblast growth factor 21, fibroblast growth factor acidic,
fibroblast growth factor basic, glial cell line-derived neutrophic
factor receptor .alpha.1, glial cell line-derived neutrophic factor
receptor .alpha.2, growth related protein, growth related protein
.alpha., growth related protein .beta., growth related protein
.gamma., heparin binding epidermal growth factor, hepatocyte growth
factor, hepatocyte growth factor receptor, hepatoma-derived growth
factor, insulin-like growth factor I, insulin-like growth factor
receptor, insulin-like growth factor II, insulin-like growth factor
binding protein, keratinocyte growth factor, leukemia inhibitory
factor, leukemia inhibitory factor receptor .alpha., nerve growth
factor nerve growth factor receptor, neuropoietin, neurotrophin-3,
neurotrophin-4, oncostatin M (OSM), placenta growth factor,
placenta growth factor 2, platelet-derived endothelial cell growth
factor, platelet derived growth factor, platelet derived growth
factor A chain, platelet derived growth factor AA, platelet derived
growth factor AB, platelet derived growth factor B chain, platelet
derived growth factor BB, platelet derived growth factor receptor
.alpha., platelet derived growth factor receptor 13, pre-B cell
growth stimulating factor, stem cell factor (SCF), stem cell factor
receptor, TNF, including TNF0, TNF1, TNF2, transforming growth
factor .alpha., transforming growth factor 13, transforming growth
factor 131, transforming growth factor 131.2, transforming growth
factor .beta.2, transforming growth factor .beta.3, transforming
growth factor .beta.5, latent transforming growth factor .beta.1,
transforming growth factor .beta. binding protein I, transforming
growth factor .beta. binding protein II, transforming growth factor
.beta. binding protein III, thymic stromal lymphopoietin (TSLP),
tumor necrosis factor receptor type I, tumor necrosis factor
receptor type II, urokinase-type plasminogen activator receptor,
vascular endothelial growth factor, and chimeric proteins and
biologically or immunologically active fragments thereof.
[0074] In certain embodiments, the therapeutic protin is alpha-,
beta-, and gamma-interferons, colony stimulating factors including
granulocyte colony stimulating factors, fibroblast growth factors,
platelet derived growth factors, phospholipase-activating protein
(PUP), insulin, plant proteins such as lectins and ricins, tumor
necrosis factors and related alleles, soluble forms of tumor
necrosis factor receptors, interleukin receptors and soluble forms
of interleukin receptors, growth factors such as tissue growth
factors, such as TGF.alpha.s or TGF.beta.s and epidermal growth
factors, hormones, somatomedins, pigmentary hormones, hypothalamic
releasing factors, antidiuretic hormones, prolactin, chorionic
gonadotropin, follicle-stimulating hormone, thyroid-stimulating
hormone, tissue plasminogen activator, and immunoglobulins such as
IgG, IgE, IgM, IgA, and IgD, a galactosidase,
.alpha.-galactosidase, .beta.-galactosidase, DNAse, fetuin,
leutinizing hormone, estrogen, corticosteroids, insulin, albumin,
lipoproteins, fetoprotein, transferrin, thrombopoietin, urokinase,
DNase, integrins, thrombin, hematopoietic growth actors, leptin,
glycosidases, Humira (adalimumab), Prolia (denosumab), Enbrel
(etanercept), and fragments thereof, or any fusion proteins
comprising any of the above mentioned proteins or fragments
thereof. In addition to the aforementioned proteins, the following
Table 1 provides therapeutic proteins contemplated by the present
invention:
TABLE-US-00001 TABLE 1 Follicular dendritic cell secreted peptide
Angiotensin-converting enzyme Dermokine Antithrombin-III Secreted
frizzled-related protein 1 Apolipoprotein B-100 Ectodysplasin-A
Apolipoprotein D Secreted frizzled-related protein 2 Apolipoprotein
E Resistin Beta-1,4-galactosyltransferase 1 Osteopontin Bone
morphogenetic protein 7 Secreted frizzled-related protein 5
Complement C1q subcomponent subunit B Secreted frizzled-related
protein 4 C4b-binding protein alpha chain Secreted phosphoprotein
24 Calreticulin Glypican-6 Corticosteroid-binding globulin Secreted
frizzled-related protein 3 Carboxypeptidase A1 C-C motif chemokine
4 Carboxypeptidase A2 Melanocyte protein Pmel 17 Eotaxin Secreted
Ly-6/uPAR-related protein 1 C-C motif chemokine 13
Beta-microseminoprotein C-C motif chemokine 18 Glypican-4 C-C motif
chemokine 20 Tumor necrosis factor ligand superfamily Triggering
receptor expressed on myeloid member 15 cells 2 Resistin-like beta
C-C motif chemokine 2 Tumor necrosis factor ligand superfamily
Transforming growth factor-beta-induced member 12 protein ig-h3
SPARC CD40 ligand Glypican-5 Corneodesmosin Anterior gradient
protein 2 homolog Complement factor D Protein canopy homolog 2
Chromogranin-A Glypican-1 Collagen alpha-1(I) chain von Willebrand
factor A domain-containing Disintegrin and metalloproteinase
domain- protein 2 containing protein 18 WNT1-inducible-signaling
pathway Cysteine-rich secretory protein LCCL protein 1
domain-containing 1 C-C motif chemokine 1 Collagen alpha-4(IV)
chain SPARC-related modular calcium-binding Keratinocyte
differentiation-associated protein 2 protein C-type lectin domain
family 11 member A Complement C4-B Secreted Ly-6/uPAR-related
protein 2 Collagen alpha-2(V) chain Glypican-3 Complement C5
Secreted and transmembrane protein 1 Collagen alpha-1(VII) chain
Testis-expressed sequence 264 protein Complement component C7
Glypican-2 Complement component C8 beta chain Serine protease 23
Complement component C8 gamma chain 39S ribosomal protein L55,
mitochondrial Collagen alpha-1(XV) chain Protein NipSnap homolog 3A
Collagen alpha-1(XVI) chain Fibronectin Collagen alpha-1(XVIII)
chain Neudesin Collagen alpha-1(XIX) chain Fibroblast growth factor
receptor 2 Cartilage oligomeric matrix protein Carbonic anhydrase 6
C-reactive protein Deleted in malignant brain tumors 1 protein
Granulocyte colony-stimulating factor SPARC-related modular
calcium-binding Granulocyte-macrophage colony- protein 1
stimulating factor Amyloid beta A4 protein Protein CYR61 Tumor
necrosis factor receptor superfamily Complement component receptor
1-like member 6 protein Gamma-aminobutyric acid type B receptor
Stem cell growth factor; lymphocyte subunit 1 secreted C-type
lectin Pro-neuregulin-1, membrane-bound
CMP-N-acetylneuraminate-beta- isoform
galactosamide-alpha-2,3-sialyltransferase Glycoprotein hormone
alpha-2 Dipeptidyl peptidase 4 Membrane metallo-endopeptidase-like
1 Dentin sialophosphoprotein Fc receptor-like A Endothelin-1 C-C
motif chemokine 4-like Ephrin-B1 Epithelial discoidin
domain-containing Epidermis-specific serine protease-like receptor
1 protein Mucin-1 EMILIN-1 Vascular endothelial growth factor A
Endoplasmin Fibulin-1 Ephrin type-A receptor 3 Prolactin receptor
Ephrin type-B receptor 6 Proprotein convertase subtilisin/kexin
Glycosyltransferase 1 domain-containing type 6 protein 1 CD209
antigen Coagulation factor X Collagen alpha-2(XI) chain Coagulation
factor VIII Granulocyte-macrophage colony- Complement C1q tumor
necrosis factor- stimulating factor receptor subunit alpha related
protein 7 Elastin Fibrillin-2 Interleukin-15 receptor subunit alpha
Alpha-2-HS-glycoprotein Midkine Fibroblast growth factor 10
Integrin alpha-7 Fibrinogen alpha chain Mucin-4 Fibrinogen beta
chain Peptidyl-glycine alpha-amidating Long palate, lung and nasal
epithelium monooxygenase carcinoma-associated protein 1
Apolipoprotein A-I Gastrin Proteoglycan 4 Glycoprotein hormones
alpha chain Tumor necrosis factor receptor superfamily
N-acetylglucosamine-1- member 25 phosphotransferase subunits
alpha/beta Attractin Granzyme A Prostate-associated
microseminoprotein Hepatocyte growth factor-like protein
Alpha-amylase 1 Insulin-like growth factor-binding protein 1
Brain-derived neurotrophic factor Insulin-like growth
factor-binding protein 2 C-type lectin domain family 4 member M
Insulin-like growth factor-binding protein 4 Granulocyte
colony-stimulating factor Tumor necrosis factor receptor
superfamily receptor member 10D Insulin-like growth factor II
Interferon alpha-1/13 Carcinoembryonic antigen-related cell
Interferon-induced helicase C domain- adhesion molecule 1
containing protein 1 C-type lectin domain family 7 member A
Interferon alpha-2 CMRF35-like molecule 1 Interferon beta Choline
transporter-like protein 4 Interferon gamma Pulmonary
surfactant-associated protein A1 Insulin-like growth factor IB
Spermine oxidase Indian hedgehog protein
CMP-N-acetylneuraminate-beta-1,4- Neural cell adhesion molecule
galactoside alpha-2,3-sialyltransferase L1-like protein
Kallikrein-8 Interleukin-13 Tissue-type plasminogen activator
Interleukin-2 Peroxisomal N(1)-acetyl- Chymotrypsin-like elastase
family spermine/spermidine oxidase member 2A Probable
palmitoyltransferase ZDHHC4 Inhibin beta A chain Cholesteryl ester
transfer protein Pancreatic secretory trypsin inhibitor HLA class I
histocompatibility antigen, A-2 Tumor necrosis factor receptor
superfamily alpha chain member 21 Collagen alpha-1(II) chain
Inter-alpha-trypsin inhibitor heavy chain H1 Pro-interleukin-16
Inter-alpha-trypsin inhibitor heavy chain H2 Leptin receptor
Inter-alpha-trypsin inhibitor heavy chain H3 Decorin
Prostate-specific antigen Stromal cell-derived factor 1
Kallikrein-4 Tenascin Plasma kallikrein Disintegrin and
metalloproteinase domain- Calcium-activated chloride channel
containing protein 12 regulator 4 A disintegrin and
metalloproteinase with Bactericidal/permeability-increasing
thrombospondin motifs 13 protein-like 1 T-cell surface glycoprotein
CD3 alpha chain Leptin EGFR-coamplified and overexpressed A
disintegrin and metalloproteinase with protein thrombospondin
motifs 4 Autophagy-related protein 16-1 Hepatic triacylglycerol
lipase Breast cancer anti-estrogen resistance Lymphocyte antigen 6
complex locus protein 3 protein G6c Cadherin-23 Eosinophil
lysophospholipase Macrophage colony-stimulating factor 1 Lutropin
subunit beta Folate receptor alpha Microfibrillar-associated
protein 1 Low-density lipoprotein receptor-related Mesencephalic
astrocyte-derived protein 8 neurotrophic factor E3
ubiquitin-protein ligase LRSAM1 Matrix Gla protein Neural cell
adhesion molecule 1 72 kDa type IV collagenase Neuroligin-4,
X-linked Stromelysin-1 Netrin-G1 Neutrophil collagenase GPI
transamidase component PIG-T Mesothelin Kit ligand Mucin-5AC
Seizure 6-like protein Mucin-6 SLAM family member 7 Norrin Tumor
necrosis factor Oxytocin-neurophysin 1 Uromodulin Beta-nerve growth
factor Tumor necrosis factor ligand superfamily Tumor necrosis
factor ligand superfamily member 13 member 18 Protein CREG1
Neurotrophin-3 EGF-like domain-containing protein 8
Platelet-derived growth factor subunit A Aminoacyl tRNA synthetase
complex- Phosphopantothenoylcysteine interacting multifunctional
protein 1 decarboxylase ADAMTS-like protein 4 Plasminogen activator
inhibitor 1 Coagulation factor XI Plasminogen activator inhibitor 2
Interleukin-22 receptor subunit alpha-2 Procollagen C-endopeptidase
enhancer 1 Deformed epidermal autoregulatory factor 1 Transmembrane
and ubiquitin-like domain- homolog containing protein 2
Prostaglandin-H2 D-isomerase Protein disulfide-isomerase
Alpha-1-antitrypsin Pigment epithelium-derived factor
Alpha-1-antichymotrypsin Pepsin A Acyl-CoA-binding protein
Gastricsin Complement factor B Sonic hedgehog protein
Choriogonadotropin subunit beta Peptidoglycan recognition protein
I-alpha Versican core protein Biglycan Epidermal growth factor
receptor Prolactin-inducible protein Ecto-NOX disulfide-thiol
exchanger 2 Platelet factor 4 Hyaluronidase-1 Plasminogen
Interleukin-1 receptor antagonist protein Serum
paraoxonase/arylesterase 1 Interleukin-6 receptor subunit beta
Alkaline phosphatase, placental type Interleukin-1 receptor-like 1
Peptidyl-prolyl cis-trans isomerase B Insulin Bone marrow
proteoglycan Glycodelin Basic salivary proline-rich protein 1
Parathyroid hormone-related protein Pulmonary surfactant-associated
protein C Nurim Parathyroid hormone Prolyl 4-hydroxylase subunit
alpha-2 Serum amyloid P-component CD276 antigen Secretogranin-1
Cysteine-rich with EGF-like domain Basement membrane-specific
heparan protein 1 sulfate proteoglycan core protein CUB and sushi
domain-containing protein 1 Antileukoproteinase Ficolin-2
Stabilin-1 Fc receptor-like protein 5 Extracellular superoxide
dismutase [Cu--Zn] Protein GPR89 Somatotropin Junctional adhesion
molecule A Serpin B5 Leucine-rich repeat-containing protein 8A
Spondin-1 Multiple inositol polyphosphate Structural maintenance of
chromosomes phosphatase 1 protein 3 Neuropilin-1 Syntaxin-1A
Plexin-A4 Tetranectin Plexin-B1 Transforming growth factor beta-1
Periostin Thyroglobulin Protein RIC-3 Metalloproteinase inhibitor 1
SLIT and NTRK-like protein 2 Metalloproteinase inhibitor 2
Sulfatase-modifying factor 1 Metalloproteinase inhibitor 3
Sulfatase-modifying factor 2 Urokinase-type plasminogen activator
Transmembrane protease, serine 6 Lactotransferrin Lymphotoxin-alpha
Trypsin-1 Tumor necrosis factor receptor superfamily Submaxillary
gland androgen-regulated member 10B protein 3B Urokinase
plasminogen activator surface Tumor necrosis factor receptor
superfamily receptor member 1A V-set domain-containing T-cell
activation Vascular endothelial growth factor inhibitor 1 receptor
1 Glucagon Vitamin D-binding protein N-acetylmuramoyl-L-alanine
amidase Vitronectin Sulfhydryl oxidase 1 von Willebrand factor
Dehydrogenase/reductase SDR family Lymphocyte antigen 6 complex
locus member 4 protein G5c Interleukin-18-binding protein
Zinc-alpha-2-glycoprotein Kin of IRRE-like protein 2
Uncharacterized protein C14orf93 Myeloid-associated differentiation
marker Retinoschisin Chordin Alpha-1,3-mannosyltransferase ALG2
1-acyl-sn-glycerol-3-phosphate C-type lectin domain family 11,
member A, acyltransferase gamma isoform CRA_b Advanced
glycosylation end product- Major facilitator superfamily domain-
specific receptor containing protein 7 NLR family CARD
domain-containing Leucine-rich repeat transmembrane protein 4
neuronal protein 1 Pro-neuregulin-2, membrane-bound NADH
dehydrogenase [ubiquinone] 1 beta isoform subcomplex subunit 11,
mitochondrial Sperm-associated antigen 11A UPF0546 membrane protein
C1orf91 Oocyte-secreted protein 1 homolog Carbonic
anhydrase-related protein 10 Serum albumin Cholecystokinin Cochlin
Codanin-1 Plasma protease C1 inhibitor Uncharacterized protein
C6orf89 Interleukin-7 receptor subunit alpha Chondroitin sulfate
glucuronyltransferase Inter-alpha-trypsin inhibitor heavy chain H5
Chitinase domain-containing
protein 1 Platelet-derived growth factor D Transmembrane protein
C9orf7 Protein S100-A7 CMRF35-like molecule 9 Sialic acid-binding
Ig-like lectin 10 Cytochrome P450 2S1 Tubulointerstitial nephritis
antigen-like Crumbs protein homolog 3 Tumor necrosis factor ligand
superfamily Dehydrogenase/reductase SDR family member 13B member 7
Long-chain-fatty-acid--CoA ligase 5 Protein ENED Claudin-14
Complement factor H-related protein 4 Leucine-rich
repeat-containing protein 20 Leucine-rich repeat LGI family member
3 Interleukin-1 family member 7 Gliomedin Lymphocyte antigen 6
complex locus Glycerophosphodiester phosphodiesterase protein G5b
domain-containing protein 5 Acetylcholinesterase Probable G-protein
coupled receptor 113 Amelogenin, X isoform Probable G-protein
coupled receptor 114 Angiogenin Glycerol-3-phosphate
acyltransferase 4 Anthrax toxin receptor 2 Gremlin-1 Annexin A2
Potassium channel subfamily K member 17 Apolipoprotein C-III KDEL
motif-containing protein 2 Apolipoprotein L1 Layilin Complement C1q
subcomponent subunit A Leucine-rich repeat-containing protein 8B
Complement C1q subcomponent subunit C Leucine-rich
repeat-containing protein 8D Calcitonin Sialic acid-binding Ig-like
lectin 6 Soluble calcium-activated nucleotidase 1
Pregnancy-specific beta-1-glycoprotein 2 C-C motif chemokine 15
Ly6/PLAUR domain-containing protein 1 CD97 antigen ( Ly6/PLAUR
domain-containing protein 5 Contactin-4 MLN64 N-terminal domain
homolog Complement C2 Macrophage migration inhibitory factor
Collagen alpha-6(IV) chain 2-acylglycerol O-acyltransferase 3
Collagen alpha-2(VI) chain Mitochondrial carrier homolog 1 Collagen
alpha-1(XI) chain Apolipoprotein L6 Crumbs homolog 1 Protocadherin
alpha-6 Cystatin-C Protocadherin gamma-A12 Neutrophil defensin 1
Voltage-gated hydrogen channel 1 Endothelin-3 All-trans-retinol
13,14-reductase Low affinity immunoglobulin epsilon Regulator of
microtubule dynamics Fc receptor protein 2 Fibroblast growth factor
receptor 3 R-spondin-4 Fibroblast growth factor receptor 4
Long-chain fatty acid transport protein 3 Growth arrest-specific
protein 6 Vesicle-trafficking protein SEC22c Growth hormone
receptor Claudin-1 Bifunctional UDP-N-acetylglucosamine 2-
Leucine-rich repeats and immunoglobulin-
epimerase/N-acetylmannosamine kinase like domains protein 3
Immunoglobulin superfamily member 8 SLAM family member 9
Interleukin-4 receptor alpha chain Transthyretin Kallikrein-14
Serine/threonine-protein kinase 32B Kallikrein-6 Platelet-derived
growth factor subunit B Laminin subunit beta-3 Noggin
Leucyl-cystinyl aminopeptidase Tryptase alpha-1 Mannan-binding
lectin serine protease 1 Tetratricopeptide repeat protein 14
Mannan-binding lectin serine protease 2 XTP3-transactivated gene B
protein Neutrophil gelatinase-associated lipocalin
Palmitoyltransferase ZDHHC15 Neuropeptide Y Zona pellucida
sperm-binding protein 3 Aggrecan core protein Leucine-rich
repeat-containing protein 39 Pulmonary surfactant-associated
protein B Pancreatic triacylglycerol lipase Poliovirus
receptor-related protein 1 Transmembrane protein 139 Renin Leukemia
inhibitory factor Ribonuclease pancreatic Galectin-1 Semenogelin-1
C-C motif chemokine 21 Signaling lymphocytic activation molecule
CD5 antigen-like Tissue factor pathway inhibitor Carbohydrate
sulfotransferase 9 Usherin Lipopolysaccharide-binding protein
Fibroblast growth factor 23 Cysteine-rich motor neuron 1 protein
Interleukin-23 subunit alpha Connective tissue growth factor
Epididymal secretory protein E1 Protein eyes shut homolog
ADAMTS-like protein 1 Mucin-like protein 1 Chemokine-like factor
Fibroblast growth factor 19 EGF-like domain-containing protein 7
Follistatin-related protein 3 Tectonic-1 Hedgehog-interacting
protein Transmembrane protein 25 Interleukin-17 receptor B
UDP-GalNAc: beta-1,3-N- FXYD domain-containing ion transport
acetylgalactosaminyltransferase 1 regulator 5 Interleukin-15
(IL-15) Endothelial lipase Multiple epidermal growth factor-like
EGF-containing fibulin-like extracellular domains 11 matrix protein
2 Mucin and cadherin-like protein Otoraplin Ribonuclease 4 Group 3
secretory phospholipase A2 SH2 domain-containing protein 3C Group
XV phospholipase A2 CMP-N-acetylneuraminate-beta- Tumor necrosis
factor ligand superfamily galactosamide-alpha-2,3-sialyltransferase
member 14 Transmembrane protein 9 Plexin-A2 WAP four-disulfide core
domain protein 2 Papilin Adenosine A3 receptor Prokineticin-1
Gamma-secretase subunit APH-1A Ribonuclease 7 Basigin Kunitz-type
protease inhibitor 1 Baculoviral IAP repeat-containing protein 7
Spondin-2 Calumenin Testican-2 Alpha-S1-casein Inactive serine
protease PAMR1 Cyclin-L1 Torsin-2A Complement factor H Vasohibin-1
Chorionic somatomammotropin hormone Vasorin Coxsackievirus and
adenovirus receptor Xylosyltransferase 1 Ectonucleotide
Ectonucleotide pyrophosphatase/phosphodiesterase family
pyrophosphatase/phosphodiesterase family member 2 member 6
ERO1-like protein alpha Oncostatin-M Coagulation factor IX Derlin-1
Low affinity immunoglobulin gamma Fc HERV-FRD_6p24.1 provirus
ancestral Env region receptor III-B polyprotein Ficolin-3 Prostasin
Fc receptor-like protein 2 Transmembrane protease, serine 11E
Leucine-rich repeat transmembrane protein HLA class I
histocompatibility antigen, FLRT3 Cw-16 alpha chain Gelsolin Wnt
inhibitory factor 1 Granulysin C-type natriuretic peptide
Transmembrane glycoprotein NMB Angiopoietin-2 Granulins
Deoxyribonuclease gamma Heparanase Carboxypeptidase A5 Ig mu chain
C region C-C motif chemokine 14 Interleukin-1 alpha Interleukin-5
Interleukin-31 receptor A Interleukin-10 Junctional adhesion
molecule B C-X-C motif chemokine 2 Lipocalin-1 C-X-C motif
chemokine 5 Leucine-rich repeat-containing G-protein A disintegrin
and metalloproteinase with coupled receptor 6 thrombospondin motifs
6 Latent-transforming growth factor beta- Polypeptide binding
protein 1 N-acetylgalactosaminyltransferase 1 Matrilin-3 Fibulin-2
Myelin protein zero-like protein 1 Ficolin-1 Neurobeachin-like
protein 2 SL cytokine Nicastrin Follistatin ADP-ribose
pyrophosphatase, FRAS1-related extracellular matrix mitochondrial
protein 1 Protocadherin-15 Enamelin Placenta growth factor
Hyaluronan and proteoglycan link protein 1 Protein O-linked-mannose
beta-1,2-N- Leukocyte immunoglobulin-like receptor
acetylglucosaminyltransferase 1 subfamily A member 3 Probable
hydrolase PNKD Interleukin-17F Pleiotrophin Interleukin-1 receptor
accessory protein Poliovirus receptor Serine protease inhibitor
Kazal-type 5 Reticulon-4 receptor Kallikrein-15 Serum amyloid A
protein Interferon alpha-14 Sex hormone-binding globulin
Pregnancy-specific beta-1-glycoprotein 4 SLAM family member 6
Collagenase 3 Sarcolemmal membrane-associated protein Matrix
metalloproteinase-16 Sushi, von Willebrand factor type A, EGF
Pituitary adenylate cyclase-activating and pentraxin
domain-containing protein 1 polypeptide Thyroxine-binding globulin
Prokineticin-2 Transmembrane and coiled-coil domain-
Latent-transforming growth factor beta- containing protein 1
binding protein 3 Transmembrane protease, serine 3 Somatoliberin
Tumor necrosis factor receptor superfamily Thrombospondin type-1
domain-containing member 10C protein 1 Tumor necrosis factor
receptor superfamily Angiogenic factor with G patch and FHA member
11B domains 1 Serotransferrin TGF-beta receptor type III Tryptase
beta-2 Thyrotropin subunit beta Protein YIPF5 Uncharacterized
protein C19orf36 Vesicle-associated membrane protein- Complement
C1q tumor necrosis associated protein B/C factor-related protein 2
cDNA, FLJ96669, highly similar to Homo Ectonucleotide sapiens
secreted protein, acidic, cysteine-
pyrophosphatase/phosphodiesterase family rich (osteonectin)(SPARC),
mRNA member 5 cDNA FLJ77519, highly similar to Homo Polypeptide N-
sapiens secreted frizzled related protein
acetylgalactosaminyltransferase-like mRNA protein 2 T-cell
differentiation antigen CD6 Slit homolog 1 protein Pikachurin
Growth hormone variant Fibrinogen-like protein 1
Angiopoietin-related protein 3 Interleukin-32 Angiopoietin-related
protein 7 Matrilin-4 Ecto-ADP-ribosyltransferase 5 Sperm-associated
antigen 11B Carbonic anhydrase-related protein 11 Coagulation
factor XII Probable ribonuclease 11 Hepcidin Probable
carboxypeptidase X1 Klotho Protein FAM3D Serglycin C-X-C motif
chemokine 14 Tomoregulin-2 Beta-defensin 127 Chordin-like protein 2
Beta-defensin 129 Tumor necrosis factor receptor superfamily
Cysteine-rich secretory protein LCCL member 6B domain-containing 2
UPF0414 transmembrane protein C20orf30 Fibroblast growth factor 21
C-type lectin domain family 4 member C Plasma alpha-L-fucosidase
UPF0317 protein C14orf159, mitochondrial Gastrokine-1 Netrin-G2
Gastrokine-2 Metalloreductase STEAP2 Glutathione peroxidase 7 Sushi
domain-containing protein 4 HHIP-like protein 1 Protein YIF1B
Interferon kappa Apolipoprotein M Apolipoprotein C-I C4b-binding
protein beta chain Procollagen C-endopeptidase enhancer 2 T-cell
surface glycoprotein CD8 beta chain Left-right determination factor
1 C-C motif chemokine 3-like 1 Leucine-rich repeat LGI family
member 4 Fibroblast growth factor 8 BRCA1-A complex subunit Abraxas
Sialomucin core protein 24 Leucine zipper protein 2 Programmed cell
death 1 ligand 2 Neurexophilin-3 Secreted and transmembrane 1
Osteomodulin Complement C1q tumor necrosis factor- Kazal-type
serine protease inhibitor related protein 6 domain-containing
protein 1 EGF-like module-containing mucin-like Sperm acrosome
membrane-associated hormone receptor-like 3 protein 3 Noelin-3
Secretoglobin family 3A member 1 Odorant-binding protein 2b
Tsukushin Urotensin-2 Claudin-2 (SP82) Vitrin Complement factor
H-related protein 2 WNT1-inducible-signaling pathway Immunoglobulin
superfamily containing protein 3 leucine-rich repeat protein cDNA
FLJ75759, highly similar to Homo Leucine-rich repeat and
immunoglobulin- sapiens follistatin-like 3 (secreted like
domain-containing nogo receptor- glycoprotein) (FSTL3), mRNA
interacting protein 1 Angiotensin-converting enzyme 2 Kin of
IRRE-like protein 3 Adiponectin Hematopoietic cell signal
transducer Angiopoietin-related protein 4 Follitropin subunit beta
Apolipoprotein A-V Melanoma inhibitory activity protein 3 Asporin
Leucine-rich repeat-containing protein 4 Bactericidal
permeability-increasing protein Zinc transporter 5 CUB
domain-containing protein 1 Leucine-rich repeat neuronal protein 1
Cartilage intermediate layer protein 1 Apical endosomal
glycoprotein Beta-Ala-His dipeptidase Serum amyloid A-4 protein
Collagen alpha-1(V) chain Probetacellulin Collagen alpha-1(XXV)
chain Beta-1,4-galactosyltransferase 7 Estradiol
17-beta-dehydrogenase 11 3-hydroxybutyrate dehydrogenase type 2
DnaJ homolog subfamily C member 10 C1GALT1-specific chaperone 1
EGF-like domain-containing protein 6 Beta-casein Coagulation factor
XIII A chain Kappa-casein Glucose-6-phosphate isomerase
Transmembrane protein C2orf18 Appetite-regulating hormone
Carboxypeptidase N catalytic chain Interleukin-12 subunit beta
CD320 antigen Interleukin-22 Chondroitin sulfate synthase 1
lntelectin-1 Chondroitin sulfate synthase 2 Leucine-rich
glioma-inactivated protein 1 CMRF35-like molecule 7 Lymphocyte
antigen 96 Protein canopy homolog 3 Matrilysin Short-chain
dehydrogenase/reductase 3 Mucin-20 Delta-like protein 4 Proprotein
convertase subtilisin/kexin Delta and Notch-like epidermal
growth type 9 factor-related receptor Peptidoglycan recognition
protein Dolichol kinase Interferon-induced 17 kDa protein
Endothelin-converting enzyme-like 1 Protein Wnt-4 Integral membrane
protein 2B Allograft inflammatory factor 1-like Insulin-like growth
factor-binding protein 5 Armadillo repeat-containing X-linked
Endothelial cell-selective adhesion protein 3 molecule Chondroitin
sulfate N- Signal peptide, CUB and EGF-like domain-
acetylgalactosaminyltransferase 1 containing protein 1
Chitotriosidase-1 Complement factor H-related protein 3 Claudin
domain-containing protein 1 Prorelaxin H1 Erlin-2
Follistatin-related protein 1 Glycosyltransferase 8
domain-containing Globoside alpha-1,3-N- protein 1
acetylgalactosaminyltransferase 1 Golgi membrane protein 1
Gamma-glutamyl hydrolase Probable G-protein coupled receptor 125
Cadherin-24 Interleukin-20 receptor alpha chain
Glycerol-3-phosphate acyltransferase 3 Galectin-7 G-protein coupled
receptor 56 NKG2D ligand 4 Hyaluronan-binding protein 2
L-amino-acid oxidase Proheparin-binding EGF-like growth factor
Prolyl 3-hydroxylase 1 Histidine-rich glycoprotein GPI ethanolamine
phosphate transferase 2 Carbohydrate sulfotransferase 14 GPI
ethanolamine phosphate transferase 3 Interleukin-20 receptor beta
chain Calcium-binding mitochondrial carrier Ectonucleotide protein
SCaMC-2 (Small calcium-binding pyrophosphatase/phosphodiesterase
mitochondrial carrier protein 2) family member 3 Pulmonary
surfactant-associated protein A2 Insulin-like growth factor-binding
protein 7 Splicing factor, arginine/serine-rich 16 Kallistatin
Alpha-N-acetylgalactosaminide alpha-2,6- Fibronectin type III
domain-containing sialyltransferase 6 protein 3B Single Ig
IL-1-related receptor Leukemia inhibitory factor receptor
Tectonic-3 Lin-7 homolog B Tumor necrosis factor ligand superfamily
Thioredoxin-related transmembrane member 11 protein 1 Tumor
necrosis factor receptor superfamily Disintegrin and
metalloproteinase domain- member 19 containing protein 32
Palmitoyltransferase ZDHHC9 Ly6/PLAUR domain-containing protein 3
Fibulin-5 C-type lectin domain family 14 member A Protein
Z-dependent protease inhibitor Protein cornichon homolog
Alpha-2-macroglobulin Protein FAM151A Agouti-related protein
FK506-binding protein 14 Pancreatic alpha-amylase Neuropilin and
tolloid-like protein 2 Natriuretic peptides B Protocadherin beta-13
Atrial natriuretic factor Prenylcysteine oxidase 1 Neutral
ceramidase Peflin Beta-2-microglobulin Peptidyl-prolyl cis-trans
isomerase-like 1 Bone morphogenetic protein 4 Prostate stem cell
antigen Biotinidase Protein patched homolog 2 Scavenger receptor
cysteine-rich type 1 Chitobiosyldiphosphodolichol beta- protein
M130 mannosyltransferase Carboxypeptidase B2 Protein sel-1 homolog
1 Carboxypeptidase Z ProSAAS C-C motif chemokine 5 Sialic
acid-binding Ig-like lectin 9 C-C motif chemokine 7 SLIT and
NTRK-like protein 1 C-C motif chemokine 8 Statherin CD59
glycoprotein Testisin Complement factor I Transmembrane
channel-like protein 5 Clusterin Transmembrane protease, serine 4
Collagen alpha-2(I) chain Metastasis-suppressor KiSS-1 Collagen
alpha-1(III) chain Islet amyloid polypeptide Collagen alpha-1(IV)
chain Trem-like transcript 2 protein Collagen alpha-3(IV) chain
Thioredoxin domain-containing protein 12 Collagen alpha-5(IV) chain
Vascular endothelial growth factor B Collagen alpha-3(VI) chain
Vascular endothelial growth factor C Complement component C6
Reticulocalbin-3 Collagen alpha-1(IX) chain Fibrillin-1 Collagen
alpha-1(X) chain Protein FAM3A Collagen alpha-1(XVII) chain Protein
G7c Collagen alpha-1(XXI) chain Neuropilin and tolloid-like protein
1 Coatomer subunit alpha Pregnancy-specific beta-1-glycoprotein 11
Complement receptor type 1 Serpin B4 Cystatin-SN ADAM DEC1
Deoxyribonuclease-1 ADP-dependent glucokinase Extracellular matrix
protein 1 Alpha-amylase 2B Low affinity immunoglobulin gamma
UDP-GlcNAc: betaGal beta-1,3-N- Fc region receptor III-A
acetylglucosaminyltransferase 3 Alpha-fetoprotein Calcitonin
gene-related peptide 2 Heparin-binding growth factor 2
Carboxypeptidase E Fibrinogen gamma chain Cardiotrophin-like
cytokine factor 1 Growth/differentiation factor 5 Collagen
alpha-2(VIII) chain Glial cell line-derived neurotrophic factor
Crumbs homolog 2 Insulin-like growth factor-binding protein 3
Dentin matrix acidic phosphoprotein 1 Insulin-like growth factor IA
Down syndrome cell adhesion molecule Ig gamma-1 chain C region
Immunoglobulin superfamily member 1 Ig gamma-2 chain C region
Interleukin-4 Ig gamma-3 chain C region Interleukin-6 receptor
subunit alpha Insulin-like 3 Interleukin-24 Inter-alpha-trypsin
inhibitor heavy chain Ladinin-1 UPF0378 protein KIAA0100 Lipase
member I Kininogen-1 Pancreatic lipase-related protein 1 Laminin
subunit alpha-2 Leucine-rich alpha-2-glycoprotein Laminin subunit
alpha-4 Matrix-remodeling-associated protein 5 Laminin subunit
beta-1 Netrin-4 Protein-lysine 6-oxidase Hepatocyte growth factor
receptor Multimerin-1 C-C motif chemokine 22
Vasopressin-neurophysin 2-copeptin Nyctalopin Nidogen-1 Osteocalcin
Phospholipase A2, Basic salivary proline-rich protein 3 Perforin-1
Pregnancy-specific beta-1-glycoprotein 10
Phosphatidylinositol-glycan-specific Leucine-rich repeat
transmembrane protein phospholipase D FLRT2 Fibrocystin R-spondin-3
Phospholipid transfer protein Sialoadhesin Prostatic acid
phosphatase Trypsin-3 Vitamin K-dependent protein Z Dipeptidase 2
Salivary acidic proline-rich Collagen and calcium-binding EGF
phosphoprotein 1/2 domain-containing protein 1 Pregnancy zone
protein Germ cell-specific gene 1-like protein Prorelaxin H2
Leucine-rich repeat-containing protein 31 Semaphorin-4D
Apolipoprotein O Slit homolog 2 protein Dystroglycan Alpha-tectorin
Neutrophil defensin 4 Tenascin-X Amphoterin-induced protein 3
Trefoil factor 3 Gamma-secretase subunit APH-1B Transferrin
receptor protein 1 Apolipoprotein C-IV Protransforming growth
factor alpha Arylsulfatase G Transforming growth factor beta-2
Glia-activating factor Tumor necrosis factor ligand superfamily
Caspase recruitment domain-containing member 6 protein 18 Tumor
necrosis factor receptor superfamily Heparan sulfate glucosamine
3-O- member 1B sulfotransferase 3A1 Tumor necrosis factor receptor
superfamily Thyrotropin-releasing hormone-degrading member 5
ectoenzyme Thrombopoietin Guanylin VIP peptides Choline
transporter-like protein 3 Acidic mammalian chitinase
17-beta-hydroxysteroid dehydrogenase 14 Cysteine-rich secretory
protein 2 Immunoglobulin lambda-like polypeptide 1
Haptoglobin-related protein DnaJ homolog subfamily B member 14 C-C
motif chemokine 26 F-box only protein 8 Collectin-11 Fibroleukin
Cysteine-rich with EGF-like domain Methionine-R-sulfoxide reductase
B3, protein 2 mitochondrial C-X-C motif chemokine 16 Leucine-rich
repeat LGI family member 2 Fibroblast growth factor-binding protein
1 Vesicle transport protein GOT1B Interleukin-1 family member 5
Integral membrane protein GPR177 Interleukin-1 family member 9
Probable G-protein coupled receptor 78 Kallikrein-5 HEPACAM family
member 2 Matrilin-2 Interleukin-27 receptor subunit alpha Cell
surface glycoprotein CD200 receptor 1 Proenkephalin-A
Lysophosphatidic acid phosphatase type 6 Integrin alpha-10
Nucleotide exchange factor SIL1 KIEL motif-containing protein 1
Thrombospondin type-1 domain-containing Leukocyte
immunoglobulin-like receptor protein 4 subfamily A member 5
WNT1-inducible-signaling pathway protein Leucine-rich repeat and
fibronectin type-III 2 domain-containing protein 3
Bromodomain-containing protein 9 Uteroglobin CD99 antigen-like
protein 2 Netrin-G1 ligand Uncharacterized protein C1orf159
Pannexin-1 Carbohydrate sulfotransferase 12 Protocadherin-12
Probable serine carboxypeptidase CPVL Protocadherin alpha-10
Mucin-3A Protocadherin beta-10 CUB and zona pellucida-like domain-
Osteopetrosis-associated transmembrane containing protein 1 protein
1 Polypeptide N- Beta-galactoside alpha-2,6-
acetylgalactosaminyltransferase 14 sialyltransferase 1 Galectin-9
GPI transamidase component PIG-S Leucine-rich repeat-containing
protein 17 Proline-rich transmembrane protein 3 Leucine-rich repeat
neuronal protein 2 Sulfhydryl oxidase 2 Bifunctional heparan
sulfate N- A disintegrin and metalloproteinase with
deacetylase/N-sulfotransferase 3 thrombospondin motifs 16 Tuftelin
SH2 domain-containing protein 3A Brain mitochondrial carrier
protein SHC-transforming protein 4 Signal peptide, CUB and EGF-like
domain- Disintegrin and metalloproteinase domain- containing
protein 3 containing protein 23 14-3-3 protein sigma Transducin
beta-like protein 2 Alpha-1-acid glycoprotein 1 Tudor
domain-containing protein 10 Alpha-1-acid glycoprotein 2
Transmembrane 9 superfamily member 3 von Willebrand factor A
domain-containing Von Willebrand factor D and EGF domain- protein 1
containing protein Disintegrin and metalloproteinase domain- A
disintegrin and metalloproteinase with containing protein 9
thrombospondin motifs 17 Angiotensinogen Transmembrane channel-like
protein 2 Apolipoprotein A-II (Apo-AII) (ApoA-II)
Pregnancy-specific beta-1-glycoprotein 3 Apolipoprotein A-IV
(Apo-AIV) (ApoA-IV) Tenomodulin Apolipoprotein C-II (Apo-CII)
(ApoC-II) Tetraspanin-6 Beta-2-glycoprotein 1 Thioredoxin
domain-containing protein 5 Apoptosis-related protein 3 Vascular
endothelial growth factor D Beta-secretase 2 Pregnancy-specific
beta-1-glycoprotein 9 Histo-blood group ABO system transferase
Semaphorin-3F Cathepsin L2 Acid phosphatase-like protein 2 C-C
motif chemokine 3 Apolipoprotein O-like C-type lectin domain family
1 member B Beta-defensin 119 Calcium-activated chloride channel A
disintegrin and metalloproteinase with regulator 1 thrombospondin
motifs 12 Chymase Protein FAM131A Collagen alpha-1(VI) chain
Protein FAM3B Complement component C8 alpha chain
Beta-galactosidase-1-like protein Complement component C9 Lysozyme
g-like protein 1 Glucose-fructose oxidoreductase domain-
Inter-alpha-trypsin inhibitor heavy chain containing protein 2
H5-like protein DnaJ homolog subfamily B member 11 Sperm
acrosome-associated protein 5 Ectonucleotide Leucine-rich repeat
and immunoglobulin- pyrophosphatase/phosphodiesterase family like
domain-containing nogo receptor- member 7 interacting protein 2
Endoplasmic reticulum aminopeptidase 1 Surfactant-associated
protein 2 Receptor tyrosine-protein kinase erbB-3 Adiponectin
receptor protein 1 Endoplasmic reticulum resident protein Multiple
epidermal growth factor-like ERp44 domains 6 IgGFc-binding protein
Neuroendocrine protein 7B2 Complement factor H-related protein 1
Alpha-1B-glycoprotein Polypeptide N- WAP, kazal, immunoglobulin,
kunitz and acetylgalactosaminyltransferase 2 NTR domain-containing
protein 2 Hemopexin Arylacetamide deacetylase-like 1 Hepatocyte
growth factor activator Histatin-3 Major histocompatibility complex
class I- Pro-neuregulin-3, membrane-bound related gene protein
isoform Insulin-like growth factor-binding protein 6
Agouti-signaling protein Ig delta chain C region Claudin-8
Interleukin-1 beta UPF0454 protein C12orf49 Low-density lipoprotein
receptor-related von Willebrand factor A domain-containing protein
10 protein 5B1 Junctional adhesion molecule C Cadherin-6
Uncharacterized protein KIAA0319 Cathelicidin antimicrobial peptide
Laminin subunit alpha-5 Laminin subunit gamma-1 Fibronectin type
III domain-containing Dehydrogenase/reductase SDR family protein 4
member 7B Lipoprotein lipase C-C motif chemokine 16 Interstitial
collagenase C-C motif chemokine 24 Matrix metalloproteinase-9 HEAT
repeat-containing protein C7orf27 Mucin-16 Collagen alpha-2(IX)
chain Mucin-2 Collagen alpha-3(IX) chain
Mucin-5B Colipase Myocilin Collagen alpha-1(XXVII) chain Oxidized
low-density lipoprotein receptor 1 Carboxypeptidase N subunit 2
Prostate tumor overexpressed gene 1 Leucine-rich repeat
transmembrane protein neuronal protein 4 Receptor-interacting
serine/threonine- Collagen triple helix repeat-containing protein
kinase 2 protein 1 Equilibrative nucleoside transporter 3
Endothelin-2 Selenoprotein P Fibromodulin Pulmonary
surfactant-associated protein D Fc receptor-like B Stimulated by
retinoic acid gene 6 protein Zinc finger RAD18 domain-containing
homolog protein C1orf124 Trefoil factor 1 Growth/differentiation
factor 15 Tissue factor pathway inhibitor 2 Glia-derived nexin
Prothrombin Progonadoliberin-1 Toll-like receptor 9 Granzyme K
Intercellular adhesion molecule 4 Interferon alpha-17
Interleukin-19 Interferon alpha-21 lsthmin-2 Interferon alpha-8 Kin
of IRRE-like protein 1 Interferon omega-1 Kallikrein-10 Early
placenta insulin-like peptide Latent-transforming growth factor
beta- EGF, latrophilin and seven transmembrane binding protein 4
domain-containing protein 1 Paired immunoglobulin-like type 2
receptor Fibronectin type 3 and ankyrin repeat alpha domains
protein 1 Regenerating islet-derived protein 3 alpha Lysyl oxidase
homolog 4 E3 ubiquitin-protein ligase RNF5 Lumican Protachykinin-1
Adropin Secreted frizzled-related protein 1, isoform Leucine-rich
repeat transmembrane protein CRA_a FLRT1 Plasminogen-related
protein B Nucleobindin-2 Probable palmitoyltransferase ZDHHC16
Phospholipase A2 Angiopoietin-related protein 1 Proenkephalin-B
UPF0510 protein C19orf63 Peptidoglycan recognition protein I-beta
Scavenger receptor cysteine-rich type 1 Immunoglobulin superfamily
containing protein M160 leucine-rich repeat protein 2 ER
degradation-enhancing alpha- V-set and immunoglobulin domain-
mannosidase-like 2 containing protein 2 Beta-galactosidase-1-like
protein 2 Peptide YY Interleukin-17 receptor E Retinol-binding
protein 3 Interleukin-20 Atherin Interleukin-25 Translocation
protein SEC63 homolog PDZ domain-containing protein 11 Transforming
growth factor beta-3 Relaxin-3 Protein Wnt-10b Retinoid-inducible
serine carboxypeptidase Renalase Short palate, lung and nasal
epithelium Proprotein convertase subtilisin/kexin
carcinoma-associated protein 2 type 4 WAP four-disulfide core
domain protein 5 Carboxypeptidase A4 Platelet-derived growth factor
C Olfactomedin-4 Disintegrin and metalloproteinase domain-
Insulin-like growth factor-binding protein containing protein 33
complex acid labile chain BSD domain-containing protein 1
Amelogenin, Y isoform Cell adhesion molecule 3 Arylsulfatase F
CDC45-related protein Choriogonadotropin subunit beta variant 2
Chondrolectin Beta-defensin 104 Diacylglycerol O-acyltransferase 2
Beta-defensin 105 3-keto-steroid reductase Beta-defensin 107
Interleukin-17 receptor C Protein WFDC11 Interleukin-17 receptor D
WAP four-disulfide core domain protein 6 Integrator complex subunit
1 Epigen Junctional adhesion molecule-like Protein FAM19A5 E3
ubiquitin-protein ligase LNX Claudin-6 Leucine-rich repeat
transmembrane Carcinoembryonic antigen-related cell neuronal
protein 3 adhesion molecule 19 Methionine adenosyltransferase 2 A
disintegrin and metalloproteinase with subunit beta thrombospondin
motifs 1 Podocalyxin-like protein 2 Protein COQ10 A, mitochondrial
Prominin-2 Uncharacterized protein C19orf41 Plexin
domain-containing protein 2 Uncharacterized protein C21orf63
Roundabout homolog 4 Protein delta homolog 2 Lactosylceramide
alpha-2,3- Cocaine- and amphetamine-regulated sialyltransferase
transcript protein SID1 transmembrane family member 2 Lipoma HMGIC
fusion partner-like 1 protein Sushi domain-containing protein 1
Leucine-rich repeat-containing protein 18 Serine/threonine-protein
kinase TAO2 Leucine-rich repeat-containing protein 25 Transmembrane
protease, serine 2 Leucine-rich repeat-containing protein 3B
UDP-glucuronic acid decarboxylase 1 Leucine-rich repeat-containing
protein 3 Uncharacterized protein C10orf58 Ly6/PLAUR
domain-containing protein 4 Thioredoxin-related transmembrane
Vitamin K epoxide reductase complex protein 2 subunit 1
CMP-N-acetylneuraminate-beta- A disintegrin and metalloproteinase
with galactosamide-alpha-2,3-sialyltransferase thrombospondin
motifs 20 Putative uncharacterized protein Putative uncharacterized
protein ENSP00000380674 ENSP00000381830 Transmembrane protein 119
Cat eye syndrome critical region protein 1 Transmembrane protein 98
Testis-expressed protein 101 Pre-B lymphocyte protein 3
Xylosyltransferase 2 Putative uncharacterized protein C14orf144
Protein FAM20A Membrane-bound transcription factor site-1
Transmembrane and immunoglobulin protease domain-containing protein
1 Ficolin (Collagen/fibrinogen domain Putative killer cell
immunoglobulin-like containing) 3 (Hakata antigen) (NL3)
receptor-like protein KIR3DX1 (Leukocyte (Ficolin
(Collagen/fibrinogen domain receptor cluster member 12) containing)
3 (Hakata antigen), isoform CRA_b) Interleukin-1 family member 6
Herstatin Prostate and testis expressed protein 2 Leucine-rich
repeat-containing protein 28 Group XIIA secretory phospholipase A2
LRRN4 C-terminal-like protein Collagen alpha-3(V) chain Ly6/PLAUR
domain-containing protein 2 Alpha-2-macroglobulin-like protein 1
Transmembrane protein 81 Dermatopontin Myelin protein zero-like
protein 3 Cartilage-associated protein Protein notum homolog Desert
hedgehog protein UDP-glucuronosyltransferase 3A2 Extracellular
matrix protein 2 Protocadherin alpha-1 Gastric intrinsic factor
Phospholipase D4 Interleukin-33 Retinol dehydrogenase 10 Bone
morphogenetic protein 2 Sialic acid-binding Ig-like lectin 14 Bone
morphogenetic protein 6 Transmembrane protein 161A Uncharacterized
protein KIAA0564 Transmembrane protein 161B Cerberus Transmembrane
protein 182 Carbohydrate sulfotransferase 8 Protein FAM24B
Contactin-associated protein-like 3 Transmembrane protein 52 Group
XIIB secretory phospholipase A2- Major facilitator superfamily
domain- like protein containing protein 4 Corticoliberin
UDP-glucuronosyltransferase 2A3 A disintegrin and metalloproteinase
with Odontogenic ameloblast-associated thrombospondin motifs 19
protein UPF0556 protein C19orf10 Neurosecretory protein VGF C-X-C
motif chemokine 3 Secreted phosphoprotein 2, 24 kDa Cystatin-M
Protein FAM150B Defensin-5 Growth/differentiation factor 9
Defensin-6 Clusterin-like protein 1 A disintegrin and
metalloproteinase with Transmembrane and immunoglobulin
thrombospondin motifs 18 domain-containing protein 2 A disintegrin
and metalloproteinase with C-type lectin domain-containing protein
thrombospondin motifs 3 UNQ5810/PRO19627 Dickkopf-related protein 4
Epididymal-specific lipocalin-10 A disintegrin and
metalloproteinase with A disintegrin and metalloproteinase with
thrombospondin motifs 5 thrombospondin motifs 8 Mammalian
ependymin-related protein 1 Epididymal-specific lipocalin-8
Fibrillin-3 Basic proline-rich peptide P-E Fetuin-B Putative
uncharacterized protein C10orf99 Fibroblast growth factor 6
Uncharacterized protein C17orf77 Keratinocyte growth factor
Arylacetamide deacetylase-like 2 Growth/differentiation factor 8
Epididymal-specific lipocalin-12 Gastric inhibitory polypeptide B
melanoma antigen 2 Glycoprotein hormone beta-5 B melanoma antigen 3
Granzyme M Bovine seminal plasma protein homolog 1
Gastrin-releasing peptide Complement C1q-like protein 3 Serine
protease HTRA1 UPF0565 protein C2orf69 Interferon alpha-4 UPF0669
protein C6orf120 Interferon alpha-5 Colipase-like protein C6orf127
Interferon alpha-7 Uncharacterized protein C7orf69 A disintegrin
and metalloproteinase with Platelet-derived growth factor
receptor-like thrombospondin motifs 7 protein Immunoglobulin
superfamily member 10 Chondroadherin-like protein
Protease-associated domain-containing Putative uncharacterized
protein protein of 21 kDa UNQ6490/PRO21339 Abhydrolase
domain-containing protein Putative uncharacterized protein FAM108A1
UNQ6493/PRO21345 A disintegrin and metalloproteinase with Putative
uncharacterized protein thrombospondin motifs 9 UNQ5815/PRO19632
Interleukin-9 receptor Cystatin-A Interleukin-9 Peptidase inhibitor
R3HDML Inhibin beta B chain Cystatin-9 Serine protease inhibitor
Kazal-type 2 DAN domain family member 5 BMP-binding endothelial
regulator Insulin-like growth factor-binding protein- protein like
1 Keratinocyte-associated protein 2 Epididymal sperm-binding
protein 1 Laminin subunit alpha-1 Elafin Leukocyte cell-derived
chemotaxin-2 Protein FAM55A Gastric triacylglycerol lipase
Growth/differentiation factor 6 Leucine-rich repeat and calponin
Glucose-fructose oxidoreductase domain- homology domain-containing
protein 3 containing protein 1 Pancreatic lipase-related protein 2
Erythropoietin Epididymis-specific alpha-mannosidase Glutathione
peroxidase 6 Fibronectin type III domain-containing Uncharacterized
protein protein 7 UNQ511/PRO1026 Microfibrillar-associated protein
5 Beta-defensin 128 Muellerian-inhibiting factor Interleukin-31
Matrix metalloproteinase-21 Interleukin-34 Matrix
metalloproteinase-17 Plasma kallikrein-like protein 4 Matrix
metalloproteinase-20 Epididymal-specific lipocalin-9
N-acetylglucosamine-1- cDNA FLJ60957, highly similar to
phosphotransferase subunit gamma Secreted frizzled-related protein
4 Multimerin-2 Lipase member M Promotilin CLECSF12 FRAS1-related
extracellular matrix Putative inactive group IIC secretory protein
3 phospholipase A2 Protein kinase C-binding protein NELL1 Serine
protease MPN2 Protein kinase C-binding protein NELL2 Netrin-5
Neurotrypsin NHL repeat-containing protein 3 Neuroserpin
Olfactomedin-like protein 2B Nidogen-2 Ovochymase-2 Abhydrolase
domain-containing protein Putative uncharacterized protein FAM108B1
UNQ3029/PRO9830 Neurotrophin-4 Ovochymase-1 Epididymal secretory
glutathione Putative pregnancy-specific beta-1- peroxidase
glycoprotein 7 Group 10 secretory phospholipase A2 Ovostatin
homolog 2 Group IID secretory phospholipase A2 Orexigenic
neuropeptide QRFP Lactoperoxidase Lymphocyte antigen 6K p53
apoptosis effector related to PMP-22 Prostate and testis expressed
protein 1 Placenta-specific protein 1 Putative phospholipase B-like
1 Tuberoinfundibular peptide of Putative uncharacterized protein 39
residues FLJ42147 Prolargin Otogelin Secretogranin-2 Ribonuclease 8
Endonuclease domain-containing 1 Nuclear pore complex-interacting
protein- protein like 2 Semaphorin-3B Proactivator polypeptide-like
1 Somatostatin Protein spinster homolog 2 Dehydrogenase/reductase
SDR family von Willebrand factor C domain- member 4-like 2
containing protein 2-like Transcobalamin-1 Urotensin-2B Trefoil
factor 2 Tetraspanin-18 Testican-1 UPF0514 membrane protein FAM159A
Serum paraoxonase/lactonase 3 Latherin Tolloid-like protein 2
Methyltransferase-like protein 7B Trypsin-2 Protein TEX261 RING
finger and SPRY domain- Alkylated DNA repair protein alkB
containing protein 1 homolog 7 Calcium-binding and coiled-coil
domain- Transmembrane emp24 domain- containing protein 1 containing
protein 6 Protein Wnt-2 XK-related protein 5 Ectonucleoside
triphosphate Putative V-set and immunoglobulin diphosphohydrolase 8
domain-containing protein 7 Protein Wnt-8b Insulin growth
factor-like family member 3 UDP-GlcNAc: betaGal beta-1,3-N- Nuclear
pore complex-interacting protein- acetylglucosaminyltransferase 4
like 1 EMI domain-containing protein 1 Secreted phosphoprotein 1
Uncharacterized protein C6orf15 Collagen alpha-5(VI) chain
Collectin-10 B melanoma antigen 5 Long-chain-fatty-acid--CoA ligase
WAP four-disulfide core domain protein ACSBG2 10A
Oncoprotein-induced transcript 3 protein UPF0369 protein C6orf57
Peptidase inhibitor 15 Putative uncharacterized protein C10orf31
Proline-rich acidic protein 1 Putative uncharacterized protein
C11orf45 Urocortin Uncharacterized protein C12orf28 Trypsin-X3 (EC
3.4.21.4) Uncharacterized protein C17orf67 HHIP-like protein 2
Beta-defensin 121 Fractalkine Beta-defensin 130 Protein Wnt-11
Histidine triad nucleotide-binding protein 2 Protein Wnt-7a Apelin
FCH and double SH3 domains protein 1 Placenta-specific protein 9
Hepatoma-derived growth factor-related Hepatocellular
carcinoma-associated protein 2 protein TD26 Interleukin-12 subunit
alpha Persephin UPF0577 protein KIAA1324 Regulated
endocrine-specific protein 18 Complement C1q tumor necrosis factor-
Complement C1q tumor necrosis factor- related protein 9 related
protein 8 Mucin-17 Bone morphogenetic protein 8A Lysosomal protein
NCU-G1 Protein WFDC13 Prolyl 4-hydroxylase subunit alpha-3 Protein
Wnt-8a Peptidyl-prolyl cis-trans isomerase Ig-like
domain-containing protein SDCCAG10 ENSP00000270642 Peptidase
inhibitor 16 Abhydrolase domain-containing protein 15 Poliovirus
receptor-related protein 4 Ribonuclease-like protein 9 Solute
carrier family 22 member 15 Uncharacterized protein C2orf66 GPI
inositol-deacylase Uncharacterized protein C17orf99 Transmembrane
protein 43 Protein FAM150A Angiopoietin-related protein 2
Placenta-specific 1-like protein Angiopoietin-related protein 6
Uncharacterized protein C18orf20 Arylsulfatase K Beta-defensin 110
Augurin Neuritin-like protein Brain-specific serine protease 4
Histidine-rich carboxyl terminus protein 1 DBH-like monooxygenase
protein 1 C-type lectin domain family 2 member A Uncharacterized
protein C1orf56 Leucine-rich repeat-containing protein 70
Cerebellin-3 Serpin A13 Cerebellin-4 BTB/POZ domain-containing
protein 17 Colipase-like protein C6orf126 Uncharacterized protein
C12orf53 Uncharacterized protein C11orf83 C-type lectin domain
family 9 member A Uncharacterized protein C16orf89 Complement
C1q-like protein 4 Carboxypeptidase-like protein X2 CMRF35-like
molecule 4 Cystatin-9-like Protein FAM151B Dehydrogenase/reductase
SDR family Abhydrolase domain-containing protein member 13
FAM108A2/A3 Beta-defensin 123 Osteocrin Beta-defensin 132
Transmembrane protease, serine 11E2 Cytokine-like protein 1
Transmembrane protein 14E Dickkopf-related protein 2 Transmembrane
protein 207 Dickkopf-like protein 1 TOMM20-like protein 1
Epididymal secretory protein E3-beta Uncharacterized protein
C3orf41 EGF-like repeat and discoidin I-like Submaxillary gland
androgen-regulated domain-containing protein 3 protein 3A Protein
FAM55D B melanoma antigen 1 Fibroblast growth factor 17 Inactive
carboxylesterase 4 Fibroblast growth factor 22 Four-jointed box
protein 1 Fibroblast growth factor-binding protein 2 Protein HSN2
Growth/differentiation factor 3 Humanin GLIPR1-like protein 1
Kielin/chordin-like protein Serine protease inhibitor Kazal-type 6
UPF0624 protein C6orf186 Interleukin-17B Putative neurofibromin
1-like protein 4/6 Interleukin-17C Peroxidasin-like protein
Interleukin-17D SCO-spondin Hyaluronan and proteoglycan link
Putative uncharacterized protein protein 3 UNQ9165/PRO28630
Vitelline membrane outer layer protein 1 Calcium-activated chloride
channel homolog regulator family member 3 Choriogonadotropin
subunit beta Probable serine protease variant 1 UNQ9391/PRO34284
Lysozyme-like protein 1 Uncharacterized protein C4orf26 Matrix
metalloproteinase-28 Uncharacterized protein C4orf40 Nephronectin
Uncharacterized protein C5orf55 WAP four-disulfide core domain
Putative macrophage-stimulating protein protein 12 MSTP9
Olfactomedin-like protein 1 Uncharacterized protein C15orf61
Olfactomedin-like protein 2A Chymotrypsinogen B2 Serine protease 27
Beta-defensin 108A Secretoglobin family 3A member 2 Beta-defensin
111 A disintegrin and metalloproteinase with Putative V-set and
immunoglobulin thrombospondin motifs 2 domain-containing protein 6
Disintegrin and metalloproteinase Serine protease inhibitor
Kazal-type domain-containing protein 28 5-like 3
Bactericidal/permeability-increasing Putative serine protease
inhibitor Kazal- protein-like 2 type 5-like 2 Acid
sphingomyelinase-like Dehydrogenase/reductase SDR family
phosphodiesterase 3b member 7C Serine protease inhibitor Kazal-type
7 Beta-defensin 131 Neurexophilin-4 Beta-defensin 134 Protein
Wnt-9b Beta-defensin 136 Zymogen granule protein 16 homolog B
Beta-defensin 116 Semaphorin-3D Protein FAM132A Apolipoprotein L4
Protein FAM132B Transmembrane protease, serine 11D Beta-defensin
115 Scrapie-responsive protein 1 Beta-defensin 114 Putative annexin
A2-like protein Serine protease inhibitor Kazal-type 9 Bone
morphogenetic protein 10 Lipase member N Secretogranin-3 Pancreatic
lipase-related protein 3 Complement C1q tumor necrosis factor-
Testis, prostate and placenta-expressed related protein 4 protein
Uncharacterized protein C1orf54 Neuromedin-S Carboxypeptidase A6
Neuropeptide S C-C motif chemokine 19 Neuronal pentraxin-like
protein C16orf38 C-C motif chemokine 25 Otolin-1 Chymotrypsin-like
elastase family Iron/zinc purple acid phosphatase-like member 2B
protein Protein CEI Ovostatin homolog 1 Uncharacterized protein
C6orf1 Plasminogen-related protein A Uncharacterized protein
C7orf34 Polyserase-3 Keratinocyte-associated protein 3 Putative
peptide YY-2 Uncharacterized protein C9orf47 Putative peptide YY-3
Collagen alpha-1(VIII) chain Ribonuclease-like protein 10
Uncharacterized protein C18orf54 Ribonuclease-like protein 12
Cystatin-like 1 Ribonuclease-like protein 13 C2 domain-containing
protein 2 Serpin A11 DDRGK domain-containing protein 1 Kunitz-type
protease inhibitor 4 Protein FAM55C Meteorin-like protein Collagen
alpha-1(XXVI) chain Putative testis serine protease 2 Protein
FAM19A2 Beta-defensin 112 Protein FAM5B Uncharacterized protein
FLJ37543 Fibroblast growth factor 5 Protein FAM24A Probable serine
protease HTRA3 Secreted frizzled-related protein 4 Interleukin-1
family member 8 Complement C1q-like protein 2 Serine protease
inhibitor Kazal-type 4 Putative uncharacterized protein C17orf69
Otospiralin Putative cystatin-13 Liver-expressed antimicrobial
peptide 2 Beta-defensin 109 Lysyl oxidase homolog 1 Beta-defensin
113 Lysyl oxidase homolog 2 Beta-defensin 135 Long palate, lung and
nasal epithelium Peptidase S1 domain-containing protein
carcinoma-associated protein 4 LOC136242 Lysozyme g-like protein 2
Growth/differentiation factor 7 Endomucin IgA-inducing protein
homolog Neuropeptide B Putative lipocalin 1-like protein 1
Kinesin-like protein KIF7 Putative serine protease 29
Leukocyte-associated immunoglobulin- Putative scavenger receptor
cysteine-rich like receptor 2 domain-containing protein LOC619207
Calcium-dependent phospholipase A2 Secretoglobin-like protein
Proapoptotic caspase adapter protein Putative stereocilin-like
protein Integrin beta-like protein 1 Insulin growth factor-like
family member 2 Tolloid-like protein 1 KIR2DL4 Kunitz-type protease
inhibitor 3 Putative zinc-alpha-2-glycoprotein-like 1 Protein
TMEM155 Insulin growth factor-like family member 4 Prosalusin
Uncharacterized protein C2orf72 Protein amnionless Replication
initiation-like protein Protein WFDC10B Prostate and testis
expressed protein 3 WAP four-disulfide core domain protein 8 B
melanoma antigen 4 Protein Wnt-5b Putative uncharacterized protein
C1orf191 Protein Wnt-7b Beta-defensin 108B-like Zona
pellucida-binding protein 2 Uncharacterized protein FLJ90687 SH3
domain-binding protein 5-like Secreted frizzled-related protein 2
Adipocyte adhesion molecule Basic proline-rich peptide IB-1
Uncharacterized protein C12orf59 Fibroblast growth factor 16
Apolipoprotein A-I-binding protein Serine protease inhibitor
Kazal-type 8 Claudin-17 Uncharacterized protein KIAA0495 Inactive
caspase-12 Platelet basic protein-like 2 Uncharacterized protein
C7orf58 Serpin E3 Collagen alpha-1(XXVIII) chain CR1 receptor
Dentin matrix protein 4 Secreted phosphoprotein 1 Uncharacterized
protein C16orf48 Stress induced secreted protein 1 Carboxylesterase
3 Protein Wnt Protein FAM20B Protein Wnt (Fragment) GPN-loop GTPase
3 Putative serine protease LOC138652 GRAM domain-containing protein
1B TOM1 Phosphatidylinositol glycan anchor Putative uncharacterized
protein biosynthesis class U protein FLJ46089 Interleukin-27
subunit alpha Putative uncharacterized protein C1orf134
Pro-neuregulin-4, membrane-bound UDP-GlcNAc: betaGal beta-1,3-N-
isoform acetylglucosaminyltransferase 9 Leucine-rich repeat
neuronal protein 3 Uncharacterized protein C11orf44 NMDA
receptor-regulated protein 2 Uncharacterized protein C12orf73
NADH-cytochrome b5 reductase 1 Putative cystatin-9-like 2 Parkinson
disease 7 domain-containing Putative abhydrolase domain-containing
protein 1 protein FAM108A5 FK506-binding protein 11 Beta-defensin
133 C-type lectin domain family 12 member B Fibrosin-1 Solute
carrier family 35 member F5 Probable folate receptor delta Sialic
acid-binding Ig-like lectin 12 RPE-spondin Protein FAM19A3
NPIP-like protein ENSP00000346774 WD repeat-containing protein 82
Putative testis-specific prion protein Adipocyte enhancer-binding
protein 1 Proline-rich protein 1 ADAMTS-like protein 3 Putative
uncharacterized protein FP248 Coiled-coil domain-containing protein
80 UPF0670 protein C8orf55 Ecto-NOX disulfide-thiol exchanger 1
Putative zinc-alpha-2-glycoprotein-like 2 Neuronal growth regulator
1 SPARC protein Interphotoreceptor matrix proteoglycan 1
Otopetrin-1 cDNA FLJ36603 fis, clone cDNA FLJ55667, highly similar
to TRACH2015180, highly similar to Secreted protein acidic and rich
in Secreted frizzled-related protein 2 cysteine Lipase member H
Lipase member K Mucin-19 (MUC-19) C-type lectin domain family 18
member C Psoriasis susceptibility 1 candidate gene Putative
uncharacterized protein 2 protein UNQ6125/PRO20090 Integral
membrane protein 2A Complement C3 Vesicle transport protein SFT2B
Collagen alpha-2(IV) chain von Willebrand factor A domain-
Uncharacterized protein containing protein 3A UNQ6126/PRO20091
Protein shisa-2 homolog Serpin-like protein HMSD Signal peptidase
complex subunit 3 Prostate and testis expressed protein 4 CD164
sialomucin-like 2 protein Collagen alpha-1(XXII) chain Cadherin-16
Putative uncharacterized protein C13orf28 Cadherin-19 Cystatin-S
Cerebellin-2 R-spondin-1 Transmembrane protein C3orf1 C8orf2 Sperm
equatorial segment protein 1 Odorant-binding protein 2a
Uncharacterized protein C6orf72 Opiorphin Uncharacterized protein
C11orf24 Kidney androgen-regulated protein Acyl-CoA synthetase
family member 2, Putative uncharacterized protein mitochondrial
UNQ5830/PRO19650/PRO19816 Probable UDP-sugar transporter protein
Putative uncharacterized protein SLC35A5 UNQ6975/PRO21958 C-type
lectin domain family 1 member A Tachykinin-3 C-type lectin domain
family 3 member A Secreted phosphoprotein 1 C-type lectin domain
family 4 member E Sclerostin C-type lectin domain family 4 member G
ADAMTS-like protein 2 Probable cation-transporting Scavenger
receptor cysteine-rich domain- ATPase 13A4 containing protein
LOC284297 UPF0480 protein C15orf24 Tryptase beta-1 Zona pellucida
sperm-binding protein 4 Tryptase delta Endoplasmic reticulum
resident protein Putative cat eye syndrome critical region ERp27
protein 9 Transmembrane protein C16orf54 Plexin domain-containing
protein 1 Cytochrome P450 4F12 MC51L-53L-54L homolog (Fragment)
Cytochrome P450 4X1 COBW-like placental protein (Fragment)
Cytochrome P450 4Z1 Cytokine receptor-like factor 2 Protein CREG2
Beta-defensin 103 DnaJ homolog subfamily B member 9 Beta-defensin
106 Dipeptidase 3 Hyaluronidase-3 Membrane protein FAM174A
Interleukin-28 receptor alpha chain Thioredoxin domain-containing
Glycosyltransferase 54 domain-containing protein 15 protein Protein
FAM19A4 Chordin-like protein 1 Adenosine monophosphate-protein
Putative uncharacterized protein transferase FICD UNQ9370/PRO34162
Prenylcysteine oxidase-like Netrin receptor UNC5B Phytanoyl-CoA
hydroxylase-interacting Fibroblast growth factor receptor FGFR-1
protein-like secreted form protein (Fragment) FXYD
domain-containing ion transport Uncharacterized protein
regulator 4 ENSP00000244321 Growth/differentiation factor 11 ECE2
Cerebral dopamine neurotrophic factor EPA6 GPN-loop GTPase 2
Putative soluble interleukin 18 receptor 1 Growth hormone-inducible
Putative abhydrolase domain-containing transmembrane protein
protein FAM108A6 Glycerophosphodiester Putative V-set and
immunoglobulin phosphodiesterase domain-containing
domain-containing-like protein protein 2 ENSP00000303034 WAP,
kazal, immunoglobulin, kunitz and B cell maturation antigen
transcript variant NTR domain-containing protein 1 4 (Tumor
necrosis factor receptor superfamily member 17) KDEL
motif-containing protein 1 UPF0672 protein C3orf58 Adipophilin
Methylthioribose-1-phosphate isomerase Lactase-like protein 17-beta
hydroxysteroid dehydrogenase 13 Chondromodulin-1 Aminopeptidase B
Collagen alpha-6(VI) chain Dermcidin Leucine-rich repeat-containing
protein 33 Meteorin MANSC domain-containing protein 1
Methyltransferase-like protein 7A Lipocalin-15 NL3 Arylsulfatase I
N-acetyltransferase 15 Mesoderm development candidate 2 Ephrin-A4
Dickkopf-related protein 1 Protein Plunc Podocan Kallikrein-11
Fibronectin type III domain-containing WNT1 induced secreted
protein 1 splice protein 1 variant x (Fragment) Neurotrimin
Interleukin-1 family member 10 Olfactory receptor 10W1 PLA2G2D
Protein PARM-1 Proteoglycan 3 PDZ domain-containing protein 2
Insulin-like peptide INSL5 Proepiregulin Olfactomedin-like protein
3 Polycystic kidney disease protein 1-like 1 Extracellular
glycoprotein lacritin WLPL514 Retinol dehydrogenase 13 Matrix
metalloproteinase-26 Neutrophil defensin 3 RELT-like protein 2
GLGQ5807 Solute carrier family 35 member E3 TUFT1 Zinc transporter
ZIP9 DRLV8200 Noelin-2 IDLW5808 Seizure 6-like protein 2 UBAP2
Semaphorin-3A C1q/TNF-related protein 8 Semaphorin-4C KIR2DL4
(Fragment) Abhydrolase domain-containing protein Chemokine-like
factor super family 2 14A transcript variant 2 Ankyrin repeat
domain-containing Keratinocytes associated transmembrane protein 36
protein 1 Protein shisa-4 GKGM353 Neuromedin-U MATL2963 Nodal
homolog NINP6167 Synaptogyrin-2 POM121-like Brain-specific
angiogenesis inhibitor 1- RTFV9368 (SLE-dependent associated
protein 2-like protein 2 upregulation 1) Coiled-coil
domain-containing Leucine-rich repeat and immunoglobulin- protein
104 like domain-containing nogo receptor- interacting protein 4
Transmembrane 4 L6 family member 20 KCNQ2 Transmembrane protein 107
ELCV5929 Transmembrane protein 143 KVVM3106 Transmembrane protein
178 ISPF6484 Transmembrane protein 205 LKHP9428 Transmembrane
protein 41A VNFT9373 Transmembrane protein 50A ACAH3104
Transmembrane protein 50B RVLA1944 Interleukin-28B Wpep3002
Neuronal pentraxin-2 ZDHHC11 Collectrin AGLW2560 Transmembrane
protein 92 TSSP3028 Transmembrane protein 95 RFVG5814 Transmembrane
protein 9B SHSS3124 Probable carboxypeptidase PM20D1 MMP19
Tetraspanin-12 GSQS6193 Tetraspanin-13 VGPW2523 Tetraspanin-15
LMNE6487 UPF0513 transmembrane protein ALLA2487 Mitochondrial
uncoupling protein 4 GALI1870 Polyserase-2 FRSS1829 Probable
palmitoyltransferase ZDHHC24 MRSS6228 Zona pellucida sperm-binding
protein 1 GRPR5811 Zona pellucida sperm-binding protein 2 AVLL5809
Conserved oligomeric Golgi complex CR1 C3b/C4b receptor SCR9 (or
16) C- subunit 7 term. exon SCR = short consensus repeat
Adiponectin receptor protein 2 PIKR2786 Inhibin beta C chain S100
calcium binding protein A7-like 3 Brorin GTWW5826 (LP5085 protein)
Semaphorin-3C KTIS8219 (HCG2020043) Heparan sulfate glucosamine
3-O- Hyaluronan and proteoglycan link sulfotransferase 2 protein 4
Leptin receptor overlapping transcript- Micronovel like 1
SPARC-like protein 1 SAMK3000 Fibulin-7 VFLL3057 Protein HEG
homolog 1 CVWG5837 Fibrinogen C domain-containing VGSA5840 protein
1 Phospholipase A1 member A GHPS3125 Basic salivary proline-rich
protein 2 GRTR3118 Spermatogenesis-associated protein 6 PAMP6501
Sushi repeat-containing protein SRPX2 LTLL9335 Twisted gastrulation
protein homolog 1 VCEW9374 Torsin-1B AHPA9419 Protein Wnt-5a
MDHV1887 Acrosin-binding protein HSAL5836 C-type lectin domain
family 18 member B LHLC1946 Lysosomal-associated transmembrane Long
palate, lung and nasal epithelium protein 4A carcinoma-associated
protein 3 (Ligand- binding protein RYA3) Semaphorin-3E LPPA601
Ameloblastin PINK1 Major facilitator superfamily domain- SERH2790
containing protein 5 Angiopoietin-1 FLFF9364 Angiopoietin-4 APELIN
Multiple epidermal growth factor-like GLSH6409 domains 9 Acid
sphingomyelinase-like SFVP2550 phosphodiesterase 3a ADAMTS-like
protein 5 RRLF9220 Spexin PTML5838 Putative trypsin-6 VLGN1945
Proto-oncogene protein Wnt-1 AVPC1948 Bone morphogenetic protein 3b
AWQG2491 Bone morphogenetic protein 5 PSVL6168 Bone morphogenetic
protein 8B LCII3035 Protein FAM26D PPRR6495 C1q-related factor
RLSC6348 WAP four-disulfide core domain protein 1 CSRP2BP
Cerebellin-1 GLLV3061 Carboxypeptidase O GWSI6489 Myelin protein
zero-like protein 2 cDNA FLJ53955, highly similar to (Epithelial
V-like antigen 1) Secreted frizzled-related protein 4 Serine
protease 1-like protein 1 PPIF Coiled-coil domain-containing
protein 70 VSSW1971 C-C motif chemokine 28 KLIA6249 Uncharacterized
protein C4orf29 ALLW1950 CUB domain-containing protein 2 GVEI466
Trem-like transcript 4 protein ESFI5812 Uncharacterized protein
C6orf58 GNNC2999 Chondroadherin AAGG6488 Cartilage intermediate
layer protein 2 HHSL751 Uncharacterized protein C10orf25
Beta-defensin 108B Isthmin-1 Beta-defensin 118 Cystatin-8
Beta-defensin 124 Cardiotrophin-1 (CT-1) Beta-defensin 125
Chymotrypsinogen B Beta-defensin 126 C-X-C motif chemokine 9
Deoxyribonuclease-1-like 2 C-X-C motif chemokine 13 Stanniocalcin-2
EMILIN-3 Endothelial cell-specific molecule 1 Secretagogin
Carboxylesterase 7 Epididymal secretory protein E3-alpha Protein
NOV homolog Epiphycan UPF0528 protein FAM172A Protein FAM5C
Interleukin-27 subunit beta Fibroblast growth factor 20 Protein
FAM3C Fibroblast growth factor-binding protein 3 Stromal
cell-derived factor 2-like protein 1 Transmembrane protein 204
Butyrophilin subfamily 1 member A1 Phosphatidylethanolamine-binding
Keratinocyte-associated transmembrane protein 4 protein 2
Coagulation factor V Immunoglobulin alpha Fc receptor Coagulation
factor VII EMILIN-2 Pro-MCH Ephrin type-A receptor 10 Folate
receptor gamma Exostosin-like 2 Mucin-7 Follistatin-related protein
4 Galanin-like peptide Follistatin-related protein 5 Hemicentin-1
Transmembrane protein 66 Interleukin-6 Growth/differentiation
factor 2 Embryonic growth/differentiation factor 1 GDNF family
receptor alpha-4 Interleukin-8 Ig gamma-4 chain C region Gremlin-2
Lymphocyte antigen 86 Stromelysin-2 Inhibin beta E chain Probable
G-protein coupled receptor 171 GRAM domain-containing protein 1C
Pappalysin-2 Interferon alpha-10 Microfibril-associated
glycoprotein 4 Interferon alpha-16 Neuromedin-B Interferon alpha-6
Mimecan Immunoglobulin superfamily member 21 Matrix
metalloproteinase-19 Agrin Interleukin-11 Prolactin Interleukin-17A
Kelch-like protein 11 Interleukin-18 Protein Wnt-16 Interleukin-26
Properdin Interleukin-28A Kallikrein-13 Transmembrane emp24 domain-
1-acyl-sn-glycerol-3-phosphate containing protein 3 acyltransferase
delta Interleukin-29 Kallikrein-9 Insulin-like peptide INSL6
Vitamin K-dependent protein S Protein Wnt-2b Butyrophilin-like
protein 8 Pregnancy-specific beta-1-glycoprotein 1 Laminin subunit
beta-4 Sperm acrosome membrane-associated Lymphatic vessel
endothelial hyaluronic protein 4 acid receptor 1 Laminin subunit
gamma-3 Cystatin-SA Lysyl oxidase homolog 3 Transmembrane protein
59 Neurotensin/neuromedin N Apolipoprotein(a)-like protein 2 MAM
domain-containing protein 2 Lysozyme-like protein 2
Microfibrillar-associated protein 2 Lysozyme-like protein 4
Melanoma inhibitory activity protein 2 Reelin Matrix
metalloproteinase-24 Retinol-binding protein 4 Matrix
metalloproteinase-25 Carbonic anhydrase 14 Netrin-1
Tubulointerstitial nephritis antigen Netrin-3 Neuropeptide W
Alpha-N-acetylgalactosaminide alpha-
Alpha-1,3-mannosyl-glycoprotein 4-beta- 2,6-sialyltransferase 1
N-acetylglucosaminyltransferase B Alpha-N-acetylgalactosaminide
alpha- Transmembrane emp24 domain- 2,6-sialyltransferase 3
containing protein 5 Melanoma-derived growth regulatory Complement
C1q tumor necrosis factor- protein related protein 3
FMRFamide-related peptides Podocan-like protein 1 Otoconin-90
Pregnancy-specific beta-1-glycoprotein 5 Neurturin Keratocan
Neurexophilin-1 Group IIE secretory phospholipase A2
Neurexophilin-2 Left-right determination factor 2 Platelet factor 4
variant NKG2D ligand 2 Nociceptin Macrophage metalloelastase V-set
and transmembrane domain- Triggering receptor expressed on myeloid
containing protein 1 cells 1 Proline-rich protein 4 Cytokine
receptor-like factor 1 Prolactin-releasing peptide Secretin Serine
protease 33 Stromal cell-derived factor 2 Pregnancy-specific
beta-1-glycoprotein 8 Lysozyme-like protein 6 Retbindin Serpin A9
FMRFamide-related peptides Sclerostin domain-containing protein 1
Ribonuclease K6 Lysocardiolipin acyltransferase 1 Ribonuclease T2
Plasma glutamate carboxypeptidase Repetin Slit homolog 3 protein
Complement C1r subcomponent-like C3 and PZP-like
alpha-2-macroglobulin protein domain-containing protein 8
Uncharacterized glycosyltransferase Retinoic acid receptor
responder AER61 protein 2 Semaphorin-3G Cartilage acidic protein 1
Secretoglobin family 1C member 1 Stanniocalcin-1 Secretoglobin
family 1D member 1 Beta-tectorin Secretoglobin family 1D member 2
Post-GPI attachment to proteins factor 3 Serpin A12 Germ
cell-specific gene 1 protein Serpin I2 Interleukin-21 receptor von
Willebrand factor C and EGF V-set and immunoglobulin domain-
domain-containing protein containing protein 4 A disintegrin and
metalloproteinase with Scavenger receptor cysteine-rich domain-
thrombospondin motifs 15 containing group B protein Sodium channel
subunit beta-2 Prothyroliberin Metalloproteinase inhibitor 4
Semaphorin-4A T-cell immunomodulatory protein A disintegrin and
metalloproteinase with Tumor necrosis factor receptor
thrombospondin motifs 10 superfamily member 27 Thymic stromal
lymphopoietin Toll-like receptor 7 Transmembrane protein 130 Unique
cartilage matrix-associated Thioredoxin domain-containing protein
protein 16
Urocortin-2 Alpha-2-antiplasmin Urocortin-3 ( WAP four-disulfide
core domain protein 3 Protein AMBP Protein WFDC9 Complement C1q
tumor necrosis factor- A disintegrin and metalloproteinase with
related protein 9-like thrombospondin motifs 14 Growth inhibition
and differentiation- Adipocyte plasma membrane-associated related
protein 88 protein Protein Wnt-10a Peroxidasin homolog Protein
Wnt-3a Progressive ankylosis protein homolog Proto-oncogene protein
Wnt-3 Chitinase-3-like protein 1 Protein Wnt-6 UPF0672 protein
CXorf36 Protein Wnt-9a Arylsulfatase J Cytokine SCM-1 beta
Cortistatin Zymogen granule membrane protein 16 Ceruloplasmin Zona
pellucida-binding protein 1 Angiopoietin-related protein 5 Anterior
gradient protein 3 homolog Coiled-coil domain-containing protein
126 Amelotin CD177 antigen Uncharacterized protein C5orf46 Protein
canopy homolog 4 Uncharacterized aarF domain-containing Fibronectin
type-III domain-containing protein kinase 1 protein C4orf31 Draxin
Protein FAM180A Fibroblast growth factor 18 Platelet basic protein
C-X-C motif chemokine 11 Interferon epsilon Ly6/PLAUR
domain-containing protein 6 lntelectin-2 Chymotrypsin-like elastase
family Alpha-1,3-mannosyl-glycoprotein 4-beta- member 1
N-acetylglucosaminyltransferase A Erythropoietin receptor Matrix
extracellular phosphoglycoprotein MAM domain-containing cDNA
FLJ77863, highly similar to Homo glycosylphosphatidylinositol
anchor sapiens secreted and transmembrane 1 protein 2 (SECTM1),
mRNA Matrix metalloproteinase-27 Epididymal-specific lipocalin-6
Inactive serine protease 35 Afamin Coiled-coil domain-containing
Probable cation-transporting ATPase protein 134 13A5 Suprabasin
Glutathione peroxidase 3 Secretoglobin family 1D member 4
Claudin-18 V-set and transmembrane domain- Putative killer cell
immunoglobulin-like containing protein 2A receptor like protein
KIR3DP1 ADM Secretory phospholipase A2 receptor Uncharacterized
protein C2orf82 Haptoglobin Insulin growth factor-like family
Carcinoembryonic antigen-related cell member 1 adhesion molecule 20
Cadherin-like protein 29 Bone morphogenetic protein 3 Bone
morphogenetic protein 15 Bone marrow stromal antigen 2 Plasma
serine protease inhibitor Cytochrome P450 20A1 Carcinoembryonic
antigen-related cell Bactericidal/permeability-increasing adhesion
molecule 21 protein-like 3 Alpha-lactalbumin Protein dpy-19 homolog
2 Sister chromatid cohesion protein DCC1 Group IIF secretory
phospholipase A2 Galectin-3-binding protein Carboxypeptidase B
Dynein heavy chain domain-containing Glycosyltransferase 8
domain-containing protein 1 protein 2 C-C motif chemokine 17
Protein FAM19A1 Fatty acyl-CoA reductase 1 GDNF family receptor
alpha-like Fin bud initiation factor homolog Probable glutathione
peroxidase 8 Polymeric immunoglobulin receptor Cystatin-D
Prion-like protein doppel Cystatin-F C-X-C motif chemokine 6
Platelet-activating factor acetylhydrolase C-X-C motif chemokine 10
Pappalysin-1 Beta-defensin 1 Solute carrier family 22 member 12
Hyaluronan and proteoglycan link Chorionic somatomammotropin
hormone- protein 2 like 1 Disintegrin and metalloproteinase
Regulator of microtubule dynamics domain-containing protein 30
protein 3 Suppressor of fused homolog Retinol dehydrogenase 14
Folate receptor beta Galanin Extracellular sulfatase Sulf-2
Transcobalamin-2 Tumor necrosis factor receptor
Catechol-O-methyltransferase domain- superfamily member 14
containing protein 1 Artemin Tripeptidyl-peptidase 1 Collagen
alpha-1(XII) chain Trem-like transcript 1 protein Collagen
alpha-1(XIV) chain Guanylate cyclase activator 2B Beta-defensin 2
Inducible T-cell costimulator Interleukin-21 Interleukin-3
Interleukin-7 Notch homolog 2 N-terminal-like protein Inhibin alpha
chain Laminin subunit beta-2 Laminin subunit alpha-3 Neuropilin-2
Dehydrogenase/reductase SDR family EGF-containing fibulin-like
extracellular member on chromosome X matrix protein 1 FXYD
domain-containing ion transport Receptor-type tyrosine-protein
regulator 6 phosphatase kappa Serine incorporator 2 Regenerating
islet-derived protein 4 Stromelysin-3 Tachykinin-4 Secreted
phosphoprotein 1 Matrix metalloproteinase-23 Serine
beta-lactamase-like protein Complement C1q tumor necrosis factor-
LACTB, mitochondrial related protein 5 Galectin-3 Opticin
Pancreatic prohormone Pre-small/secreted glycoprotein
Pregnancy-specific beta-1-glycoprotein 6 Pentraxin-related protein
PTX3 Dickkopf-related protein 3 Carboxylesterase 8
Dehydrogenase/reductase SDR family Thioredoxin-related
transmembrane member 11 protein 4 Regenerating islet-derived
protein 3 Major facilitator superfamily domain- gamma containing
protein 2 RING finger protein 43 Kallikrein-12 Semenogelin-2
Brevican core protein Mucin-15 Porimin Bone sialoprotein 2
Torsin-1A Lymphotactin C-C motif chemokine 23 Growth-regulated
alpha protein Testican-3 R-spondin-2 Basic salivary proline-rich
protein 4 Transmembrane and coiled-coil domain- Tumor necrosis
factor receptor containing protein 3 superfamily member 18 VEGF
co-regulated chemokine 1 Brother of CDO ADM2
Beta-1,4-galactosyltransferase 4 Hydroxysteroid
11-beta-dehydrogenase Dehydrogenase/reductase SDR family 1-like
protein member 9 Delta-like protein 1 Eppin Ephrin-A1 Otoancorin
Fibroblast growth factor receptor-like 1 Tenascin-R GDNF family
receptor alpha-3 Growth factor Platelet receptor Gi24 Protein
TSPEAR Progonadoliberin-2 Hephaestin Kallikrein-7 Butyrophilin-like
protein 3 Apolipoprotein F Butyrophilin-like protein 9 Protein
CASC4 Laminin subunit gamma-2 VIP36-like protein Protein LMBR1L
Magnesium transporter protein 1 Mucin-21 Amiloride-sensitive amine
oxidase Endoplasmic reticulum mannosyl- [copper-containing]
oligosaccharide 1,2-alpha-mannosidase DNA damage-regulated
autophagy Pancreatic secretory granule membrane modulator protein 2
major glycoprotein GP2 Transmembrane protein C17orf87 Semaphorin-4B
Complement factor H-related protein 5 Semaphorin-5B FK506-binding
protein 7 Epsilon-sarcoglycan Serine incorporator 1
Guanylate-binding protein 5 Transmembrane and ubiquitin-like
Ectonucleoside triphosphate domain-containing protein 1
diphosphohydrolase 6 Protein ERGIC-53-like Serpin B3 Toll-like
receptor 10 Protein RMD5 homolog B Toll-like receptor 8 Scavenger
receptor class A member 5 Selenoprotein T Semaphorin-6B Sialic
acid-binding Ig-like lectin 11 Transmembrane protein 108 Sorting
nexin-24 Sushi domain-containing protein 3 Complement C1q tumor
necrosis factor- Latent-transforming growth factor beta- related
protein 1 binding protein 2 Putative uncharacterized protein
Putative uncharacterized protein UNQ6494/PRO21346 UNQ6190/PRO20217
Secreted and transmembrane 1 precusor Secreted and transmembrane 1
precusor variant variant C-type lectin domain family 18 member A
Collagen alpha-1(XX) chain Cysteine-rich secretory protein 3 Netrin
receptor UNC5D Complement C4-A Mucin-13 Putative uncharacterized
protein ATP-dependent metalloprotease YME1L1 PRO2829
Calcium-activated chloride channel Proprotein convertase
subtilisin/kexin regulator 2 type 5 Neuroblastoma suppressor of
tumorigenicity 1
[0075] The therapeutic proteins provided herein should not be
considered to be exclusive. Rather, as is apparent from the
disclosure provided herein, the methods of the invention are
applicable to any protein wherein attachment of a water soluble
polymer is desired according to the invention. For example,
therapeutic proteins are described in US 2007/0026485, incorporated
herein by reference in its entirety.
Blood Coagulation Proteins
[0076] In one aspect, the starting material of the present
invention is a blood coagulation protein, which can be derived from
human plasma, or produced by recombinant engineering techniques, as
described in patents U.S. Pat. Nos. 4,757,006; 5,733,873;
5,198,349; 5,250,421; 5,919,766; and EP 306 968.
[0077] Therapeutic polypeptides such as blood coagulation proteins
including Factor IX (FIX), Factor VIII (FVIII), Factor VIIa
(FVIIa), Von Willebrand Factor (VWF), Factor FV (FV), Factor X
(FX), Factor XI (FXI), Factor XII (FXII), thrombin (FII), protein
C, protein S, tPA, PAI-1, tissue factor (TF) and ADAMTS 13 protease
are rapidly degraded by proteolytic enzymes and neutralized by
antibodies. This reduces their half-life and circulation time,
thereby limiting their therapeutic effectiveness. Relatively high
doses and frequent administration are necessary to reach and
sustain the desired therapeutic or prophylactic effect of these
coagulation proteins. As a consequence, adequate dose regulation is
difficult to obtain and the need of frequent intravenous
administrations imposes restrictions on the patient's way of
living.
[0078] As described herein, blood coagulation proteins including,
but not limited to, Factor IX (FIX), Factor VIII (FVIII), Factor
VIIa (FVIIa), Von Willebrand Factor (VWF), Factor FV (FV), Factor X
(FX), Factor XI, Factor XII (FXII), thrombin (FII), protein C,
protein S, tPA, PAI-1, tissue factor (TF) and ADAMTS 13 protease
are contemplated by the invention. As used herein, the term "blood
coagulation protein" refers to any Factor IX (FIX), Factor VIII
(FVIII), Factor VIIa (FVIIa), Von Willebrand Factor (VWF), Factor
FV (FV), Factor X (FX), Factor XII (FXII), thrombin (FII), protein
C, protein S, tPA, PAI-1, tissue factor (TF) and ADAMTS 13 protease
which exhibits biological activity that is associated with that
particular native blood coagulation protein.
[0079] The blood coagulation cascade is divided into three distinct
segments: the intrinsic, extrinsic, and common pathways (Schenone
et al., Curr Opin Hematol. 2004; 11:272-7). The cascade involves a
series of serine protease enzymes (zymogens) and protein cofactors.
When required, an inactive zymogen precursor is converted into the
active form, which consequently converts the next enzyme in the
cascade.
[0080] The intrinsic pathway requires the clotting factors VIII,
IX, X, XI, and XII. Initiation of the intrinsic pathway occurs when
prekallikrein, high-molecular-weight kininogen, factor XI (FXI) and
factor XII (FXII) are exposed to a negatively charged surface. Also
required are calcium ions and phospholipids secreted from
platelets.
[0081] The extrinsic pathway is initiated when the vascular lumen
of blood vessels is damaged. The membrane glycoprotein tissue
factor is exposed and then binds to circulating factor VII (FVII)
and to small preexisting amounts of its activated form FVIIa. This
binding facilitates full conversion of FVII to FVIIa and
subsequently, in the presence of calcium and phospholipids, the
conversion of factor IX (FIX) to factor IXa (FIXa) and factor X
(FX) to factor Xa (FXa). The association of FVIIa with tissue
factor enhances the proteolytic activity by bringing the binding
sites of FVII for the substrate (FIX and FX) into closer proximity
and by inducing a conformational change, which enhances the
enzymatic activity of FVIIa.
[0082] The activation of FX is the common point of the two
pathways. Along with phospholipid and calcium, factors Va (FVa) and
Xa convert prothrombin to thrombin (prothrombinase complex), which
then cleaves fibrinogen to form fibrin monomers. The monomers
polymerize to form fibrin strands. Factor XIIIa (FXIIIa) covalently
bonds these strands to one another to form a rigid mesh.
[0083] Conversion of FVII to FVIIa is also catalyzed by a number of
proteases, including thrombin, FIXa, FXa, factor XIa (FXIa), and
factor XIIa (FXIIa). For inhibition of the early phase of the
cascade, tissue factor pathway inhibitor targets FVIIa/tissue
factor/FXa product complex.
Factor VIIa
[0084] FVII (also known as stable factor or proconvertin) is a
vitamin K-dependent serine protease glycoprotein with a pivotal
role in hemostasis and coagulation (Eigenbrot, Curr Protein Pept
Sci. 2002; 3:287-99).
[0085] FVII is synthesized in the liver and secreted as a
single-chain glycoprotein of 48 kD. FVII shares with all vitamin
K-dependent serine protease glycoproteins a similar protein domain
structure consisting of an amino-terminal gamma-carboxyglutamic
acid (Gla) domain with 9-12 residues responsible for the
interaction of the protein with lipid membranes, a carboxy-terminal
serine protease domain (catalytic domain), and two epidermal growth
factor-like domains containing a calcium ion binding site that
mediates interaction with tissue factor. Gamma-glutamyl carboxylase
catalyzes carboxylation of Gla residues in the amino-terminal
portion of the molecule. The carboxylase is dependent on a reduced
form of vitamin K for its action, which is oxidized to the epoxide
form. Vitamin K epoxide reductase is required to convert the
epoxide form of vitamin K back to the reduced form.
[0086] The major proportion of FVII circulates in plasma in zymogen
form, and activation of this form results in cleavage of the
peptide bond between arginine 152 and isoleucine 153. The resulting
activated FVIIa consists of a NH2-derived light chain (20 kD) and a
COOH terminal-derived heavy chain (30 kD) linked via a single
disulfide bond (Cys 135 to Cys 262). The light chain contains the
membrane-binding Gla domain, while the heavy chain contains the
catalytic domain.
[0087] The plasma concentration of FVII determined by genetic and
environmental factors is about 0.5 mg/mL (Pinotti et al., Blood.
2000; 95:3423-8). Different FVII genotypes can result in
several-fold differences in mean FVII levels. Plasma FVII levels
are elevated during pregnancy in healthy females and also increase
with age and are higher in females and in persons with
hypertriglyceridemia. FVII has the shortest half-life of all
procoagulant factors (3-6 h). The mean plasma concentration of
FVIIa is 3.6 ng/mL in healthy individuals and the circulating
half-life of FVIIa is relatively long (2.5 h) compared with other
coagulation factors.
[0088] Hereditary FVII deficiency is a rare autosomal recessive
bleeding disorder with a prevalence estimated to be 1 case per
500,000 persons in the general population (Acharya et al., J Thromb
Haemost. 2004; 2248-56). Acquired FVII deficiency from inhibitors
is also very rare. Cases have also been reported with the
deficiency occurring in association with drugs such as
cephalosporins, penicillins, and oral anticoagulants. Furthermore,
acquired FVII deficiency has been reported to occur spontaneously
or with other conditions, such as myeloma, sepsis, aplastic anemia,
with interleukin-2 and antithymocyte globulin therapy.
[0089] Reference polynucleotide and polypeptide sequences include,
e.g., GenBank Accession Nos. J02933 for the genomic sequence,
M13232 for the cDNA (Hagen et al. PNAS 1986; 83: 2412-6), and
P08709 for the polypeptide sequence (references incorporated herein
in their entireties). A variety of polymorphisms of FVII have been
described, for example see Sabater-Lleal et al. (Hum Genet. 2006;
118:741-51) (reference incorporated herein in its entirety).
Factor IX
[0090] FIX is a vitamin K-dependent plasma protein that
participates in the intrinsic pathway of blood coagulation by
converting FX to its active form in the presence of calcium ions,
phospholipids and FVIIIa. The predominant catalytic capability of
FIX is as a serine protease with specificity for a particular
arginine-isoleucine bond within FX. Activation of FIX occurs by
FXIa which causes excision of the activation peptide from FIX to
produce an activated FIX molecule comprising two chains held by one
or more disulphide bonds. Defects in FIX are the cause of recessive
X-linked hemophilia B.
[0091] Hemophilia A and B are inherited diseases characterized by
deficiencies in FVIII and FIX polypeptides, respectively. The
underlying cause of the deficiencies is frequently the result of
mutations in FVIII and FIX genes, both of which are located on the
X chromosome. Traditional therapy for hemophilias often involves
intravenous administration of pooled plasma or semi-purified
coagulation proteins from normal individuals. These preparations
can be contaminated by pathogenic agents or viruses, such as
infectious prions, HIV, parvovirus, hepatitis A, and hepatitis C.
Hence, there is an urgent need for therapeutic agents that do not
require the use of human serum.
[0092] The level of the decrease in FIX activity is directly
proportional to the severity of hemophilia B. The current treatment
of hemophilia B consists of the replacement of the missing protein
by plasma-derived or recombinant FIX (so-called FIX substitution or
replacement treatment or therapy).
[0093] Polynucleotide and polypeptide sequences of FIX can be found
for example in the UniProtKB/Swiss-Prot Accession No. P00740, U.S.
Pat. No. 6,531,298 and in FIG. 1 (SEQ ID NO: 1).
Factor VIII
[0094] Coagulation factor VIII (FVIII) circulates in plasma at a
very low concentration and is bound non-covalently to Von
Willebrand factor (VWF). During hemostasis, FVIII is separated from
VWF and acts as a cofactor for activated factor IX (FIXa)-mediated
FX activation by enhancing the rate of activation in the presence
of calcium and phospholipids or cellular membranes.
[0095] FVIII is synthesized as a single-chain precursor of
approximately 270-330 kD with the domain structure
A1-A2-B-A3-C1-C2. When purified from plasma (e.g., "plasma-derived"
or "plasmatic"), FVIII is composed of a heavy chain (A1-A2-B) and a
light chain (A3-C1-C2). The molecular mass of the light chain is 80
kD whereas, due to proteolysis within the B domain, the heavy chain
is in the range of 90-220 kD.
[0096] FVIII is also synthesized as a recombinant protein for
therapeutic use in bleeding disorders. Various in vitro assays have
been devised to determine the potential efficacy of recombinant
FVIII (rFVIII) as a therapeutic medicine. These assays mimic the in
vivo effects of endogenous FVIII. In vitro thrombin treatment of
FVIII results in a rapid increase and subsequent decrease in its
procoagulant activity, as measured by in vitro assays. This
activation and inactivation coincides with specific limited
proteolysis both in the heavy and the light chains, which alter the
availability of different binding epitopes in FVIII, e.g. allowing
FVIII to dissociate from VWF and bind to a phospholipid surface or
altering the binding ability to certain monoclonal antibodies.
[0097] The lack or dysfunction of FVIII is associated with the most
frequent bleeding disorder, hemophilia A. The treatment of choice
for the management of hemophilia A is replacement therapy with
plasma derived or rFVIII concentrates. Patients with severe
hemophilia A with FVIII levels below 1%, are generally on
prophylactic therapy with the aim of keeping FVIII above 1% between
doses. Taking into account the average half-lives of the various
FVIII products in the circulation, this result can usually be
achieved by giving FVIII two to three times a week.
[0098] Reference polynucleotide and polypeptide sequences include,
e.g., UniProtKB/Swiss-Prot P00451 (FA8 HUMAN); Gitschier J et al.,
Characterization of the human Factor VIII gene, Nature, 312(5992):
326-30 (1984); Vehar G H et al., Structure of human Factor VIII,
Nature, 312(5992):337-42 (1984); Thompson A R. Structure and
Function of the Factor VIII gene and protein, Semin Thromb Hemost,
2003:29; 11-29 (2002).
Von Willebrand Factor
[0099] Von Willebrand factor (VWF) is a glycoprotein circulating in
plasma as a series of multimers ranging in size from about 500 to
20,000 kD. Multimeric forms of VWF are composed of 250 kD
polypeptide subunits linked together by disulfide bonds. VWF
mediates initial platelet adhesion to the sub-endothelium of the
damaged vessel wall. Only the larger multimers exhibit hemostatic
activity. It is assumed that endothelial cells secrete large
polymeric forms of VWF and those forms of VWF which have a low
molecular weight (low molecular weight VWF) arise from proteolytic
cleavage. The multimers having large molecular masses are stored in
the Weibel-Pallade bodies of endothelial cells and liberated upon
stimulation.
[0100] VWF is synthesized by endothelial cells and megakaryocytes
as prepro-VWF that consists to a large extent of repeated domains.
Upon cleavage of the signal peptide, pro-VWF dimerizes through
disulfide linkages at its C-terminal region. The dimers serve as
protomers for multimerization, which is governed by disulfide
linkages between the free end termini. The assembly to multimers is
followed by the proteolytic removal of the propeptide sequence
(Leyte et al., Biochem. J. 274 (1991), 257-261).
[0101] The primary translation product predicted from the cloned
cDNA of VWF is a 2813-residue precursor polypeptide (prepro-VWF).
The prepro-VWF consists of a 22 amino acid signal peptide and a 741
amino acid propeptide, with the mature VWF comprising 2050 amino
acids (Ruggeri Z. A., and Ware, J., FASEB J., 308-316 (1993).
[0102] Defects in VWF are causal to Von Willebrand disease (VWD),
which is characterized by a more or less pronounced bleeding
phenotype. VWD type 3 is the most severe form, in which VWF is
completely missing, and VWD type 1 relates to a quantitative loss
of VWF and its phenotype can be very mild. VWD type 2 relates to
qualitative defects of VWF and can be as severe as VWD type 3. VWD
type 2 has many sub forms, some being associated with the loss or
the decrease of high molecular weight multimers. Von Willebrand
disease type 2a (VWD-2A) is characterized by a loss of both
intermediate and large multimers. VWD-2B is characterized by a loss
of highest-molecular-weight multimers. Other diseases and disorders
related to VWF are known in the art.
[0103] The polynucleotide and amino acid sequences of prepro-VWF
are available at GenBank Accession Nos. NM_000552 and NP_000543,
respectively.
[0104] Other blood coagulation proteins according to the present
invention are described in the art, e.g. Mann K G, Thromb Haemost,
1999; 82:165-74.
A. Polypeptides
[0105] In one aspect, the starting material of the present
invention is a protein or polypeptide. As described herein, the
term therapeutic protein refers to any therapeutic protein molecule
which exhibits biological activity that is associated with the
therapeutic protein. In one embodiment of the invention, the
therapeutic protein molecule is a full-length protein.
[0106] Therapeutic protein molecules contemplated include
full-length proteins, precursors of full length proteins,
biologically active subunits or fragments of full length proteins,
as well as biologically active derivatives and variants of any of
these forms of therapeutic proteins. Thus, therapeutic protein
include those that (1) have an amino acid sequence that has greater
than about 60%, about 65%, about 70%, about 75%, about 80%, about
85%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98% or about 99% or greater amino
acid sequence identity, over a region of at least about 25, about
50, about 100, about 200, about 300, about 400, or more amino
acids, to a polypeptide encoded by a referenced nucleic acid or an
amino acid sequence described herein; and/or (2) specifically bind
to antibodies, e.g., polyclonal or monoclonal antibodies, generated
against an immunogen comprising a referenced amino acid sequence as
described herein, an immunogenic fragment thereof, and/or a
conservatively modified variant thereof.
[0107] According to the present invention, the term "recombinant
therapeutic protein" includes any therapeutic protein obtained via
recombinant DNA technology. In certain embodiments, the term
encompasses proteins as described herein.
[0108] As used herein, "endogenous therapeutic protein" includes a
therapeutic protein which originates from the mammal intended to
receive treatment. The term also includes therapeutic protein
transcribed from a transgene or any other foreign DNA present in
said mammal. As used herein, "exogenous therapeutic protein"
includes a blood coagulation protein which does not originate from
the mammal intended to receive treatment.
[0109] As used herein, "plasma-derived blood coagulation protein"
or "plasmatic" includes all forms of the protein found in blood
obtained from a mammal having the property participating in the
coagulation pathway.
[0110] As used herein "biologically active derivative" or
"biologically active variant" includes any derivative or variant of
a molecule having substantially the same functional and/or
biological properties of said molecule, such as binding properties,
and/or the same structural basis, such as a peptidic backbone or a
basic polymeric unit.
[0111] An "analog," such as a "variant" or a "derivative," is a
compound substantially similar in structure and having the same
biological activity, albeit in certain instances to a differing
degree, to a naturally-occurring molecule. For example, a
polypeptide variant refers to a polypeptide sharing substantially
similar structure and having the same biological activity as a
reference polypeptide. Variants or analogs differ in the
composition of their amino acid sequences compared to the
naturally-occurring polypeptide from which the analog is derived,
based on one or more mutations involving (i) deletion of one or
more amino acid residues at one or more termini of the polypeptide
and/or one or more internal regions of the naturally-occurring
polypeptide sequence (e.g., fragments), (ii) insertion or addition
of one or more amino acids at one or more termini (typically an
"addition" or "fusion") of the polypeptide and/or one or more
internal regions (typically an "insertion") of the
naturally-occurring polypeptide sequence or (iii) substitution of
one or more amino acids for other amino acids in the
naturally-occurring polypeptide sequence. By way of example, a
"derivative" is a type of analog and refers to a polypeptide
sharing the same or substantially similar structure as a reference
polypeptide that has been modified, e.g., chemically.
[0112] A variant polypeptide is a type of analog polypeptide and
includes insertion variants, wherein one or more amino acid
residues are added to a therapeutic protein amino acid sequence of
the invention. Insertions may be located at either or both termini
of the protein, and/or may be positioned within internal regions of
the therapeutic protein amino acid sequence. Insertion variants,
with additional residues at either or both termini, include for
example, fusion proteins and proteins including amino acid tags or
other amino acid labels. In one aspect, the blood coagulation
protein molecule optionally contains an N-terminal Met, especially
when the molecule is expressed recombinantly in a bacterial cell
such as E. coli.
[0113] In deletion variants, one or more amino acid residues in a
therapeutic protein polypeptide as described herein are removed.
Deletions can be effected at one or both termini of the therapeutic
protein polypeptide, and/or with removal of one or more residues
within the therapeutic protein amino acid sequence. Deletion
variants, therefore, include fragments of a therapeutic protein
polypeptide sequence.
[0114] In substitution variants, one or more amino acid residues of
a therapeutic protein polypeptide are removed and replaced with
alternative residues. In one aspect, the substitutions are
conservative in nature and conservative substitutions of this type
are well known in the art. Alternatively, the invention embraces
substitutions that are also non-conservative. Exemplary
conservative substitutions are described in Lehninger,
[Biochemistry, 2nd Edition; Worth Publishers, Inc., New York
(1975), pp. 71-77] and are set out immediately below.
Conservative Substitutions
TABLE-US-00002 [0115] SIDE CHAIN CHARACTERISTIC AMINO ACID
Non-polar (hydrophobic): A. Aliphatic A L I V P B. Aromatic F W C.
Sulfur-containing M D. Borderline G Uncharged-polar: A. Hydroxyl S
T Y B. Amides N Q C. Sulfhydryl C D. Borderline G Positively
charged (basic) K R H Negatively charged (acidic) D E
[0116] Alternatively, exemplary conservative substitutions are set
out immediately below.
Conservative Substitutions II
TABLE-US-00003 [0117] EXEMPLARY ORIGINAL RESIDUE SUBSTITUTION Ala
(A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg
Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln,
Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met,
Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,
Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr
(Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala
B. Polynucleotides
[0118] Nucleic acids encoding a therapeutic protein of the
invention include, for example and without limitation, genes,
pre-mRNAs, mRNAs, cDNAs, polymorphic variants, alleles, synthetic
and naturally-occurring mutants.
[0119] Polynucleotides encoding a therapeutic protein of the
invention also include, without limitation, those that (1)
specifically hybridize under stringent hybridization conditions to
a nucleic acid encoding a referenced amino acid sequence as
described herein, and conservatively modified variants thereof; (2)
have a nucleic acid sequence that has greater than about 95%, about
96%, about 97%, about 98%, about 99%, or higher nucleotide sequence
identity, over a region of at least about 25, about 50, about 100,
about 150, about 200, about 250, about 500, about 1000, or more
nucleotides (up to the full length sequence of 1218 nucleotides of
the mature protein), to a reference nucleic acid sequence as
described herein. Exemplary "stringent hybridization" conditions
include hybridization at 42.degree. C. in 50% formamide,
5.times.SSC, 20 mM Na.PO4, pH 6.8; and washing in 1.times.SSC at
55.degree. C. for 30 minutes. It is understood that variation in
these exemplary conditions can be made based on the length and GC
nucleotide content of the sequences to be hybridized. Formulas
standard in the art are appropriate for determining appropriate
hybridization conditions. See Sambrook et al., Molecular Cloning: A
Laboratory Manual (Second ed., Cold Spring Harbor Laboratory Press,
1989) .sctn..sctn. 9.47-9.51.
[0120] A "naturally-occurring" polynucleotide or polypeptide
sequence is typically derived from a mammal including, but not
limited to, primate, e.g., human; rodent, e.g., rat, mouse,
hamster; cow, pig, horse, sheep, or any mammal. The nucleic acids
and proteins of the invention can be recombinant molecules (e.g.,
heterologous and encoding the wild type sequence or a variant
thereof, or non-naturally occurring).
C. Production of Therapeutic Proteins
[0121] Production of a therapeutic protein includes any method
known in the art for (i) the production of recombinant DNA by
genetic engineering, (ii) introducing recombinant DNA into
prokaryotic or eukaryotic cells by, for example and without
limitation, transfection, electroporation or microinjection, (iii)
cultivating said transformed cells, (iv) expressing therapeutic
protein, e.g. constitutively or upon induction, and (v) isolating
said blood coagulation protein, e.g. from the culture medium or by
harvesting the transformed cells, in order to obtain purified
therapeutic protein.
[0122] In other aspects, the therapeutic protein is produced by
expression in a suitable prokaryotic or eukaryotic host system
characterized by producing a pharmacologically acceptable blood
coagulation protein molecule. Examples of eukaryotic cells are
mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, and
HepG2.
[0123] A wide variety of vectors are used for the preparation of
the therapeutic protein and are selected from eukaryotic and
prokaryotic expression vectors. Examples of vectors for prokaryotic
expression include plasmids such as, and without limitation, pRSET,
pET, and pBAD, wherein the promoters used in prokaryotic expression
vectors include one or more of, and without limitation, lac, trc,
trp, recA, or araBAD. Examples of vectors for eukaryotic expression
include: (i) for expression in yeast, vectors such as, and without
limitation, pAO, pPIC, pYES, or pMET, using promoters such as, and
without limitation, AOX1, GAP, GAL1, or AUG1; (ii) for expression
in insect cells, vectors such as and without limitation, pMT, pAc5,
pIB, pMIB, or pBAC, using promoters such as and without limitation
PH, p10, MT, Ac5, OpIE2, gp64, or polh, and (iii) for expression in
mammalian cells, vectors such as and without limitation pSVL, pCMV,
pRc/RSV, pcDNA3, or pBPV, and vectors derived from, in one aspect,
viral systems such as and without limitation vaccinia virus,
adeno-associated viruses, herpes viruses, or retroviruses, using
promoters such as and without limitation CMV, SV40, EF-1, UbC, RSV,
ADV, BPV, and .beta.-actin.
D. Administration
[0124] In one embodiment a conjugated therapeutic protein of the
present invention may be administered by injection, such as
intravenous, intramuscular, or intraperitoneal injection.
[0125] To administer compositions comprising a conjugated
therapeutic protein of the present invention to human or test
animals, in one aspect, the compositions comprise one or more
pharmaceutically acceptable carriers. The terms "pharmaceutically"
or "pharmacologically acceptable" refer to molecular entities and
compositions that are stable, inhibit protein degradation such as
aggregation and cleavage products, and in addition do not produce
allergic, or other adverse reactions when administered using routes
well-known in the art, as described below. "Pharmaceutically
acceptable carriers" include any and all clinically useful
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like,
including those agents disclosed above.
[0126] As used herein, "effective amount" includes a dose suitable
for treating a disease or disorder or ameliorating a symptom of a
disease or disorder. In one embodiment, "effective amount" includes
a dose suitable for treating a mammal having a bleeding disorder as
described herein.
[0127] The compositions may be administered orally, topically,
transdermally, parenterally, by inhalation spray, vaginally,
rectally, or by intracranial injection. The term parenteral as used
herein includes subcutaneous injections, intravenous,
intramuscular, intracisternal injection, or infusion techniques.
Administration by intravenous, intradermal, intramuscular,
intramammary, intraperitoneal, intrathecal, retrobulbar,
intrapulmonary injection and or surgical implantation at a
particular site is contemplated as well. Generally, compositions
are essentially free of pyrogens, as well as other impurities that
could be harmful to the recipient.
[0128] Single or multiple administrations of the compositions can
be carried out with the dose levels and pattern being selected by
the treating physician. For the prevention or treatment of disease,
the appropriate dosage will depend on the type of disease to be
treated, as described above, the severity and course of the
disease, whether drug is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the drug, and the discretion of the attending
physician.
[0129] The present invention also relates to a pharmaceutical
composition comprising an effective amount of a conjugated
therapeutic protein as defined herein. The pharmaceutical
composition may further comprise a pharmaceutically acceptable
carrier, diluent, salt, buffer, or excipient. The pharmaceutical
composition can be used for treating the above-defined bleeding
disorders. The pharmaceutical composition of the invention may be a
solution or a lyophilized product. Solutions of the pharmaceutical
composition may be subjected to any suitable lyophilization
process.
[0130] As an additional aspect, the invention includes kits which
comprise a composition of the invention packaged in a manner which
facilitates its use for administration to subjects. In one
embodiment, such a kit includes a compound or composition described
herein (e.g., a composition comprising a conjugated therapeutic
protein), packaged in a container such as a sealed bottle or
vessel, with a label affixed to the container or included in the
package that describes use of the compound or composition in
practicing the method. In one embodiment, the kit contains a first
container having a composition comprising a conjugated therapeutic
protein and a second container having a physiologically acceptable
reconstitution solution for the composition in the first container.
In one aspect, the compound or composition is packaged in a unit
dosage form. The kit may further include a device suitable for
administering the composition according to a specific route of
administration. Preferably, the kit contains a label that describes
use of the therapeutic protein or peptide composition.
Water Soluble Polymers
[0131] In one aspect, a therapeutic protein derivative (i.e., a
conjugated therapeutic protein) molecule provided is bound to a
water-soluble polymer including, but not limited to, polyethylene
glycol (PEG), branched PEG, polysialic acid (PSA), hydroxyalkyl
starch (HAS), hydroxylethyl starch (HES), carbohydrate,
polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitin
sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran,
polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene
glycol (PPG) polyoxazoline, poly acryloylmorpholine, polyvinyl
alcohol (PVA), polycarboxylate, polyvinylpyrrolidone,
polyphosphazene, polyoxazoline, polyethylene-co-maleic acid
anhydride, polystyrene-co-maleic acid anhydride,
poly(l-hydroxymethylethylene hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC). In one
embodiment of the invention, the water soluble polymer is
consisting of sialic acid molecule having a molecular weight range
of 350 to 120,000, 500 to 100,000, 1000 to 80,000, 1500 to 60,000,
2,000 to 45,000 Da, 3,000 to 35,000 Da, and 5,000 to 25,000 Da. The
coupling of the water soluble polymer can be carried out by direct
coupling to the protein or via linker molecules. One example of a
chemical linker is MBPH (4-[4-N-Maleimidophenyl]butyric acid
hydrazide) containing a carbohydrate-selective hydrazide and a
sulfhydryl-reactive maleimide group (Chamow et al., J Biol Chem
1992; 267:15916-22). Other exemplary and preferred linkers are
described below.
[0132] In one embodiment, the derivative retains the full
functional activity of native therapeutic protein products, and
provides an extended half-life in vivo, as compared to native
therapeutic protein products. In another embodiment, the derivative
retains at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44. 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 110, 120, 130, 140, or 150 percent (%) biological activity
relative to native blood coagulation protein. In a related aspect,
the biological activities of the derivative and native blood
coagulation protein are determined by the ratios of chromogenic
activity to blood coagulation factor antigen value (blood
coagulation factor:Chr:blood coagulation factor:Ag). In still
another embodiment of the invention, the half-life of the construct
is decreased or increased 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,
1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold relative to the
in vivo half-life of native therapeutic protein.
A. Sialic Acid and PSA
[0133] PSAs consist of polymers (generally homopolymers) of
N-acetylneuraminic acid. The secondary amino group normally bears
an acetyl group, but it may instead bear a glycolyl group. Possible
substituents on the hydroxyl groups include acetyl, lactyl, ethyl,
sulfate, and phosphate groups.
##STR00007##
[0134] Structure of Sialic Acid (N-Acetylneuraminic Acid)
[0135] PSAs and mPSAs generally comprise linear polymers consisting
essentially of N-acetylneuraminic acid moieties linked by 2,8- or
2,9-glycosidic linkages or combinations of these (e.g. alternating
2,8- and 2,9-linkages). In particularly preferred PSAs and mPSAs,
the glycosidic linkages are .alpha.-2,8. Such PSAs and mPSAs are
conveniently derived from colominic acids, and are referred to
herein as "CAs" and "mCAs". Typical PSAs and mPSAs comprise at
least 2, preferably at least 5, more preferably at least 10 and
most preferably at least 20 N-acetylneuraminic acid moieties. Thus,
they may comprise from 2 to 300 N-acetylneuraminic acid moieties,
preferably from 5 to 200 N-acetylneuraminic acid moieties, or most
preferably from 10 to 100 N-acetylneuraminic acid moieties. PSAs
and CAs preferably are essentially free of sugar moieties other
than N-acetylneuraminic acid. Thus PSAs and CAs preferably comprise
at least 90%, more preferably at least 95% and most preferably at
least 98% N-acetylneuraminic acid moieties.
[0136] Where PSAs and CAs comprise moieties other than
N-acetylneuraminic acid (as, for example in mPSAS and mCAs) these
are preferably located at one or both of the ends of the polymer
chain. Such "other" moieties may, for example, be moieties derived
from terminal N-acetylneuraminic acid moieties by oxidation or
reduction.
[0137] For example, WO-A-0187922 describes such mPSAs and mCAs in
which the non-reducing terminal N-acetylneuraminic acid unit is
converted to an aldehyde group by reaction with sodium periodate.
Additionally, WO 2005/016974 describes such mPSAs and mCAs in which
the reducing terminal N-acetylneuraminic acid unit is subjected to
reduction to reductively open the ring at the reducing terminal
N-acetylneuraminic acid unit, whereby a vicinal diol group is
formed, followed by oxidation to convert the vicinal diol group to
an aldehyde group.
[0138] Sialic acid rich glycoproteins bind selectin in humans and
other organisms. They play an important role in human influenza
infections. E.g., sialic acid can hide mannose antigens on the
surface of host cells or bacteria from mannose-binding lectin. This
prevents activation of complement. Sialic acids also hide the
penultimate galactose residue thus preventing rapid clearance of
the glycoprotein by the galactose receptor on the hepatic
parenchymal cells.
##STR00008##
[0139] Structure of Colominic Acid (Homopolymer of
N-Acetylneuraminic Acid)
[0140] Colominic acids (a sub-class of PSAs) are homopolymers of
N-acetylneuraminic acid (NANA) with .alpha. (2.fwdarw.8) ketosidic
linkage, and are produced, inter alia, by particular strains of
Escherichia coli possessing K1 antigen. Colominic acids have many
physiological functions. They are important as a raw material for
drugs and cosmetics.
[0141] Comparative studies in vivo with polysialylated and
unmodified asparaginase revealed that polysialylation increased the
half-life of the enzyme (Fernandes and Gregoriadis, Biochimica
Biophysica Acta 1341: 26-34, 1997).
[0142] As used herein, "sialic acid moieties" includes sialic acid
monomers or polymers ("polysaccharides") which are soluble in an
aqueous solution or suspension and have little or no negative
impact, such as side effects, to mammals upon administration of the
PSA-blood coagulation protein conjugate in a pharmaceutically
effective amount. The polymers are characterized, in one aspect, as
having 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,
300, 400, or 500 sialic acid units. In certain aspects, different
sialic acid units are combined in a chain.
[0143] In one embodiment of the invention, the sialic acid portion
of the polysaccharide compound is highly hydrophilic, and in
another embodiment the entire compound is highly hydrophilic.
Hydrophilicity is conferred primarily by the pendant carboxyl
groups of the sialic acid units, as well as the hydroxyl groups.
The saccharide unit may contain other functional groups, such as,
amine, hydroxyl or sulphate groups, or combinations thereof. These
groups may be present on naturally-occurring saccharide compounds,
or introduced into derivative polysaccharide compounds.
[0144] The naturally occurring polymer PSA is available as a
polydisperse preparation showing a broad size distribution (e.g.
Sigma C-5762) and high polydispersity (PD). Because the
polysaccharides are usually produced in bacteria carrying the
inherent risk of copurifying endotoxins, the purification of long
sialic acid polymer chains may raise the probability of increased
endotoxin content. Short PSA molecules with 1-4 sialic acid units
can also be synthetically prepared (Kang S H et al., Chem Commun.
2000; 227-8; Ress D K and Linhardt R J, Current Organic Synthesis.
2004; 1:31-46), thus minimizing the risk of high endotoxin levels.
However PSA preparations with a narrow size distribution and low
polydispersity, which are also endotoxin-free, can now be
manufactured. Polysaccharide compounds of particular use for the
invention are, in one aspect, those produced by bacteria. Some of
these naturally-occurring polysaccharides are known as glycolipids.
In one embodiment, the polysaccharide compounds are substantially
free of terminal galactose units.
B. Polyethylene Glycol (PEG) and Pegylation
[0145] In certain aspects, therapeutic proteins are conjugated to a
water soluble polymer by any of a variety of chemical methods
(Roberts J M et al., Advan Drug Delivery Rev 2002; 54:459-76). For
example, in one embodiment a therapeutic protein is modified by the
conjugation of PEG to free amino groups of the protein using
N-hydroxysuccinimide (NHS) esters. In another embodiment the water
soluble polymer, for example PEG, is coupled to free SH groups
using maleimide chemistry or the coupling of PEG hydrazides or PEG
amines to carbohydrate moieties of the therapeutic protein after
prior oxidation.
[0146] The conjugation is in one aspect performed by direct
coupling (or coupling via linker systems) of the water soluble
polymer to a therapeutic protein under formation of stable bonds.
In addition degradable, releasable or hydrolysable linker systems
are used in certain aspects the present invention (Tsubery et al. J
Biol Chem 2004; 279:38118-24/Greenwald et al., J Med Chem 1999;
42:3657-67/Zhao et al., Bioconj Chem 2006;
17:341-51/WO2006/138572A2/U.S. Pat. No. 7,259,224B2/U.S. Pat. No.
7,060,259B2).
[0147] In one embodiment of the invention, a therapeutic protein is
modified via lysine residues by use of polyethylene glycol
derivatives containing an active N-hydroxysuccinimide ester (NHS)
such as succinimidyl succinate, succinimidyl glutarate or
succinimidyl propionate. These derivatives react with the lysine
residues of the therapeutic protein under mild conditions by
forming a stable amide bond. In one embodiment of the invention,
the chain length of the PEG derivative is 5,000 Da. Other PEG
derivatives with chain lengths of 500 to 2,000 Da, 2,000 to 5,000
Da, greater than 5,000 up to 10,000 Da or greater than 10,000 up to
20,000 Da, or greater than 20,000 up to 150,000 Da are used in
various embodiments, including linear and branched structures.
[0148] Alternative methods for the PEGylation of amino groups are,
without limitation, the chemical conjugation with PEG carbonates by
forming urethane bonds, or the reaction with aldehydes or ketones
by reductive amination forming secondary amide bonds.
[0149] In one embodiment of the present invention a therapeutic
protein molecule is chemically modified using PEG derivatives that
are commercially available. These PEG derivatives in alternative
aspects have linear or branched structures. Examples of
PEG-derivatives containing NHS groups are listed below.
[0150] The following PEG derivatives are non-limiting examples of
those commercially available from Nektar Therapeutics (Huntsville,
Ala.; see www.nektar.com/PEG reagent catalog; Nektar Advanced
PEGylation, price list 2005-2006):
##STR00009##
##STR00010##
[0151] This reagent with branched structure is described in more
detail by Kozlowski et al. (BioDrugs 2001; 5:419-29).
[0152] Other non-limiting examples of PEG derivatives are
commercially available from NOF Corporation (Tokyo, Japan; see
www.nof.co.jp/english: Catalogue 2005)
##STR00011##
[0153] Structures of Branched PEG-Derivatives (NOF Corp.):
##STR00012##
[0154] These propane derivatives show a glycerol backbone with a
1,2 substitution pattern. In the present invention branched PEG
derivatives based on glycerol structures with 1,3 substitution or
other branched structures described in US2003/0143596A1 are also
contemplated.
[0155] PEG derivatives with degradable (for example, hydrolysable)
linkers as described by Tsubery et al. (J Biol Chem 2004;
279:38118-24) and Shechter et al. (WO04089280A3) are also
contemplated.
[0156] Surprisingly, the PEGylated therapeutic protein of this
invention exhibits functional activity, combined with an extended
half-life in vivo. In addition the PEGylated rFVIII, FVIIa, FIX, or
other blood coagulation factor seems to be more resistant against
thrombin inactivation.
C. Hydroxyalkyl Starch (HAS) and Hydroxylethyl Starch (HES)
[0157] In various embodiments of the present invention, a
therapeutic protein molecule is chemically modified using
hydroxyalkyl starch (HAS) or hydroxylethyl starch (HES) or
derivatives thereof.
[0158] HES is a derivative of naturally occurring amylopectin and
is degraded by alpha-amylase in the body. HES is a substituted
derivative of the carbohydrate polymer amylopectin, which is
present in corn starch at a concentration of up to 95% by weight.
HES exhibits advantageous biological properties and is used as a
blood volume replacement agent and in hemodilution therapy in the
clinics (Sommermeyer et al., 1987, Krankenhauspharmazie, 8 (8),
271-278; and Weidler et al., 1991, Arzneim.-Forschung/Drug Res. g
419 494-498).
[0159] Amylopectin consists of glucose moieties, wherein in the
main chain alpha-1,4-glycosidic bonds are present and at the
branching sites alpha-1, 6-glycosidic bonds are found. The
physical-chemical properties of this molecule are mainly determined
by the type of glycosidic bonds. Due to the nicked
alpha-1,4-glycosidic bond, helical structures with about six
glucose-monomers per turn are produced. The physico-chemical as
well as the biochemical properties of the polymer can be modified
via substitution. The introduction of a hydroxyethyl group can be
achieved via alkaline hydroxyethylation. By adapting the reaction
conditions it is possible to exploit the different reactivity of
the respective hydroxy group in the unsubstituted glucose monomer
with respect to a hydroxyethylation. Owing to this fact, the
skilled person is able to influence the substitution pattern to a
limited extent.
[0160] HAS refers to a starch derivative which has been substituted
by at least one hydroxyalkyl group. Therefore, the term
hydroxyalkyl starch is not limited to compounds where the terminal
carbohydrate moiety comprises hydroxyalkyl groups R1, R2, and/or
R3, but also refers to compounds in which at least one hydroxy
group present anywhere, either in the terminal carbohydrate moiety
and/or in the remaining part of the starch molecule, HAS', is
substituted by a hydroxyalkyl group R1, R2, or R3.
##STR00013##
[0161] The alkyl group may be a linear or branched alkyl group
which may be suitably substituted. Preferably, the hydroxyalkyl
group contains 1 to 10 carbon atoms, more preferably from 1 to 6
carbon atoms, more preferably from 1 to 4 carbon atoms, and even
more preferably 2-4 carbon atoms. "Hydroxyalkyl starch" therefore
preferably comprises hydroxyethyl starch, hydroxypropyl starch and
hydroxybutyl starch, wherein hydroxyethyl starch and hydroxypropyl
starch are particularly preferred.
[0162] Hydroxyalkyl starch comprising two or more different
hydroxyalkyl groups is also comprised in the present invention. The
at least one hydroxyalkyl group comprised in HAS may contain two or
more hydroxy groups. According to one embodiment, the at least one
hydroxyalkyl group comprised HAS contains one hydroxy group.
[0163] The term HAS also includes derivatives wherein the alkyl
group is mono- or polysubstituted. In one embodiment, the alkyl
group is substituted with a halogen, especially fluorine, or with
an aryl group, provided that the HAS remains soluble in water.
Furthermore, the terminal hydroxy group a of hydroxyalkyl group may
be esterified or etherified. HAS derivatives are described in
WO/2004/024776, which is incorporated by reference in its
entirety.
D. Methods of Attachment
[0164] A therapeutic protein may be covalently linked to the
polysaccharide compounds by any of various techniques known to
those of skill in the art. In various aspects of the invention,
sialic acid moieties are bound to a therapeutic protein, e.g., FIX,
FVIII, FVIIa or VWF, for example by the method described in U.S.
Pat. No. 4,356,170, which is herein incorporated by reference.
[0165] Other techniques for coupling PSA to polypeptides are also
known and contemplated by the invention. For example, US
Publication No. 2007/0282096 describes conjugating an amine or
hydrazide derivative of, e.g., PSA, to proteins. In addition, US
Publication No. 2007/0191597 describes PSA derivatives containing
an aldehyde group for reaction with substrates (e.g., proteins) at
the reducing end. These references are incorporated by reference in
their entireties.
[0166] Various methods are disclosed at column 7, line 15, through
column 8, line 5 of U.S. Pat. No. 5,846,951 (incorporated by
reference in its entirety). Exemplary techniques include linkage
through a peptide bond between a carboxyl group on one of either
the blood coagulation protein or polysaccharide and an amine group
of the blood coagulation protein or polysaccharide, or an ester
linkage between a carboxyl group of the blood coagulation protein
or polysaccharide and a hydroxyl group of the therapeutic protein
or polysaccharide. Another linkage by which the therapeutic protein
is covalently bonded to the polysaccharide compound is via a Schiff
base, between a free amino group on the blood coagulation protein
being reacted with an aldehyde group formed at the non-reducing end
of the polysaccharide by periodate oxidation (Jennings H J and
Lugowski C, J Immunol. 1981; 127:1011-8; Fernandes A I and
Gregoriadis G, Biochim Biophys Acta. 1997; 1341; 26-34). The
generated Schiff base is in one aspect stabilized by specific
reduction with NaCNBH3 to form a secondary amine. An alternative
approach is the generation of terminal free amino groups in the PSA
by reductive amination with NH4C1 after prior oxidation.
Bifunctional reagents can be used for linking two amino or two
hydroxyl groups. For example, PSA containing an amino group is
coupled to amino groups of the protein with reagents like BS3
(Bis(sulfosuccinimidyl)suberate/Pierce, Rockford, Ill.). In
addition heterobifunctional cross linking reagents like Sulfo-EMCS
(N-.epsilon.-Maleimidocaproyloxy) sulfosuccinimide ester/Pierce) is
used for instance to link amine and thiol groups.
[0167] In another approach, a PSA hydrazide is prepared and coupled
to the carbohydrate moiety of the protein after prior oxidation and
generation of aldehyde functions.
[0168] As described above, a free amine group of the therapeutic
protein reacts with the 1-carboxyl group of the sialic acid residue
to form a peptidyl bond or an ester linkage is formed between the
1-carboxylic acid group and a hydroxyl or other suitable active
group on a blood coagulation protein. Alternatively, a carboxyl
group forms a peptide linkage with deacetylated 5-amino group, or
an aldehyde group of a molecule of a therapeutic protein forms a
Schiff base with the N-deacetylated 5-amino group of a sialic acid
residue.
[0169] Alternatively, the polysaccharide compound is associated in
a non-covalent manner with a therapeutic protein. For example, the
polysaccharide compound and the pharmaceutically active compound
are in one aspect linked via hydrophobic interactions. Other
non-covalent associations include electrostatic interactions, with
oppositely charged ions attracting each other.
[0170] In various embodiments, the therapeutic protein is linked to
or associated with the polysaccharide compound in stoichiometric
amounts (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:7, 1:8, 1:9, or
1:10, etc.). In various embodiments, 1-6, 7-12 or 13-20
polysaccharides are linked to the blood coagulation protein. In
still other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20 or more polysaccharides are linked to
the blood coagulation protein.
[0171] In various embodiments, the therapeutic protein is modified
to introduce glycosylation sites (i.e., sites other than the native
glycosylation sites). Such modification may be accomplished using
standard molecular biological techniques known in the art.
Moreover, the therapeutic protein, prior to conjugation to a water
soluble polymer via one or more carbohydrate moieties, may be
glycosylated in vivo or in vitro. These glycosylated sites can
serve as targets for conjugation of the proteins with water soluble
polymers (US Patent Application No. 20090028822, US Patent
Application No. 2009/0093399, US Patent Application No.
2009/0081188, US Patent Application No. 2007/0254836, US Patent
Application No. 2006/0111279, and DeFrees S. et al., Glycobiology,
2006, 16, 9, 833-43). For example, a protein that is not naturally
glycoslyated in vivo (e.g., a protein that is not a glycoprotein)
may be modified as described above.
E. Aminooxy Linkage
[0172] In one embodiment of the invention, the reaction of
hydroxylamine or hydroxylamine derivatives with aldehydes (e.g., on
a carbohydrate moiety following oxidation by sodium periodate) to
form an oxime group is applied to the preparation of conjugates of
blood coagulation protein. For example, a glycoprotein (e.g., a
therapeutic protein according to the present invention) is first
oxidized with a oxidizing agent such as sodium periodate (NaIO4)
(Rothfus J A et Smith E L., J Biol Chem 1963, 238, 1402-10; and Van
Lenten L and Ashwell G., J Biol Chem 1971, 246, 1889-94). The
periodate oxidation of glycoproteins is based on the classical
Malaprade reaction described in 1928, the oxidation of vicinal
diols with periodate to form an active aldehyde group (Malaprade
L., Analytical application, Bull Soc Chim France, 1928, 43,
683-96). Additional examples for such an oxidizing agent are lead
tetraacetate (Pb(OAc)4), manganese acetate (MnO(Ac)3), cobalt
acetate (Co(OAc)2), thallium acetate (TlOAc), cerium sulfate
(Ce(SO4)2) (U.S. Pat. No. 4,367,309) or potassium perruthenate
(KRuO4) (Marko et al., J Am Chem Soc 1997, 119, 12661-2). By
"oxidizing agent" a mild oxidizing compound which is capable of
oxidizing vicinal diols in carbohydrates, thereby generating active
aldehyde groups under physiological reaction conditions is
meant.
[0173] The second step is the coupling of the polymer containing an
aminooxy group to the oxidized carbohydrate moiety to form an oxime
linkage. In one embodiment of the invention, this step can be
carried out in the presence of catalytic amounts of the
nucleophilic catalyst aniline or aniline derivatives (Dirksen A et
Dawson P E, Bioconjugate Chem. 2008; Zeng Y et al., Nature Methods
2009; 6:207-9). The aniline catalysis dramatically accelerates the
oxime ligation allowing the use of very low concentrations of the
reagents. In another embodiment of the invention the oxime linkage
is stabilized by reduction with NaCNBH3 to form an alkoxyamine
linkage (FIG. 2). Additional catalysts are described below.
[0174] Additional information on aminooxy technology can be found
in the following references, each of which is incorporated in their
entireties: EP 1681303A1 (HASylated erythropoietin); WO 2005/014024
(conjugates of a polymer and a protein linked by an oxime linking
group); WO96/40662 (aminooxy-containing linker compounds and their
application in conjugates); WO 2008/025856 (Modified proteins);
Peri F et al., Tetrahedron 1998, 54, 12269-78; Kubler-Kielb J et.
Pozsgay V., J Org Chem 2005, 70, 6887-90; Lees A et al., Vaccine
2006, 24(6), 716-29; and Heredia K L et al., Macromoecules 2007,
40(14), 4772-9.
[0175] In various embodiments of the invention, the water soluble
polymer which is linked according to the aminooxy technology
described herein to an oxidized carbohydrate moiety of a
therapeutic protein (e.g., FVIII, FVIIa, or FIX) include, but are
not limited to polyethylene glycol (PEG), branched PEG, polysialic
acid (PSA), carbohydrate, polysaccharides, pullulane, chitosan,
hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch,
dextran, carboxymethyl-dextran, polyalkylene oxide (PAO),
polyalkylene glycol (PAG), polypropylene glycol (PPG)
polyoxazoline, poly acryloylmorpholine, polyvinyl alcohol (PVA),
polycarboxylate, polyvinylpyrrolidone, polyphosphazene,
polyoxazoline, polyethylene-co-maleic acid anhydride,
polystyrene-co-maleic acid anhydride, poly(l-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC).
Nucleophilic Catalysts
[0176] As described herein, the conjugation of water soluble
polymers to therapeutic proteins can be catalyzed by aniline.
Aniline strongly catalyzes aqueous reactions of aldehydes and
ketones with amines to form stable imines such as hydrazones and
oximes. The following diagram compares an uncatalyzed versus the
aniline-catalyzed oxime ligation reaction (Kohler J J, ChemBioChem
2009; 10:2147-50):
##STR00014##
[0177] However, considering the numerous health risks associated
with aniline, alternative catalysts are desirable. The present
invention provides aniline derivatives as alternative oxime
ligation catalysts. Such aniline derivatives include, but are not
limited to, o-amino benzoic acid, m-amino benzoic acid, p-amino
benzoic acid, sulfanilic acid, o-aminobenzamide, o-toluidine,
m-toluidine, p-toluidine, o-anisidine, m-anisidine, and
p-anisidine.
[0178] In one embodiment of the invention, m-toluidine (aka
meta-toluidine, m-methylaniline, 3-methylaniline, or
3-amino-1-methylbenzene) is used to catalyze the conjugation
reactions described herein. M-toluidine and aniline have similar
physical properties and essentially the same pKa value
(m-toluidine: pKa 4.73, aniline: pKa 4.63).
[0179] The nucleophilic catalysts of the invention are useful for
oxime ligation (e.g, using aminooxy linkage) or hydrazone formation
(e.g., using hydrazide chemistry). In various embodiments of the
invention, the nucleophilic catalyst is provided in the conjugation
reaction at a concentration of of 0.1, 0.2, 0.3, 0.5, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 mM. In one
embodiment, the nucleophilic catalyst is provided between 1 to 10
mM. In various embodiments of the invention, the pH range of
conjugation reaction is 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5. In
one embodiment, the pH is between 5.5 to 6.5.
Purification of Conjugated Proteins
[0180] In various embodiments, purification of a protein that has
been incubated with an oxidizing agent and/or a therapeutic protein
that has been conjugated with a water soluble polymer according to
the present disclosure, is desired. Numerous purification
techniques are known in the art and include, without limitation,
chromatographic methods such as ion-exchange chromatography,
hydrophobic interaction chromatography, size exclusion
chromatography and affinity chromatography or combinations thereof,
filtration methods, and precipitation methods (Guide to Protein
Purification, Meth. Enzymology Vol 463 (edited by Burgess R R and
Deutscher M P), 2.sup.nd edition, Academic Press 2009).
[0181] The following examples are not intended to be limiting but
only exemplary of specific embodiments of the invention.
EXAMPLES
Example 1
Preparation of the Homobifunctional Linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.2ONH.sub.2
[0182] The homobifunctional linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.2ONH.sub.2
##STR00015##
[0183] (3-oxa-pentane-1,5-dioxyamine) containing two active
aminooxy groups was synthesized according to Boturyn et al.
(Tetrahedron 1997; 53:5485-92) in a two step organic reaction
employing a modified Gabriel-Synthesis of primary amines (FIG. 3).
In the first step, one molecule of 2,2-chlorodiethylether was
reacted with two molecules of
Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in dimethylformamide
(DMF). The desired homobifunctional product was prepared from the
resulting intermediate by hydrazinolysis in ethanol.
Example 2
Preparation of the Homobifunctional Linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.4ONH.sub.2
[0184] The Homobifunctional Linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.4ONH.sub.2
##STR00016##
[0185] (3,6,9-trioxa-undecane-1,11-dioxyamine) containing two
active aminooxy groups was synthesized according to Boturyn et al.
(Tetrahedron 1997; 53:5485-92) in a two step organic reaction
employing a modified Gabriel-Synthesis of primary amines (FIG. 3).
In the first step one molecule of
Bis-(2-(2-chlorethoxy)-ethyl)-ether was reacted with two molecules
of Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. The
desired homobifunctional product was prepared from the resulting
intermediate by hydrazinolysis in ethanol.
Example 3
Preparation of the Homobifunctional Linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.6ONH.sub.2
[0186] The Homobifunctional Linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.6ONH.sub.2
##STR00017##
[0187] (3,6,9,12,15-penatoxa-heptadecane-1,17-dioxyamine)
containing two active aminooxy groups was synthesized according to
Boturyn et al. (Tetrahedron 1997; 53:5485-92) in a two step organic
reaction employing a modified Gabriel-Synthesis of primary amines.
In the first step one molecule of hexaethylene glycol dichloride
was reacted with two molecules of
Endo-N-hydroxy-5-norbornene-2,3-dicarboximide in DMF. The desired
homobifunctional product was prepared from the resulting
intermediate by hydrazinolysis in ethanol.
Example 4
Detailed Synthesis of the Aminooxy-PSA Reagent
[0188] 3-oxa-pentane-1,5 dioxyamine was synthesized according to
Botyryn et al (Tetrahedron 1997; 53:5485-92) in a two step organic
synthesis as outlined in Example 1.
Step 1:
[0189] To a solution of
Endo-N-hydroxy-5-norbonene-2,3-dicarboxiimide (59.0 g; 1.00 eq) in
700 ml anhydrous N,N-dimetylformamide anhydrous K.sub.2CO.sub.3
(45.51 g; 1.00 eq) and 2,2-dichlorodiethylether (15.84 ml; 0.41 eq)
were added. The reaction mixture was stirred for 22 h at 50.degree.
C. The mixture was evaporated to dryness under reduced pressure.
The residue was suspended in 2 L dichloromethane and extracted two
times with saturated aqueous NaCl-solution (each 1 L). The
Dichloromethane layer was dried over Na.sub.2SO.sub.4 and then
evaporated to dryness under reduced pressure and dried in high
vacuum to give 64.5 g of
3-oxapentane-1,5-dioxy-endo-2',3'-dicarboxydiimidenorbornene as a
white-yellow solid (intermediate 1).
Step 2:
[0190] To a solution of intermediate 1 (64.25 g; 1.00 eq) in 800 ml
anhydrous Ethanol, 31.0 ml Hydrazine hydrate (4.26 eq) were added.
The reaction mixture was then refluxed for 2 hrs. The mixture was
concentrated to the half of the starting volume by evaporating the
solvent under reduced pressure. The occurring precipitate was
filtered off. The remaining ethanol layer was evaporated to dryness
under reduced pressure. The residue containing the crude product
3-oxa-pentane-1,5-dioxyamine was dried in vacuum to yield 46.3 g.
The crude product was further purified by column chromatography
(Silicagel 60; isocratic elution with Dichloromethane/Methanol
mixture, 9/1) to yield 11.7 g of the pure final product
3-oxa-pentane-1,5-dioxyamine.
Example 5
Preparation of Aminooxy-PSA
[0191] 1000 mg of oxidized PSA (MW=20 kD) obtained from the Serum
Institute of India (Pune, India) was dissolved in 16 ml 50 mM
phospate buffer pH 6.0. Then 170 mg 3-oxa-pentane-1,5-dioxyamine
was given to the reaction mixture. After shaking for 2 hrs at RT
78.5 mg sodium cyanoborohydride was added and the reaction was
performed for 18 hours over night. The reaction mixture was then
subjected to a ultrafiltration/diafiltration procedure (UF/DF)
using a membrane with a 5 kD cut-off made of regenerated cellulose
(50 cm.sup.2, Millipore).
Example 6
Preparation of Aminooxy-PSA Employing a Chromatographic
Purification Step
[0192] 1290 mg of oxidized PSA (MW=20 kD) obtained from the Serum
Institute of India (Pune, India) was dissolved in 25 ml 50 mM
phosphate buffer pH 6.0 (Buffer A). Then 209 mg
3-oxa-pentane-1,5-dioxyamine was given to the reaction mixture.
After shaking for 1 h at RT 101 mg sodium cyanoborohydride was
added and the reaction was performed for 3 hours. Then the mixture
was then subjected to a weak anion exchange chromatography step
employing a Fractogel EMD DEAE 650-M chromatography gel (column
dimension: XK26/135). The reaction mixture was diluted with 110 ml
Buffer A and loaded onto the DEAE column pre-equilibrated with
Buffer A at a flow rate of 1 cm/min. Then the column was washed
with 20 CV Buffer B (20 mM Hepes, pH 6.0) to remove free
3-oxa-pentane-1,5-dioxyamine and cyanide at a flow rate of 2
cm/min. The aminooxy-PSA reagent was then eluted with a step
gradient consisting of 67% Buffer B and 43% Buffer C (20 mM Hepes,
1M NaCl, pH 7.5). The eluate was concentrated by UF/DF using a 5 kD
membrane made of polyether sulfone (50 cm.sup.2, Millipore). The
final diafiltration step was performed against Buffer D (20 mM
Hepes, 90 mM NaCl, pH 7.4). The preparation was analytically
characterized by measuring total PSA (Resorcinol assay) and total
aminooxy groups (TNBS assay) to determine the degree of
modification. Furthermore the polydispersity as well as free
3-oxa-pentane-1,5-dioxyamine and cyanide was determined.
Example 7
Preparation of Aminooxy-PSA without a Reduction Step
[0193] 573 mg of oxidized PSA (MW=20 kD) obtained from the Serum
Institute of India (Pune, India) was dissolved in 11.3 ml 50 mM
phosphate buffer pH 6.0 (Buffer A). Then 94 mg
3-oxa-pentane-1,5-dioxyamine was given to the reaction mixture.
After shaking for 5 h at RT the mixture was then subjected to a
weak anion exchange chromatography step employing a Fractogel EMD
DEAE 650-M chromatography gel (column dimension: XK16/105). The
reaction mixture was diluted with 50 ml Buffer A and loaded onto
the DEAE column pre-equilibrated with Buffer A at a flow rate of 1
cm/min. Then the column was washed with 20 CV Buffer B (20 mM
Hepes, pH 6.0) to remove free 3-oxa-pentane-1,5-dioxyamine and
cyanide at a flow rate of 2 cm/min. The aminooxy-PSA reagent was
the eluted with a step gradient consisting of 67% Buffer B and 43%
Buffer C (20 mM Hepes, 1 M NaCl, pH 7.5). The eluate was
concentrated by UF/DF using a 5 kD membrane made of polyether
sulfone (50 cm.sup.2, Millipore). The final diafiltration step was
performed against Buffer D (20 mM Hepes, 90 mM NaCl, pH 7.4). The
preparation was analytically characterized by measuring total PSA
(Resorcinol assay) and total aminooxy groups (TNBS assay) to
determine the degree of modification. Furthermore the
polydispersity as well as free 3-oxa-pentane-1,5-dioxyamine was
determined.
Example 8
Preparation of Aminooxy-PSA without a Reduction Step in the
Presence of the Nucleophilic Catalyst m-Toluidine
[0194] 573 mg of oxidized PSA (MW=20 kD) obtained from the Serum
Institute of India (Pune, India) is dissolved in 9 ml 50 mM
phosphate buffer pH 6.0 (Buffer A). Then 94 mg
3-oxa-pentane-1,5-dioxyamine is given to this solution.
Subsequently 2.3 ml of a 50 mM m-toluidine stock solution are added
to this reaction mixture. After shaking for 2 h at RT the mixture
is then subjected to a weak anion exchange chromatography step
employing a Fractogel EMD DEAE 650-M chromatography gel (column
dimension: XK16/105). The reaction mixture is diluted with 50 ml
Buffer A and loaded onto the DEAE column pre-equilibrated with
Buffer A at a flow rate of 1 cm/min. Then the column is washed with
20CV Buffer B (20 mM Hepes, pH 6.0) to remove free
3-oxa-pentane-1,5-dioxyamine and cyanide at a flow rate of 2
cm/min. The aminooxy-PSA reagent is the eluted with a step gradient
consisting of 67% Buffer B and 43% Buffer C (20 mM Hepes, 1 M NaCl,
pH 7.5). The eluate is concentrated by UF/DF using a 5 kD membrane
made of polyether sulfone (50 cm.sup.2, Millipore). The final
diafiltration step is performed against Buffer D (20 mM Hepes, 90
mM NaCl, pH 7.4). The preparation is analytically characterized by
measuring total PSA (Resorcinol assay) and total aminooxy groups
(TNBS assay) to determine the degree of modification. Furthermore
the polydispersity as well as free 3-oxa-pentane-1,5-dioxyamine is
determined.
Example 9
Preparation of Aminooxy-PSA Reagent
[0195] An Aminooxy-PSA reagent was prepared according to the
Examples 4-8. After diafiltration, the product was frozen at
-80.degree. C. and lyophilized. After lyophilization the reagent
was dissolved in the appropriate volume of water and used for
preparation of PSA-protein conjugates via carbohydrate
modification.
Example 10
Evaluation of the Efficacy of Different Alternative Nucleophilic
Catalysts
[0196] rFIX was incubated with sodium periodate, aminooxy-PSA
reagent under standardized conditions (1 mg/ml rFIX in 20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl.sub.2), pH 6.0, 5-fold molar
aminooxy-PSA reagent excess, 100 .mu.M NaIO.sub.4) using different
nucleophilic catalysts (aniline, m-toluidine, o-anisidine,
m-anisidine, o-aminobenzoic acid, m-aminobenzoic acid,
p-aminobenzoic acid, p-aminobenzamide, sulfanilic acid/standard
concentration: 10 mM) The reaction was carried out for 2 hrs in the
dark at room temperature under gentle stirring and quenched for 15
min at room temperature by the addition of aqueous cysteine
solution with a final concentration of 1 mM.
[0197] The coupling efficiency was determined by SDS-PAGE using an
Invitrogen X-cell mini system. Samples were spiked with lithium
dodecyl sulfate (LDS) buffer and denatured for 10 min at 70.degree.
C. Then the samples were applied on 3-8% TRIS-acetate gels and ran
at 150 V for 60 min. Subsequently the gels were stained with
Coomassie.
[0198] In addition the samples were characterized by use of a
SEC-HPLC system using a Agilent 1200 HPLC system equipped with a
Shodex KW 803 column under conditions as previously described
(Kolarich et al, Transfusion 2006; 46:1959-77).
[0199] 50 .mu.l of samples were injected undiluted and eluted
isocratically with a 0.22 .mu.m filtered solution of 20 mM NaH2PO4,
50 mM Na2SO4, pH 6.1 at a flow rate of 0.5 ml/min. The elution
pattern was recorded at 280 nm.
[0200] The results are summarized in FIGS. 5A-C and 6 (SDS PAGE)
and Table 2 (SEC-HPLC results). The catalytic effect of the
different preparations is demonstrated. It is shown that the use of
m-toluidine leads to equivalent results as obtained with
aniline.
TABLE-US-00004 TABLE 2 di-PSAylated mono- free nucleophilic
catalysts rFIX PSAylated rFIX rFIX no catalyst 4.5% 24.9% 70.6% 10
mM aniline 47.7% 33.6% 18.7% 10 mM m-toluidine 31.4% 40.8% 27.8% 10
mM o-aminobenzioc acid 30.9% 38.5% 30.6% 10 mM m-aminobenzioc acid
27.6% 38.0% 34.4% 10 mM p-aminobenzioc acid 18.1% 39.3% 42.6% 10 mM
o-aminobenzamide 15.9% 38.4% 45.7% 10 mM sulfanilic acid 11.8%
35.8% 52.4%
Example 11
Polysialylation of rFIX Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0201] 12.3 mg rFIX was dissolved in 6.1 ml histidine buffer, pH
6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl2). 254 .mu.l of an
aqueous sodium periodate solution (5 mM) was then added and the
reaction mixture is incubated for 1 h in the dark at 4.degree. C.
under gentle stirring and quenched for 15 min at room temperature
by the addition of 6.5 .mu.l of a 1 M aqueous cysteine solution.
The mixture was subsequently subjected to UF/DF employing Vivaspin
15R 10 kD centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.
[0202] The retentate (8.8 ml), containing oxidized rFIX was mixed
with 2.46 ml of an aqueous m-toluidine solution (50 mM) and
incubated for 30 min at room temperature. Then aminooxy-PSA reagent
with a MW of 20 kD (described above) was added to give a 5-fold
molar reagent excess. This mixture was incubated for 2.5 h at RT in
the dark under gentle stirring.
[0203] The free rFIX was removed by means of anion exchange
chromatography (AEC). The reaction mixture was diluted with 15 ml
Buffer A (50 mM Hepes, 5 mM CaCl2, pH 7.5) and loaded onto a 20 ml
HiPrep QFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. The column was then eluted with
Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl2, pH 7.5). Free rFIX
elutes at a conductivity between 12-25 mS/cm and the conjugate
between 27-45 mS/cm. The conductivity of the conjugate containing
fractions was subsequently raised to 190 mS/cm with Buffer C (50 mM
Hepes, 5M NaCl, 5 mM CaCl2, pH 6.9) and loaded onto a 20 ml HiPrep
Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, 5 mM CaCl2,
pH 6.9). Free aminooxy-PSA reagent was washed out within 5 CV
Buffer D. Subsequently the conjugate is eluted with 100% Buffer E
(50 mM Hepes, 5 mM CaCl2, pH 7.4). The conjugate containing
fractions were concentrated by UF/DF using Vivaspin 15R 10 kD
centrifugal filtrator. The final diafiltration step was performed
against histidine buffer, pH 7.2 containing 150 mM NaCl and 5 mM
CaCl2. The preparation was analytically characterized by measuring
total protein (Bradford) and FIX chromogenic activity. The PSA-rFIX
conjugate showed a specific activity of >50% in comparison to
native rFIX is determined.
Method 2:
[0204] 12.3 mg rFIX is dissolved in in L-histidine buffer, pH 6.0
(20 mM L-histidine, 150 mM NaCl, 5 mM CaCl2) to get a final protein
concentration of 1 mg rFIX/ml. A 5 mM aqueous sodium periodate
solution is added to get a final concentration of 100 .mu.M and the
reaction mixture is incubated for 1 hour in the dark at 4.degree.
C. under gentle stirring at pH 6.0 and quenched for 15 min at room
temperature by the addition of an 1 M aqueous L-cysteine solution
(or other quenching reagents) to get a final concentration of 10
mM. The mixture is subsequently subjected to UF/DF employing
Vivaspin 15R 10 kD centrifugal filtrators to remove excess
periodate, quencher and the byproducts thereof.
[0205] The obtained retentate (8.8 ml), containing oxidized rFIX,
is mixed with an aqueous m-toluidine solution (50 mM) to give a
final concentration of 10 mM and incubated for 30 min at room
temperature. Then aminooxy-PSA reagent with a MW of 20 kD
(described above) is added to give a 5-fold molar reagent excess.
This mixture was incubated at pH 6.0 for 2.5 hours at room
temperature; 0.5 hours to 18 hours at +4.degree. C.) in the dark
under gentle stirring.
[0206] The free rFIX is removed by means of anion exchange
chromatography (AEC). The reaction mixture is diluted with
appropriate amounts of Buffer A (50 mM Hepes, 5 mM CaCl2, pH 7.5)
to correct the solutions conductivity and pH prior to load onto a
20 ml HiPrep QFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with buffer A. Then the column is eluted with
Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl2, pH 7.5). Free rFIX is
eluted by a step gradient using 25% of Buffer B, which results in a
conductivity between 12-25 mS/cm in the obtained fraction and the
conjugate using a step gradient of 50% Buffer B, which results in a
conductivity between between 27-45 mS/cm in the conjugate fraction.
The conductivity of the conjugate containing fraction is
subsequently raised to 190 mS/cm with Buffer C (50 mM Hepes, 5 M
NaCl, 5 mM CaCl2, pH 6.9 or by use of anti-chaotropic salts e.g.
ammonium sulphate, ammonium acetate etc.) and loaded onto a 20 ml
HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn. or
comparable HIC media) pre-equilibrated with Buffer D (50 mM Hepes,
3 M NaCl, 5 mM CaCl2, pH 6.9). Free aminooxy-PSA reagent is washed
out within 5 CV Buffer D. Subsequently, the conjugate is eluted
with 100% Buffer E (50 mM Hepes, 5 mM CaCl2, pH 7.4). The conjugate
containing fractions are concentrated by UF/DF using a 10 kD
membrane made of regenerated cellulose (88 cm2, cut-off 10 kD,
Millipore). The final diafiltration step is performed against
L-histidine buffer, pH 7.2 containing 150 mM NaCl and 5 mM CaCl2.
The preparation is analytically characterized by measuring total
protein (Bradford and BCA procedure) and FIX chromogenic- and
clotting activity. For the PSA-rFIX conjugate a specific activity
of >50% in comparison to native rFIX is determined.
Method 3:
[0207] 25.4 mg rFIX was dissolved in 18.7 ml histidine buffer, pH
6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl2). 531 .mu.l of an
aqueous sodium periodate solution (5 mM) and 5.07 ml of an aqueous
m-toluidine solution (50 mM) were then added. Subsequently, the
aminooxy-PSA reagent with a MW of 20 kD (described above) was added
to give a 5-fold molar reagent excess. The mixture was incubated
for 2 h in the dark at room temperature under gentle stirring and
quenched for 15 min at room temperature by the addition of 25 .mu.l
of 1 M aqueous cysteine solution.
[0208] The free rFIX was removed by means of anion exchange
chromatography (AEC). The reaction mixture was diluted with 20 ml
Buffer A (50 mM Hepes, 5 mM CaCl2, pH 7.5) and loaded onto a 20 ml
HiPrep QFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column was eluted with
Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl2, pH 7.5). Free rFIX
eluted at a conductivity between 12-25 mS/cm and the conjugate
between 27-45 mS/cm. The conductivity of the conjugate containing
fractions was subsequently raised to 190 mS/cm with Buffer C (50 mM
Hepes, 5 M NaCl, 5 mM CaCl2, pH 6.9) and loaded onto a 20 ml HiPrep
Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, 5 mM CaCl2,
pH 6.9). Free aminooxy-PSA reagent was washed out within 5 CV
Buffer D. Subsequently, the conjugate was eluted with 100% Buffer E
(50 mM Hepes, 5 mM CaCl2, pH 7.4). The conjugate containing
fractions were concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD, Millipore). The final
diafiltration step was performed against histidine buffer, pH 7.2
containing 150 mM NaCl and 5 mM CaCl2. The preparation was
analytically characterized by measuring total protein (Bradford)
and FIX chromogenic activity. For the PSA-rFIX conjugate a specific
activity of >50% in comparison to native rFIX was determined.
The conjugate was additionally analytically characterized by Size
Exclusion HPLC using a Agilent 1200 HPLC system equipped with a
Shodex KW 803 column under conditions as previously described
(Kolarich et al, Transfusion 2006; 46:1959-77). It was shown that
the preparation contains no free FIX. The conjugate consisted of
57% mono-polysialylated and 31% di-polysialylated and 12%
tri-polysialyated product.
Method 4:
[0209] 25.4 mg rFIX was dissolved in L-histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl, 5 mM CaCl2) to get a final protein
concentration of 2 mg rFIX/ml. Subsequently an 5 mM aqueous sodium
periodate solution was added within 15 minutes to give a final
concentration of 100 .mu.M, followed by addition of an 50 mM
aqueous m-toluidine solution to get a final concentration of 10 mM
within a time period of 30 minutes. Then the aminooxy-PSA reagent
with a MW of 20 kD (described above) was added to give a 5-fold
molar reagent excess. After correction of the pH to 6.0 the mixture
was incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of a 1 M aqueous L-cysteine solution to give a final
concentration of 10 mM.
[0210] The free rFIX was removed by means of ion exchange
chromatography (IEC). The reaction mixture was diluted with
appropriate amounts of Buffer A (50 mM Hepes, 5 mM CaCl2, pH 7.5)
to correct the solutions conductivity and pH value prior to load
onto a 20 ml HiPrep QFF 16/10 column (GE Healthcare, Fairfield,
Conn.) pre-equilibrated with Buffer A. Then the column was eluted
with Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl2, pH 7.5). Free
rFIX was eluted by a step gradient using 25% of Buffer B, which
results in a conductivity between 12-25 mS/cm in the obtained
fraction and the conjugate using a step gradient of 50% Buffer B,
which results in a conductivity between 27-45 mS/cm in the
conjugate fraction. The conductivity of the conjugate containing
fraction was subsequently raised to 190 mS/cm with Buffer C (50 mM
Hepes, 5 M NaCl, 5 mM CaCl2, pH 6.9; by use of anti-chaotropic
salts e.g. ammonium acetate) and loaded onto a 20 ml HiPrep Butyl
FF 16/10 column (GE Healthcare, Fairfield, Conn.; or comparable HIC
media) pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, 5 mM
CaCl2, pH 6.9). Free aminooxy-PSA reagent was washed out within 5
CV Buffer D. Subsequently the conjugate was eluted with 100% Buffer
E (50 mM Hepes, 5 mM CaCl2, pH 7.4). The conjugate containing
fractions were concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD, Millipore). The final
diafiltration step was performed against L-histidine buffer, pH 7.2
containing 150 mM NaCl and 5 mM CaCl2. The preparation was
analytically characterized by measuring total protein (Bradford and
BCA procedure) and FIX chromogenic- and clotting activity. For the
PSA-rFIX conjugate a specific activity of >50% in comparison to
native rFIX was determined. The conjugate was additionally
analytically characterized by Size Exclusion HPLC using a Agilent
1200 HPLC system equipped with a Shodex KW 803 column under
conditions as previously described (Kolarich et al, Transfusion
2006; 46:1959-77). It was shown that the preparation contains no
free FIX. The conjugate consisted of 57% mono-polysialylated and
31% di-polysialylated and 12% tri-polysialyated product.
Example 12
Polysialylation of rFVIII Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0211] 50 mg rFVIII was transferred into reaction buffer (50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 was added to give a final concentration of 200
.mu.M. The oxidation was carried at RT for 30 min in the dark under
gentle shaking. Then the reaction was quenched with cysteine (final
concentration: 10 mM) for 60 min at RT. The solution was subjected
to an IEX column with a volume of 20 ml (Merck EMD TMAE (M)) which
was equilibrated with Buffer A (20 mM Hepes, 5 mM CaCl.sub.2), pH
7.0). The column was equilibrated with 5 CV Buffer A. Then the
oxidized rFVIII was eluted with Buffer B (20 mM Hepes, 5 mM
CaCl.sub.2), 1M NaCl, pH 7.0). The rFVIII containing fractions were
collected. The protein content was determined (Coomassie, Bradford)
and adjusted to 1 mg/ml with reaction buffer and adjusted to pH 6.0
by dropwise addition of 0.5 M HCl. Then a 50-fold molar excess of a
aminooxy-PSA reagent with a MW of 20 kD (described above) was added
followed by m-toluidine as a nucleophilic catalyst (final
concentration: 10 mM). The coupling reaction was performed for 2
hours in the dark under gentle shaking at room temperature. The
excess of aminooxy-PSA reagent was removed by means of HIC. The
conductivity of the reaction mixture was raised to 130 mS/cm by
adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently, the conjugate was eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl.sub.2). Finally, the
PSA-rFVIII containing fractions were collected and subjected to
UF/DF by use of a 30 kD membrane made of regenerated cellulose (88
cm.sup.2, Millipore). The preparation was analytically
characterized by measuring total protein (Coomassie, Bradford) and
FVIII chromogenic activity. The PSA-rFVIII conjugate showed a
specific activity of >70% in comparison to native rFVIII was
determined.
Method 2:
[0212] 58 mg of recombinant factor VIII (rFVIII) derived from the
ADVATE process in Hepes buffer (50 mM HEPES, .about.350 mM sodium
chloride, 5 mM calcium chloride, 0.1% Polysorbate 80, pH 7.4) is
dissolved in reaction buffer (50 mM Hepes, 350 mM sodium chloride,
5 mM calcium chloride, pH 6.0) to get a final protein concentration
of 1.0+/-0.25 mg/ml. Then the pH of the solution is corrected to
6.0 by drop wise addition of a 0.5 N aqueous HCl solution.
Subsequently, a 40 mM aqueous sodium periodate solution is added
within 10 minutes to give a concentration of 200 .mu.M. The
oxidation reaction is carried out for 30+/-5 min at a temperature
(T) of T=+22+/-2.degree. C. Then the reaction is stopped by
addition of an aqueous L-cysteine solution (1 M) within 15 minutes
at T=+22+/-2.degree. C. to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0213] The oxidized rFVIII is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl.sub.2), pH 6.5) to give a
conductivity of 5 ms/cm. This solution is loaded onto the IEX
column (bed height: 5.4 cm) with a column volume of 10 ml using a
flow rate of 1.5 cm/min. This column is subsequently washed (flow
rate: 1.5 cm/min) with 5 CV of a 92:8 mixture (w/w) of Buffer A and
Buffer B (20 mM Hepes, 5 mM CaCl.sub.2), 1.0 M NaCl, pH 7.0). Then
the oxidized rFVIII is eluted with a 50:50 (w/w) mixture of Buffer
A and Buffer B followed by a postelution step with 5 CV of Buffer
B. The elution steps are carried out by use of a flow rate of 1.0
cm/min.
[0214] Subsequently, the aminooxy-polysialic acid (PSA-ONH.sub.2)
reagent is added in a 50-fold molar excess to the eluate containing
the purified oxidized rFVIII within a maximum time period (t) of 15
minutes under gentle stirring. Then an aqueous m-toluidine solution
(50 mM) is added within 15 minutes to get a final concentration of
10 mM. The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking.
[0215] The obtained PSA-rFVIII conjugate is purified by Hydrophobic
Interaction Chromatography (HIC) using a Phenyl Sepharose FF low
sub resin (GE Healthcare) packed into a column manufactured by GE
Healthcare with a bed height (h) of 15 cm and a resulting column
volume (CV) of 81 ml.
[0216] The reaction mixture is spiked with ammonium acetate by
addition of 50 mM Hepes buffer, containing 350 mM sodium chloride,
8 M ammonium acetate, 5 mM calcium chloride, pH 6.9. Two volumes of
the reaction mixture are mixed with 1 volume of the ammonium
acetate containing buffer system and the pH value is corrected to
pH 6.9 by drop wise addition of a 0.5 N aqueous NaOH solution. This
mixture is loaded onto the HIC column at flow rate of 1 cm/min
followed by a washing step using >3 CV equilibration buffer (50
mM Hepes, 350 mM sodium chloride, 2.5 M ammonium acetate, 5 mM
calcium chloride, pH 6.9).
[0217] For removal of reaction by-products and anti-chaotropic salt
a second washing step is performed with >5CV washing buffer 1
(50 mM Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9)
in upflow mode at a flow rate of 2 cm/min. Then elution of purified
PSA-rFVIII conjugate is performed in down flow mode using a step
gradient of 40% washing buffer 2 (50 mM Hepes, 1.5 M sodium
chloride, 5 mM calcium chloride, pH 6.9) and 60% elution buffer (20
mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow rate of 1
cm/min. The elution of the PSA-rFVIII conjugate is monitored at UV
280 nm and the eluate containing the conjugate is collected within
<4 CV. The post elution step is performed with >3 CV elution
buffer under the same conditions to separate minor and/or non
modified rFVIII from the main product.
[0218] Finally the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with a molecular weight cut off 30 kD (88 cm.sup.2,
Millipore).
[0219] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein, FVIII
chromogenic activity and determination of the polysialyation degree
by measuring the PSA content (resorcinol assay). For the conjugate
obtained a specific activity >50% and a PSA degree >5.0 is
calculated.
Method 3:
[0220] 50 mg rFVIII was transferred into reaction buffer (50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. A 50-fold
molar excess of aminooxy-PSA reagent with a MW of 20 kD (described
above) was added followed by m-toluidine as a nucleophilic catalyst
(final concentration: 10 mM) and NaIO4 (final concentration: 400
.mu.M). The coupling reaction was performed for 2 hours in the dark
under gentle shaking at room temperature. Subsequently, the
reaction was quenched with cysteine for 60 min at RT (final
concentration: 10 mM). Then the conductivity of the reaction
mixture was raised to 130 mS/cm by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column
filled with 80 ml Phenyl Sepharose FF (GE Healthcare, Fairfield,
Conn.) pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate,
350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH
6.9. Subsequently, the conjugate was eluted with 50 mM Hepes, 5 mM
calcium chloride, pH 7.5. Finally, the PSA-rFVIII containing
fractions were collected and subjected to UF/DF by use of a 30 kD
membrane made of regenerated cellulose (88 cm.sup.2, Millipore).
The preparation was analytically characterized by measuring total
protein (Bradford) and FVIII chromogenic activity. For the
PSA-rFVIII conjugate a specific activity of >70% in comparison
to native rFVIII was determined.
Method 4:
[0221] 50 mg recombinant factor VIII (rFVIII) derived from the
ADVATE process in 50 mM Hepes buffer (50 mM HEPES, .about.350 mM
sodium chloride, 5 mM calcium chloride, 0.1% Polysorbate 80, pH
7.4) was dissolved in reaction buffer (50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution was
corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution.
[0222] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent was added in a 50-fold molar excess to this rFVIII solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) was added
within 15 minutes to get a final concentration of 10 mM. Finally, a
40 mM aqueous sodium periodate solution was added to give a
concentration of 400 .mu.M.
[0223] The reaction mixture was incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction was stopped by the addition of an
aqueous L-cysteine solution (1 M) to give a final concentration of
10 mM in the reaction mixture and incubation for 60+/-5 min.
[0224] The obtained PSA-rFVIII conjugate was purified by
Hydrophobic Interaction Chromatography (HIC) using a Phenyl
Sepharose FF low sub resin (GE Healthcare) packed into a column
manufactured by GE Healthcare with a bed height (h) of 15 cm and a
resulting column volume (CV) of 81 ml.
[0225] The reaction mixture was spiked with ammonium acetate by
addition of of 50 mM Hepes buffer, containing 350 mM sodium
chloride, 8 M ammonium acetate, 5 mM calcium chloride, pH 6.9. Two
volumes of the reaction mixture was mixed with 1 volume of the
ammonium acetate containing buffer system and the pH value was
corrected to pH 6.9 by drop wise addition of an 0.5 N aqueous NaOH
solution. This mixture was loaded onto the HIC column using a flow
rate of 1 cm/min followed by a washing step using >3CV
equilibration buffer (50 mM Hepes, 350 mM sodium chloride, 2.5 M
ammonium acetate, 5 mM calcium chloride, pH 6.9).
[0226] For removal of reaction by-products and anti-chaotropic salt
a second washing step was performed with >5CV washing buffer 1
(50 mM Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9)
in upflow mode at a flow rate of 2 cm/min. Then elution of purified
rFVIII conjugate was performed in down flow mode using a step
gradient of 40% washing buffer 2 (50 mM Hepes, 1.5 M sodium
chloride, 5 mM calcium chloride, pH 6.9) and 60% elution buffer (20
mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow rate of 1
cm/min. The elution of the PSA-rFVIII conjugate was monitored at UV
280 nm and the eluate containing the conjugate was collected within
<4 CV. The post elution step was performed with >3 CV elution
buffer under the same conditions to separate minor and/or non
modified rFVIII from the main product.
[0227] Finally, the purified conjugate was concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with a molecular weight cut off 30 kD (88 cm2,
Millipore).
[0228] The conjugates prepared by use of this procedure were
analytically characterized by measuring total protein, FVIII
chromogenic activity and determination of the polysialyation degree
by measuring the PSA content (resorcinol assay). [0229] Analytical
data (mean of 6 consecutive batches): [0230] Process yield
(Bradford): 58.9% [0231] Process yield (FVIII chrom.): 46.4% [0232]
Specific activity: (FVIII chrom./mg protein): 4148 IU/mg [0233]
Specific activity (% of starting material): 79.9% [0234] PSA degree
(mol/mol): 8.1
Example 13
PEGylation of r FVIII Using an Aminooxy-PEG Reagent and m-Toluidine
as a Nucleophilic Catalyst
Method 1:
[0235] rFVIII is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). 14.7 mg rFVIII is dissolved in 7.0 ml histidine buffer, pH
6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl2). Then 296 .mu.l of
an aqueous sodium periodate solution (5 mM) is added and the
reaction mixture is incubated for 1 h in the dark at 4.degree. C.
under gentle stirring and quenched for 15 min at room temperature
by the addition of 7.5 .mu.l of a 1 M aqueous cysteine solution.
The mixture was subsequently subjected to UF/DF employing Vivaspin
15R 10 kD centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.
[0236] The retentate (10.9 ml), containing oxidized rFVIII, is
mixed with 2.94 ml of an aqueous m-toluidine solution (50 mM) and
incubated for 30 min at room temperature. Then aminooxy-PEG reagent
with a MW of 20 kD is added to give a 5-fold molar reagent excess.
This mixture was incubated for 2.5 h at room temperature in the
dark under gentle stirring.
[0237] Finally, the PEG-rFVIII conjugate is purified by
ion-exchange chromatography on Q Sepharose FF. 1.5 mg protein/ml
gel is loaded on the column equilibrated with 50 mM Hepes buffer,
pH 7.4 containing 5 mM CaCl2. The conjugate is eluted with 50 mM
Hepes buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH
7.4 and is then subjected to UF/DF using a 30 kD membrane (50 cm2,
Millipore). The preparation is analytically characterized by
measuring total protein (Coomassie, Bradford) and FVIII chromogenic
activity. It is expected that the PEG-rFVIII conjugate will
demonstrate a specific activity of >70% in comparison to native
rFVIII was determined.
Method 2:
[0238] rFVIII is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). A starting weight or concentration of rFVIII is dissolved
in or transferred to a reaction buffer (50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently a 40 mM aqueous sodium periodate solution is
added within 10 minutes to give a concentration of 200 .mu.M. The
oxidation reaction is carried out for 30+/-5 min at a temperature
(T) of T=+22+/-2.degree. C. Then the reaction is stopped by
addition of an aqueous L-cysteine solution (1 M) within 15 minutes
at T=+22+/-2.degree. C. to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0239] The oxidized rFVIII is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 6.5) to give a conductivity
of 5 ms/cm. This solution is loaded onto the IEX column (bed
height: 5.4 cm) with a column volume of 10 ml using a flow rate of
1.5 cm/min. This column is subsequently washed (flow rate: 1.5
cm/min) with 5 CV of a 92:8 mixture (w/w) of Buffer A and Buffer B
(20 mM Hepes, 5 mM CaCl2, 1.0 M NaCl, pH 7.0). Then the oxidized
rFVIII is eluted with a 50:50 (w/w) mixture of Buffer A and Buffer
B followed by a postelution step with 5 CV of Buffer B. The elution
steps are carried out by use of a flow rate of 1.0 cm/min.
[0240] Subsequently, the aminooxy-PEG reagent with a MW of 20 kD
reagent is added in a 50-fold molar excess to the eluate containing
the purified oxidized rFVIII within a maximum time period (t) of 15
minutes under gentle stirring. Then an aqueous m-toluidine solution
(50 mM) is added within 15 minutes to get a final concentration of
10 mM. The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking.
[0241] The obtained PEG-rFVIII conjugate is purified by Hydrophobic
Interaction Chromatography (HIC) using a Phenyl Sepharose FF low
sub resin (GE Healthcare) packed into a column manufactured by GE
Healthcare with a bed height (h) of 15 cm and a resulting column
volume (CV) of 81 ml.
[0242] The reaction mixture is spiked with ammonium acetate by
addition of 50 mM Hepes buffer, containing 350 mM sodium chloride,
8 M ammonium acetate, 5 mM calcium chloride, pH 6.9. Two volumes of
the reaction mixture are mixed with 1 volume of the ammonium
acetate containing buffer system and the pH value is corrected to
pH 6.9 by drop wise addition of a 0.5 N aqueous NaOH solution. This
mixture is loaded onto the HIC column using a flow rate of 1 cm/min
followed by a washing step using >3 CV equilibration buffer (50
mM Hepes, 350 mM sodium chloride, 2.5 M ammonium acetate, 5 mM
calcium chloride, pH 6.9).
[0243] For removal of reaction by-products and anti-chaotropic salt
a second washing step is performed with >5CV washing buffer 1
(50 mM Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9)
in upflow mode at a flow rate of 2 cm/min. Then elution of purified
rFVIII conjugate is performed in down flow mode using a step
gradient of 40% washing buffer 2 (50 mM Hepes, 1.5 M sodium
chloride, 5 mM calcium chloride, pH 6.9) and 60% elution buffer (20
mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow rate of 1
cm/min. The elution of the PEG-rFVIII conjugate is monitored at UV
280 nm and the eluate containing the conjugate is collected within
<4 CV. The post elution step is performed with >3 CV elution
buffer under the same conditions to separate minor and/or non
modified rFVIII from the main product.
[0244] Finally, the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with a molecular weight cut off 30 kD (Millipore).
[0245] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0246] rFVIII is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). 7.84 mg rFVIII, dissolved in 6 ml Hepes buffer (50 mM
Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) are
mixed with 314 .mu.l of an aqueous sodium periodate solution (10
mM), and 1.57 ml of an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at
room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of 8 .mu.l of aqueous cysteine
solution (1 M).
[0247] Finally the PEG-rFVIII conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded
on the column pre equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl.sub.2). The conjugate is eluted with 50 mM
Hepes buffer containing 5 mM CaCl.sub.2) and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using a 30 kD
membrane (88 cm.sup.2, Millipore). The analytical characterization
of the conjugate by FVIII chromogenic assay and determination of
total protein (Bradford) shows a specific activity of >60%
compared to the rFVIII starting material.
Method 4:
[0248] rFVIII is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of rFVIII is transferred
or dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride,
5 mM calcium chloride, pH 6.0) to get a final protein concentration
of 2 mg rFVIII/ml. Subsequently, an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration
of 100 .mu.M, followed by addition of an 50 mM aqueous m-toluidine
solution to get a final concentration of 10 mM within a time period
of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar excess. After
correction of the pH to 6.0 the mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15
min at room temperature by the addition of a 1 M aqueous L-cysteine
solution to give a final concentration of 10 mM.
[0249] The free rFVIII is removed by means of ion exchange
chromatography (IEC). The reaction mixture was diluted with
appropriate amounts of Buffer A (50 mM Hepes, 5 mM CaCl2, pH 7.5)
to correct the solutions conductivity and pH value prior to load
onto a 20 ml HiPrep QFF 16/10 column (GE Healthcare, Fairfield,
Conn.) pre-equilibrated with Buffer A. Then the column was eluted
with Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl2, pH 7.5). Free
rFVIII was eluted by a step gradient using 25% of Buffer B, which
results in a conductivity between 12-25 mS/cm in the obtained
fraction and the conjugate using a step gradient of 50% Buffer B,
which results in a conductivity between 27-45 mS/cm in the
conjugate fraction. The conductivity of the conjugate containing
fraction is subsequently raised with Buffer C (50 mM Hepes, 5 M
NaCl, 5 mM CaCl2, pH 6.9; by use of anti-chaotropic salts e.g.
ammonium acetate, ammonium sulphate etc.) and loaded onto a 20 ml
HiPrep Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn.; or
comparable HIC media) pre-equilibrated with Buffer D (50 mM Hepes,
3 M NaCl, 5 mM CaCl2, pH 6.9). Free PEG-reagent was washed out
within 5 CV Buffer D. Subsequently, the conjugate was eluted with
100% Buffer E (50 mM Hepes, 5 mM CaCl2, pH 7.4). The conjugate
containing fractions are concentrated by UF/DF using a 10 kD
membrane made of regenerated cellulose (88 cm2, cut-off 10 kD,
Millipore). The final diafiltration step is performed against Hepes
buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).
[0250] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 14
Polysialylation of rFVIIa Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0251] A starting concentration or weight of recombinant factor
VIIa (rFVIIa) is transferred or dissolved in reaction buffer (50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous NaOH solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 50 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0252] The oxidized rFVIIa is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl.sub.2), pH 6.5) to give a
conductivity of 5 ms/cm. This solution is loaded onto the IEX
column (bed height: 5.4 cm) with a column volume of 10 ml using a
flow rate of 1.5 cm/min. This column is subsequently washed (flow
rate: 1.5 cm/min) with 5 CV of a 92:8 mixture (w/w) of Buffer A and
Buffer B (20 mM Hepes, 5 mM CaCl.sub.2), 1.0 M NaCl, pH 7.0). Then
the oxidized rFVIIa is eluted with a 50:50 (w/w) mixture of Buffer
A and Buffer B followed by a postelution step with 5 CV of Buffer
B. The elution steps are carried out by use of a flow rate of 1.0
cm/min.
[0253] Subsequently, the aminooxy-polysialic acid (PSA-ONH.sub.2)
reagent is added in a 50-fold molar excess to the eluate containing
the purified oxidized rFVIIa within a maximum time period (t) of 15
minutes under gentle stirring. Then an aqueous m-toluidine solution
(50 mM) is added within 15 minutes to get a final concentration of
10 mM. The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking.
[0254] The obtained PSA-rFVIIa conjugate is purified by Hydrophobic
Interaction Chromatography (HIC) using a Phenyl Sepharose FF low
sub resin (GE Healthcare) packed into a column manufactured by GE
Healthcare with a bed height (h) of 15 cm and a resulting column
volume (CV) of 81 ml.
[0255] The reaction mixture is spiked with ammonium acetate by
addition of 50 mM Hepes buffer, containing 350 mM sodium chloride,
8 M ammonium acetate, 5 mM calcium chloride, pH 6.9. Two volumes of
the reaction mixture are mixed with 1 volume of the ammonium
acetate containing buffer system and the pH value is corrected to
pH 6.9 by drop wise addition of a 0.5 N aqueous NaOH solution. This
mixture is loaded onto the HIC column using a flow rate of 1 cm/min
followed by a washing step using >3 CV equilibration buffer (50
mM Hepes, 350 mM sodium chloride, 2.5 M ammonium acetate, 5 mM
calcium chloride, pH 6.9).
[0256] For removal of reaction by-products and anti-chaotropic salt
a second washing step is performed with >5CV washing buffer 1
(50 mM Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9)
in upflow mode at a flow rate of 2 cm/min. Then elution of purified
rFVIIa conjugate is performed in down flow mode using a step
gradient of 40% washing buffer 2 (50 mM Hepes, 1.5 M sodium
chloride, 5 mM calcium chloride, pH 6.9) and 60% elution buffer (20
mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow rate of 1
cm/min. The elution of the PSA-rFVIIa conjugate is monitored at UV
280 nm and the eluate containing the conjugate is collected within
<4 CV. The post elution step is performed with >3 CV elution
buffer under the same conditions to separate minor and/or non
modified rFVIIa from the main product.
[0257] Finally, the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with an appropriate molecular weight cut off (e.g. 10 kD
MWCO, 88 cm.sup.2, Millipore).
[0258] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 2:
[0259] A starting weight or concentration of rFVIIa is dissolved in
or transferred to a reaction buffer (50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5 N aqueous NaOH
solution.
[0260] Subsequently, the aminooxy-polysialic acid (PSA-ONH.sub.2)
reagent is added in a 50-fold molar excess to this rFVIIa solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 150 .mu.M.
[0261] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0262] The obtained PSA-rFVIIa conjugate is purified by Hydrophobic
Interaction Chromatography (HIC) using a Phenyl Sepharose FF low
sub resin (GE Healthcare) packed into a column manufactured by GE
Healthcare with a bed height (h) of 15 cm and a resulting column
volume (CV) of 81 ml.
[0263] The reaction mixture is spiked with ammonium acetate by
addition of of 50 mM Hepes buffer, containing 350 mM sodium
chloride, 8 M ammonium acetate, 5 mM calcium chloride, pH 6.9. Two
volumes of the reaction mixture is mixed with 1 volume of the
ammonium acetate containing buffer system and the pH value is
corrected to pH 6.9 by drop wise addition of an 0.5 N aqueous NaOH
solution. This mixture is loaded onto the HIC column using a flow
rate of 1 cm/min followed by a washing step using >3CV
equilibration buffer (50 mM Hepes, 350 mM sodium chloride, 2.5 M
ammonium acetate, 5 mM calcium chloride, pH 6.9).
[0264] For removal of reaction by-products and anti-chaotropic salt
a second washing step is performed with >5CV washing buffer 1
(50 mM Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9)
in upflow mode at a flow rate of 2 cm/min. Then elution of purified
rFVIIa conjugate is performed in down flow mode using a step
gradient of 40% washing buffer 2 (50 mM Hepes, 1.5 M sodium
chloride, 5 mM calcium chloride, pH 6.9) and 60% elution buffer (20
mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow rate of 1
cm/min. The elution of the PSA-rFVIIa conjugate is monitored at UV
280 nm and the eluate containing the conjugate was collected within
<4 CV. The post elution step is performed with >3 CV elution
buffer under the same conditions to separate minor and/or non
modified rFVIII from the main product.
[0265] Finally, the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose (Millipore).
[0266] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 15
PEGylation of rFIX Using an Aminooxy-PEG Reagent and m-Toluidine as
a Nucleophilic Catalyst
Method 1:
[0267] rFIX is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). A starting weight or concentration of rFIX is dissolved in
or transferred to a reaction buffer (50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5 N aqueous HCl
solution. Subsequently, a 40 mM aqueous sodium periodate solution
is added within 10 minutes to give a concentration of 200 .mu.M.
The oxidation reaction is carried out for 30+/-5 min at a
temperature (T) of T=+22+/-2.degree. C. Then the reaction is
stopped by addition of an aqueous L-cysteine solution (1 M) within
15 minutes at T=+22+/-2.degree. C. to give a final concentration of
10 mM in the reaction mixture and incubation for 60+/-5 min.
[0268] The oxidized rFVIII is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 6.5) to give a conductivity
of 5 mS/cm. This solution is loaded onto the IEX column (bed
height: 5.4 cm) with a column volume of 10 ml using a flow rate of
1.5 cm/min. This column is subsequently washed (flow rate: 1.5
cm/min) with 5 CV of a 92:8 mixture (w/w) of Buffer A and Buffer B
(20 mM Hepes, 5 mM CaCl2, 1.0 M NaCl, pH 7.0). Then the oxidized
rFIX is eluted with a 50:50 (w/w) mixture of Buffer A and Buffer B
followed by a postelution step with 5 CV of Buffer B. The elution
steps are carried out by use of a flow rate of 1.0 cm/min.
[0269] Subsequently, the aminooxy-PEG reagent with a MW of 20 kD
reagent is added in a 50-fold molar excess to the eluate containing
the purified oxidized rFIX within a maximum time period (t) of 15
minutes under gentle stirring. Then an aqueous m-toluidine solution
(50 mM) is added within 15 minutes to get a final concentration of
10 mM. The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking.
[0270] The obtained PEG-rFIX conjugate is purified by Hydrophobic
Interaction Chromatography (HIC) using a Phenyl Sepharose FF low
sub resin (GE Healthcare) packed into a column manufactured by GE
Healthcare with a bed height (h) of 15 cm and a resulting column
volume (CV) of 81 ml.
[0271] The reaction mixture is spiked with ammonium acetate by
addition of 50 mM Hepes buffer, containing 350 mM sodium chloride,
8 M ammonium acetate, 5 mM calcium chloride, pH 6.9. Two volumes of
the reaction mixture are mixed with 1 volume of the ammonium
acetate containing buffer system and the pH value is corrected to
pH 6.9 by drop wise addition of a 0.5 N aqueous NaOH solution. This
mixture is loaded onto the HIC column using a flow rate of 1 cm/min
followed by a washing step using >3 CV equilibration buffer (50
mM Hepes, 350 mM sodium chloride, 2.5 M ammonium acetate, 5 mM
calcium chloride, pH 6.9).
[0272] For removal of reaction by-products and anti-chaotropic salt
a second washing step is performed with >5CV washing buffer 1
(50 mM Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9)
in upflow mode at a flow rate of 2 cm/min. Then elution of purified
rFIX conjugate is performed in down flow mode using a step gradient
of 40% washing buffer 2 (50 mM Hepes, 1.5 M sodium chloride, 5 mM
calcium chloride, pH 6.9) and 60% elution buffer (20 mM Hepes, 5 mM
calcium chloride, pH 7.5) at a flow rate of 1 cm/min. The elution
of the PEG-rFIX conjugate is monitored at UV 280 nm and the eluate
containing the conjugate is collected within <4 CV. The post
elution step is performed with >3 CV elution buffer under the
same conditions to separate minor and/or non modified rFIX from the
main product.
[0273] Finally, the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with a molecular weight cut off 10 kD (88 cm2,
Millipore).
[0274] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 2:
[0275] rFIX is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of rFIX is transferred
or dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride,
5 mM calcium chloride, pH 6.0) to get a final protein concentration
of 2 mg rFIX/ml. Subsequently, an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration
of 100 .mu.M, followed by addition of an 50 mM aqueous m-toluidine
solution to get a final concentration of 10 mM within a time period
of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of a 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0276] The free rFIX is removed by means of ion exchange
chromatography (IEC). The reaction mixture was diluted with
appropriate amounts of Buffer A (50 mM Hepes, 5 mM CaCl2, pH 7.5)
to correct the solutions conductivity and pH value prior to load
onto a 20 ml HiPrep QFF 16/10 column (GE Healthcare, Fairfield,
Conn.) pre-equilibrated with Buffer A. Then the column was eluted
with Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl2, pH 7.5). Free
rFIX was eluted by a step gradient using 25% of Buffer B, which
results in a conductivity between 12-25 mS/cm in the obtained
fraction and the conjugate using a step gradient of 50% Buffer B,
which results in a conductivity between 27-45 mS/cm in the
conjugate fraction. The conductivity of the conjugate containing
fraction is subsequently raised with Buffer C (50 mM Hepes, 5 M
NaCl, 5 mM CaCl2, pH 6.9; by use of anti-chaotropic salts e.g.
ammonium acetate, etc) and loaded onto a 20 ml HiPrep Butyl FF
16/10 column (GE Healthcare, Fairfield, Conn.; or comparable HIC
media) pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, 5 mM
CaCl2, pH 6.9). Free aminooxy-PEG reagent was washed out within 5
CV Buffer D. Subsequently, the conjugate was eluted with 100%
Buffer E (50 mM Hepes, 5 mM CaCl2, pH 7.4). The conjugate
containing fractions are concentrated by UF/DF using a 10 kD
membrane made of regenerated cellulose (88 cm2, cut-off 10 kD,
Millipore). The final diafiltration step is performed against Hepes
buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).
[0277] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 16
PEGylation of rFVIIa Using an Aminooxy-PEG Reagent and m-Toluidine
as a Nucleophilic Catalyst
Method 1:
[0278] rFVIIa is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). A starting weight or concentration of rFVIIa is dissolved
in or transferred to a reaction buffer (50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) to get a final protein
concentration of 1.0+/-0.25 mg/ml. Then the pH of the solution is
corrected to 6.0 by drop wise addition of a 0.5 N aqueous NaOH
solution. Subsequently, a 40 mM aqueous sodium periodate solution
is added within 10 minutes to give a concentration of 50 .mu.M. The
oxidation reaction is carried out for 30+/-5 min at a temperature
(T) of T=+22+/-2.degree. C. Then the reaction is stopped by
addition of an aqueous L-cysteine solution (1 M) within 15 minutes
at T=+22+/-2.degree. C. to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0279] The oxidized rFVIIa is further purified by anion exchange
chromatography on EMD TMAE (M) (Merck). The mixture is diluted with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 6.5) to give a conductivity
of 5 mS/cm. This solution is loaded onto the IEX column (bed
height: 5.4 cm) with a column volume of 10 ml using a flow rate of
1.5 cm/min. This column is subsequently washed (flow rate: 1.5
cm/min) with 5 CV of a 92:8 mixture (w/w) of Buffer A and Buffer B
(20 mM Hepes, 5 mM CaCl2, 1.0 M NaCl, pH 7.0). Then the oxidized
rFVIIa is eluted with a 50:50 (w/w) mixture of Buffer A and Buffer
B followed by a postelution step with 5 CV of Buffer B. The elution
steps are carried out by use of a flow rate of 1.0 cm/min.
[0280] Subsequently, the aminooxy-PEG reagent with a MW of 20 kD
reagent is added in a 50-fold molar excess to the eluate containing
the purified oxidized rFVIIa within a maximum time period (t) of 15
minutes under gentle stirring. Then an aqueous m-toluidine solution
(50 mM) is added within 15 minutes to get a final concentration of
10 mM. The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking.
[0281] The obtained PEG-rFVIIa conjugate is purified by Hydrophobic
Interaction Chromatography (HIC) using a Phenyl Sepharose FF low
sub resin (GE Healthcare) packed into a column manufactured by GE
Healthcare with a bed height (h) of 15 cm and a resulting column
volume (CV) of 81 ml.
[0282] The reaction mixture is spiked with ammonium acetate by
addition of 50 mM Hepes buffer, containing 350 mM sodium chloride,
8 M ammonium acetate, 5 mM calcium chloride, pH 6.9. Two volumes of
the reaction mixture are mixed with 1 volume of the ammonium
acetate containing buffer system and the pH value is corrected to
pH 6.9 by drop wise addition of a 0.5 N aqueous NaOH solution. This
mixture is loaded onto the HIC column using a flow rate of 1 cm/min
followed by a washing step using >3 CV equilibration buffer (50
mM Hepes, 350 mM sodium chloride, 2.5 M ammonium acetate, 5 mM
calcium chloride, pH 6.9).
[0283] For removal of reaction by-products and anti-chaotropic salt
a second washing step is performed with >5CV washing buffer 1
(50 mM Hepes, 3 M sodium chloride, 5 mM calcium chloride, pH 6.9)
in upflow mode at a flow rate of 2 cm/min. Then elution of purified
rFVIIa conjugate is performed in down flow mode using a step
gradient of 40% washing buffer 2 (50 mM Hepes, 1.5 M sodium
chloride, 5 mM calcium chloride, pH 6.9) and 60% elution buffer (20
mM Hepes, 5 mM calcium chloride, pH 7.5) at a flow rate of 1
cm/min. The elution of the PEG-rFVIIa conjugate is monitored at UV
280 nm and the eluate containing the conjugate is collected within
<4 CV. The post elution step is performed with >3 CV elution
buffer under the same conditions to separate minor and/or non
modified rFVIIa from the main product.
[0284] Finally, the purified conjugate is concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with a molecular weight cut off 10 kD (Millipore).
[0285] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 2:
[0286] rFVIIa is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of rFVIIa is transferred
or dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride,
5 mM calcium chloride, pH 6.0) to get a final protein concentration
of 2 mg rFVIIa/ml. Subsequently an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration
of 100 .mu.M, followed by addition of an 50 mM aqueous m-toluidine
solution to get a final concentration of 10 mM within a time period
of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of a 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0287] The free rFVIIa is removed by means of ion exchange
chromatography (IEC). The reaction mixture was diluted with
appropriate amounts of Buffer A (50 mM Hepes, 5 mM CaCl2, pH 7.5)
to correct the solutions conductivity and pH value prior to load
onto a 20 ml HiPrep QFF 16/10 column (GE Healthcare, Fairfield,
Conn.) pre-equilibrated with Buffer A. Then the column was eluted
with Buffer B (50 mM Hepes, 1 M NaCl, 5 mM CaCl2, pH 7.5). Free
rFVIIa was eluted by a step gradient using 25% of Buffer B, which
results in a conductivity between 12-25 mS/cm in the obtained
fraction and the conjugate using a step gradient of 50% Buffer B,
which results in a conductivity between 27-45 mS/cm in the
conjugate fraction. The conductivity of the conjugate containing
fraction is subsequently raised with Buffer C (50 mM Hepes, 5 M
NaCl, 5 mM CaCl2, pH 6.9; by use of anti-chaotropic salts e.g.
ammonium acetate) and loaded onto a 20 ml HiPrep Butyl FF 16/10
column (GE Healthcare, Fairfield, Conn.; or comparable HIC media)
pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, 5 mM CaCl2,
pH 6.9). Free PEG-reagent was washed out within 5 CV Buffer D.
Subsequently the conjugate was eluted with 100% Buffer E (50 mM
Hepes, 5 mM CaCl2, pH 7.4). The conjugate containing fractions are
concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD, Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes,
5 mM CaCl2, pH 7.5).
[0288] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 17
Polysialylation of rFIX in the Presence of o-Amino Benzoic Acid
Method 1:
[0289] 8.2 mg rFIX is dissolved in 4.0 ml histidine buffer, pH 6.0
(20 mM L-histidine, 150 mM NaCl, 5 mM CaCl2). Then 82 .mu.l of an
aqueous sodium periodate solution (5 mM) is added and the reaction
mixture is incubated for 1 h in the dark at 4.degree. C. under
gentle stirring and quenched for 15 min at room temperature by the
addition of 4 .mu.l of a 1 M aqueous cysteine solution. The mixture
is subsequently subjected to UF/DF employing Vivaspin 6 10 kD
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0290] The retentate (6.5 ml), containing oxidized rFIX, is mixed
with 1.64 ml of an aqueous o-amino benzoic acid (50 mM) and
incubated for 30 min at room temperature. Then aminooxy-PSA reagent
with a MW of 20 kD (described above) is added to give a 5-fold
molar reagent excess. This mixture was incubated for 2.5 h at room
temperature in the dark under gentle stirring.
[0291] The further purification of the conjugate is carried out as
described herein.
Method 2:
[0292] A solution of 1 mg rFIX in 0.65 ml sodium phosphate buffer,
pH 6.0 containing a 5-fold molar excess of aminooxy-PSA reagent
with a MW of 20 kD (described above) was prepared. Then 333 .mu.l
of an aqueous o-amino benzoic acid solution (30 mM) was added as
nucleophilic catalyst to give a final concentration of 10 mM.
Subsequently 20 .mu.l of an aqueous solution of NaIO4 (5 mM) was
added yielding in a final concentration of 100 .mu.M. The coupling
process was performed for 2 hours in the dark under gentle shaking
at room temperature and quenched for 15 min at room temperature by
the addition of 1 .mu.l of aqueous cysteine solution (1 M). The
further purification of the conjugate is carried out as described
herein.
Example 18
Polysialylation of EPO Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0293] A starting concentration of erythropoietin (EPO) is
transferred into a reaction buffer (e.g. 50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a
protein concentration of 1 mg/ml. To this solution, NaIO4 is added
to give a final concentration of 200 .mu.M. The oxidation is
carried at RT for 30 min in the dark under gentle shaking. The
reaction is then quenched with cysteine (final concentration: 10
mM) for 60 min at RT.
[0294] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl.sub.2), pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized EPO is eluted with
Buffer B (20 mM Hepes, 5 mM CaCl.sub.2), 1M NaCl, pH 7.0). The EPO
containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M
HCl.
[0295] A 50-fold molar excess of a aminooxy-PSA reagent with a MW
of 20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy-PSA reagent is removed
by means of HIC. The conductivity of the reaction mixture is
adjusted by adding a buffer containing ammonium acetate (50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, 8 M ammonium
acetate, pH 6.9) and loaded onto a column filled with 80 ml Phenyl
Sepharose FF (GE Healthcare, Fairfield, Conn.) pre-equilibrated
with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5
mM calcium chloride, pH 6.9. Subsequently, the conjugate is eluted
with 50 mM Hepes buffer pH 7.5 containing 5 mM CaCl.sub.2). Finally
the PSA-EPO containing fractions are collected and subjected to
UF/DF by use of a membrane made of regenerated cellulose (MWCO 10
kD, 50 cm.sup.2, Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.
[0296] In an alternative embodiment, Method 1 is carried out as
follows.
[0297] 10 mg EPO is dissolved in 5 ml histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl). 100 .mu.l of an aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 50
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin 15R 10 kD
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0298] The retentate (approx. 7 ml), containing oxidized EPO, is
mixed with 2 ml of an aqueous m-toluidine solution (50 mM) and
incubated for 30 min at room temperature. Then aminooxy-PSA reagent
with a MW of 20 kD (described above) is added to give a 5-fold
molar reagent excess. This mixture is incubated for 2.5 h at RT in
the dark under gentle stirring.
[0299] The free EPO is removed by means of anion exchange
chromatography (AEC). The reaction mixture is diluted with 20 ml
Buffer A (50 mM Hepes, pH 7.5) and loaded onto a 20 ml HiPrep QFF
16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated
with Buffer A. Then the column is eluted with Buffer B (50 mM
Hepes, 1 M NaCl, pH 7.5). Free EPO is eluted by washing the column
with 25% Buffer B and the conjugate at 50% Buffer B. The
conductivity of the conjugate containing fractions is subsequently
raised to .about.190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl, pH
6.9) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GE
Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer D (50 mM
Hepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed out within 5
CV Buffer D. Subsequently, the conjugate is eluted with 100% Buffer
E (50 mM Hepes, pH 7.4). The conjugate containing fractions are
concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against histidine buffer, pH 7.2
containing 150 mM NaCl. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art. For the PSA-EPO
conjugate a specific activity of >50% in comparison to native
EPO is determined. The conjugate is additionally analytically
characterized by Size Exclusion HPLC using a Agilent 1200 HPLC
system equipped with a Shodex KW 803 column under conditions as
previously described (Kolarich et al, Transfusion 2006;
46:1959-77). It is shown that the preparation contains no free
EPO.
Method 2:
[0300] EPO is transferred or dissolved in reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0301] The oxidized EPO is further purified by ion exchange
chromatography. The oxidized EPO containing fractions of the eluate
are collected and used for the conjugation reaction.
[0302] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized EPO within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. at pH 6.0 in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0303] The obtained PSA-EPO conjugate is further purified by ion
exchange chromatography. The PSA-EPO conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose with an appropriate
molecular weight cut off (Millipore).
[0304] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0305] Erythropoietin (EPO) is transferred into reaction buffer (50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
and diluted to obtain a protein concentration of 1 mg/ml. A 50 fold
molar excess of a aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a
nucleophilic catalyst (10 mM final concentration) and NaIO4 (final
concentration: 400 .mu.M). The coupling reaction is performed for 2
hours in the dark under gentle shaking at room temperature.
Subsequently, the reaction is quenched with cysteine for 60 min at
RT (cysteine concentration: 10 mM). Then the conductivity of the
reaction mixture is adjusted by adding a buffer containing ammonium
acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column
filled with Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM
sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9.
Subsequently, the conjugate is eluted with 50 mM Hepes, 5 mM
calcium chloride, pH 7.5. Finally, the PSA-EPO containing fractions
are collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (MWCO 10 kD, 88 cm2, Millipore). The
preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
[0306] In an alternative embodiment, Method 3 is carried out as
follows. 10 mg EPO is dissolved in 8 ml histidine buffer, pH 6.0
(20 mM L-histidine, 150 mM NaCl). 200 .mu.l of an aqueous sodium
periodate solution (5 mM) and 2 ml of an aqueous m-toluidine
solution (50 mM) are then added. Subsequently, the aminooxy-PSA
reagent with a MW of 20 kD (described above) is added to give a
5-fold molar reagent excess. The mixture is incubated for 2 h in
the dark at room temperature under gentle stirring and quenched for
15 min at room temperature by the addition of 100 .mu.l of 1 M
aqueous cysteine solution.
[0307] The free EPO is removed by means of anion exchange
chromatography (AEC). The reaction mixture is diluted with 20 ml
Buffer A (50 mM Hepes, pH 7.5) and loaded onto a 20 ml HiPrep QFF
16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated
with Buffer A. Then the column is eluted with Buffer B (50 mM
Hepes, 1 M NaCl, pH 7.5). Free EPO is eluted by washing the column
with 25% Buffer B and the conjugate at 50% Buffer B. The
conductivity of the conjugate containing fractions is subsequently
raised to .about.190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl, pH
6.9) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GE
Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer D (50 mM
Hepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed out within 5
CV Buffer D. Subsequently, the conjugate is eluted with 100% Buffer
E (50 mM Hepes, pH 7.4). The conjugate containing fractions are
concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD, Millipore). The final
diafiltration step is performed against histidine buffer, pH 7.2
containing 150 mM NaCl. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art. For the PSA-EPO
conjugate a specific activity of >50% in comparison to native
EPO is determined. The conjugate is additionally analytically
characterized by Size Exclusion HPLC using a Agilent 1200 HPLC
system equipped with a Shodex KW 803 column under conditions as
previously described (Kolarich et al, Transfusion 2006;
46:1959-77). It is shown that the preparation contains no free
EPO.
Method 4:
[0308] EPO is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0309] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this EPO solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0310] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0311] The obtained PSA-EPO conjugate is purified by ion-exchange
chromatography. The PSA-EPO containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (MWCO 10 kD, 88 cm2,
Millipore).
[0312] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 19
Polysialylation of Ang-2 Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0313] A starting concentration of angiopoietin-2 (Ang-2) is
transferred into a reaction buffer (e.g. 50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a
protein concentration of 1 mg/ml. To this solution, NaIO4 is added
to give a final concentration of 200 .mu.M. The oxidation is
carried at RT for 30 min in the dark under gentle shaking. The
reaction is then quenched with cysteine (final concentration: 10
mM) for 60 min at RT.
[0314] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts, or, in the alternative, subjected to an IEX column with
a volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized Ang-2 is eluted with
Buffer B (20 mM Hepes, 5 mM CaCl2, 1 M NaCl, pH 7.0). The Ang-2
containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5
M HCl.
[0315] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently, the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the
PSA-Ang-2-containing fractions are collected and subjected to UF/DF
by use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
[0316] In an alternative embodiment, Method 1 is carried out as
follows. Angiopoietin-2 (Ang-2) is transferred into a reaction
buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and diluted to obtain a protein concentration of
1 mg/ml. To this solution, NaIO4 is added to give a final
concentration of 200 .mu.M. The oxidation is carried at RT for 30
min in the dark under gentle shaking. The reaction is then quenched
with cysteine (final concentration: 10 mM) for 60 min at R.T.
[0317] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0318] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of ion exchange chromatography. The PSA-Ang-2
conjugate-containing fractions of the eluate are collected and
subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.
Method 2:
[0319] Ang-2 is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5 N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0320] The oxidized Ang-2 is further purified by ion exchange
chromatography. The oxidized Ang-2 containing fractions of the
eluate are collected and used for the conjugation reaction.
[0321] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized Ang-2 within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. at pH 6.0 in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0322] The obtained PSA-Ang-2 conjugate is further purified by
ion-exchange chromatography
[0323] The PSA-Ang-2 conjugate containing fractions are collected
and concentrated by ultra-/diafiltration (UF/DF) using a membrane
made of regenerated cellulose with an appropriate molecular weight
cut off (Millipore).
[0324] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0325] Angiopoietin-2 (Ang-2) is transferred into reaction buffer
(50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH
6.0) and diluted to obtain a protein concentration of 1 mg/ml. A 50
fold molar excess of a PSA aminooxy reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a
nucleophilic catalyst (10 mM final concentration) and NaIO4 (final
concentration: 400 .mu.M). The coupling reaction is performed for 2
hours in the dark under gentle shaking at room temperature.
Subsequently, the reaction is quenched with cysteine for 60 min at
RT (cysteine concentration: 10 mM). Then the conductivity of the
reaction mixture is adjusted by adding a buffer containing ammonium
acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column
filled with Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM
sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9.
Subsequently, the conjugate is eluted with 50 mM Hepes, 5 mM
calcium chloride, pH 7.5. Finally, the PSA Ang-2-containing
fractions are collected and subjected to UF/DF by use of a membrane
made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0326] In an alternative embodiment, Method 3 is carried out as
follows. Angiopoietin-2 (Ang-2) is transferred into reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A
50-fold molar excess of a PSA aminooxy reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a
nucleophilic catalyst (10 mM final concentration) and NaIO4 (final
concentration: 400 .mu.M). The coupling reaction is performed for 2
hours in the dark under gentle shaking at room temperature.
Subsequently, the reaction is quenched with cysteine for 60 min at
RT (cysteine concentration: 10 mM) and the conjugate is purified by
ion exchange chromatography. PSA Ang-2-containing fractions of the
eluate are collected and subjected to UF/DF by use of a membrane
made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
Method 4:
[0327] Ang-2 is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0328] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this Ang-2 solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0329] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0330] The obtained PSA-Ang-2 conjugate is purified by ion-exchange
chromatography. The PSA-Ang-2 containing fractions of the eluate
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose (Millipore).
[0331] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 20
Polysialylation of VEGF Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0332] A starting concentration of vascular endothelial growth
factor (VEGF) is transferred into a reaction buffer (e.g., 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 is added to give a final concentration of 200
.mu.M. The oxidation is carried at RT for 30 min in the dark under
gentle shaking. The reaction is then quenched with cysteine (final
concentration: 10 mM) for 60 min at RT.
[0333] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized VEGF is eluted with
Buffer B (20 mM Hepes, 5 mM CaCl2, 1 M NaCl, pH 7.0). The VEGF
containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M
NaOH.
[0334] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently, the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the
PSA-VEGF-containing fractions are collected and subjected to UF/DF
by use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
[0335] In an alternative embodiment, Method 1 is carried out as
follows. Vascular endothelial growth factor (VEGF) is transferred
into a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride,
5 mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give
a final concentration of 200 .mu.M. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at
RT.
[0336] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0337] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of ion exchange chromatography. The PSA-VEGF-containing
fractions of the eluate are collected and subjected to UF/DF by use
of a a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
Method 2:
[0338] VEGF is transferred or dissolved in reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous HCl solution. Subsequently a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0339] The oxidized VEGF is further purified by ion exchange
chromatography. The oxidized VEGF containing fractions of the
eluate are collected and used for the conjugation reaction.
[0340] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized VEGF within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. at pH 6.0 in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0341] The obtained PSA-VEGF conjugate is further purified by ion
exchange chromatography. The PSA-VEGF conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
[0342] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0343] Vascular endothelial growth factor (VEGF) is transferred
into reaction buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. A 50-fold molar excess of a PSA aminooxy
reagent with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (10 mM final concentration)
and NaIO4 (final concentration: 400 .mu.M). The coupling reaction
is performed for 2 hours in the dark under gentle shaking at room
temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM). Then the
conductivity of the reaction mixture is adjusted by adding a buffer
containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5
mM calcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto
a column filled with Phenyl Sepharose FF (GE Healthcare, Fairfield,
Conn.) pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate,
350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH
6.9. Subsequently the conjugate is eluted with 50 mM Hepes, 5 mM
calcium chloride, pH 7.5. Finally, the PSA-VEGF containing
fractions are collected and subjected to UF/DF by use of a membrane
made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0344] In an alternative embodiment, Method 3 is carried out as
follows. Vascular endothelial growth factor (VEGF) is transferred
into reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5
mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSA
reagent with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (10 mM final concentration)
and NaIO4 (final concentration: 400 .mu.M). The coupling reaction
is performed for 2 hours in the dark under gentle shaking at room
temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM) and the conjugate
is purified by ion exchange chromatography. The PSA-VEGF containing
fractions of the eluate are collected and subjected to UF/DF by use
of a membrane made of regenerated cellulose (Millipore). The
preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
Method 4:
[0345] VEGF is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0346] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this VEGF solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0347] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0348] The obtained VEGF-conjugate is purified by ion-exchange
chromatography. The PSA-VEGF containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).
[0349] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 21
Polysialylation of EGF Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0350] A starting concentration of epidermal growth factor (EGF) is
transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to
obtain a protein concentration of 1 mg/ml. To this solution, NaIO4
is added to give a final concentration of 200 .mu.M. The oxidation
is carried at RT for 30 min in the dark under gentle shaking. The
reaction is then quenched with cysteine (final concentration: 10
mM) for 60 min at R.T.
[0351] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized EGF is eluted with
Buffer B (20 mM Hepes, 5 mM CaCl2, 1M NaCl, pH 7.0). The EGF
containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M
HCl.
[0352] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently, the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the PSA-EGF
containing fractions are collected and subjected to UF/DF by use of
a a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
[0353] In an alternative embodiment, Method 1 is carried out as
follows. Epidermal growth factor (EGF) is transferred into a
reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give
a final concentration of 200 .mu.M. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at
R.T.
[0354] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0355] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of ion exchange chromatography. The PSA-EGF containing
fractions of the eluate are collected and subjected to UF/DF by use
of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
Method 2:
[0356] EGF is transferred or dissolved in reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0357] The oxidized EGF is further purified by ion exchange
chromatography. The oxidized EGF containing fractions of the eluate
are collected and used for the conjugation reaction.
[0358] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized EGF within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. at pH 6.0 in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0359] The obtained PSA-EGF conjugate is further purified by ion
exchange chromatography. The PSA-EGF conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose with an appropriate
molecular weight cut off (Millipore).
[0360] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0361] Epidermal growth factor (EGF) is transferred into reaction
buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A
50-fold molar excess of a PSA aminooxy reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a
nucleophilic catalyst (10 mM final concentration) and NaIO4 (final
concentration: 400 .mu.M). The coupling reaction is performed for 2
hours in the dark under gentle shaking at room temperature.
Subsequently, the reaction is quenched with cysteine for 60 min at
RT (cysteine concentration: 10 mM). Then the conductivity of the
reaction mixture is adjusted by adding a buffer containing ammonium
acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column
filled with Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM
sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9.
Subsequently the conjugate is eluted with 50 mM Hepes, 5 mM calcium
chloride, pH 7.5. Finally the PSA-EGF containing fractions are
collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
[0362] In an alternative embodiment, Method 3 is carried out as
follows. Epidermal growth factor (EGF) is transferred into reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and diluted to obtain a protein concentration of
1 mg/ml. A 50-fold molar excess of a PSA aminooxy reagent with a MW
of 20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (10 mM final concentration) and NaIO4 (final
concentration: 400 .mu.M). The coupling reaction is performed for 2
hours in the dark under gentle shaking at room temperature.
Subsequently, the reaction is quenched with cysteine for 60 min at
RT (cysteine concentration: 10 mM) and the conjugate is purified by
ion exchange chromatography. The conjugate containing fractions of
the eluate are collected and subjected to UF/DF by use of a
membrane made of regenerated cellulose (Millipore). The preparation
is analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
Method 4:
[0363] EGF is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0364] Subsequently the aminooxy-polysialic acid (PSA-ONH2) reagent
is added in a 50-fold molar excess to this EGF-solution within a
maximum time period (t) of 15 minutes under gentle stirring. Then
an aqueous m-toluidine solution (50 mM) is added within 15 minutes
to get a final concentration of 10 mM. Finally a 40 mM aqueous
sodium periodate solution is added to give a concentration of 400
.mu.M.
[0365] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0366] The obtained EGF-conjugate is purified by ion-exchange
chromatography. The PSA-EGF containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).
[0367] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 22
Polysialylation of NGF Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0368] A starting concentration of nerve growth factor (NGF) is
transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to
obtain a protein concentration of 1 mg/ml. To this solution, NaIO4
is added to give a final concentration of 200 .mu.M. The oxidation
is carried at RT for 30 min in the dark under gentle shaking. The
reaction is then quenched with cysteine (final concentration: 10
mM) for 60 min at RT.
[0369] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized NGF is eluted with
Buffer B (20 mM Hepes, 5 mM CaCl2, 1M NaCl, pH 7.0). The NGF
containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M
HCl.
[0370] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently, the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the PSA-NGF
containing fractions are collected and subjected to UF/DF by use of
a a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
[0371] In an alternative embodiment, Method 1 is carried out as
follows. Nerve growth factor (NGF) is transferred into a reaction
buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and diluted to obtain a protein concentration of
1 mg/ml. To this solution, NaIO4 is added to give a final
concentration of 200 .mu.M. The oxidation is carried at RT for 30
min in the dark under gentle shaking. The reaction is then quenched
with cysteine (final concentration: 10 mM) for 60 min at RT.
[0372] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0373] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of ion exchange chromatography. The PSA-NGF containing
fractions of the eluate are collected and subjected to UF/DF by use
of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
Method 2:
[0374] NGF is transferred or dissolved in reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0375] The oxidized NGF is further purified by ion exchange
chromatography. The oxidized NGF containing fractions of the eluate
are collected and used for the conjugation reaction.
[0376] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized NGF within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. at pH 6.0 in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0377] The obtained PSA-NGF conjugate is further purified by ion
exchange chromatography. The PSA-NGF conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose with an appropriate
molecular weight cut off (Millipore).
[0378] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0379] Nerve growth factor (NGF) is transferred into reaction
buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A
50-fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a
nucleophilic catalyst (10 mM final concentration) and NaIO4 (final
concentration: 400 .mu.M). The coupling reaction is performed for 2
hours in the dark under gentle shaking at room temperature.
Subsequently, the reaction is quenched with cysteine for 60 min at
RT (cysteine concentration: 10 mM). Then the conductivity of the
reaction mixture is adjusted by adding a buffer containing ammonium
acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column
filled with Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM
sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9.
Subsequently the conjugate is eluted with 50 mM Hepes, 5 mM calcium
chloride, pH 7.5. Finally, the PSA NGF-containing fractions are
collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
[0380] In an alternative embodiment, Method 3 is carried out as
follows. Nerve growth factor (NGF) is transferred into reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and diluted to obtain a protein concentration of
1 mg/ml. A 50-fold molar excess of aminooxy-PSA reagent with a MW
of 20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (10 mM final concentration) and NaIO4 (final
concentration: 400 .mu.M). The coupling reaction is performed for 2
hours in the dark under gentle shaking at room temperature.
Subsequently, the reaction is quenched with cysteine for 60 min at
RT (cysteine concentration: 10 mM) and the conjugate is purified by
ion exchange chromatography. Then the PSA-NGF containing fractions
of the eluate are collected and subjected to UF/DF by use of a
membrane made of regenerated cellulose (Millipore). The preparation
is analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
Method 4:
[0381] NGF is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0382] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this NGF-solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0383] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0384] The obtained NGF-conjugate is purified by ion-exchange
chromatography. The PSA-NGF containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).
[0385] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 23
Polysialylation of HGH Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0386] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art.
[0387] A starting concentration of human growth hormone (HGH) is
transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to
obtain a protein concentration of 1 mg/ml. To this solution, NaIO4
is added to give a final concentration of 200 .mu.M. The oxidation
is carried at RT for 30 min in the dark under gentle shaking. The
reaction is then quenched with cysteine (final concentration: 10
mM) for 60 min at RT.
[0388] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized HGH is eluted with
Buffer B (20 mM Hepes, 5 mM CaCl2, 1 M NaCl, pH 7.0). The HGH
containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5
M HCl.
[0389] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the PSA-HGH
containing fractions are collected and subjected to UF/DF by use of
a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
[0390] In an alternative embodiment, Method 1 is carried out as
follows. As described herein, the amino acid sequence of human
growth hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art. HGH is transferred
into a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride,
5 mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give
a final concentration of 200 .mu.M. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at
RT.
[0391] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0392] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of ion exchange chromatography. The PSA-HGH containing
fractions of the eluate are collected and subjected to UF/DF by use
of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
Method 2:
[0393] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art.
[0394] HGH is transferred or dissolved in reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0395] The oxidized HGH is further purified by ion exchange
chromatography. The oxidized HGH containing fractions of the eluate
are collected and used for the conjugation reaction.
[0396] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized HGH within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. at pH 6.0 in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0397] The obtained PSA-HGH conjugate is further purified by ion
exchange chromatography. The PSA-HGH conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose with an appropriate
molecular weight cut off (Millipore).
[0398] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0399] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art.
[0400] Human growth hormone (HGH) is transferred into reaction
buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml. A
50 fold molar excess of aminooxy-PSA reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a
nucleophilic catalyst (10 mM final concentration) and NaIO4 (final
concentration: 400 .mu.M). The coupling reaction is performed for 2
hours in the dark under gentle shaking at room temperature.
Subsequently, the reaction is quenched with cysteine for 60 min at
RT (cysteine concentration: 10 mM). Then the conductivity of the
reaction mixture is adjusted by adding a buffer containing ammonium
acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column
filled with Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM
sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9.
Subsequently the conjugate is eluted with 50 mM Hepes, 5 mM calcium
chloride, pH 7.5. Finally, the PSA HGH-containing fractions are
collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
[0401] In an alternative embodiment, Method 3 is carried out as
follows. As described herein, the amino acid sequence of human
growth hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art. HGH is transferred
into reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5
mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. A 50 fold molar excess of aminooxy-PSA
reagent with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (10 mM final concentration)
and NaIO4 (final concentration: 400 .mu.M). The coupling reaction
is performed for 2 hours in the dark under gentle shaking at room
temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM) and the conjugate
is purified by ion exchange chromatography. Then the
PSA-HGH-containing fractions of the eluate are collected and
subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
Method 4:
[0402] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art.
[0403] HGH is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0404] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this HGH-solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0405] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0406] The obtained HGH-conjugate is purified by ion-exchange
chromatography. The PSA-HGH containing fractions of the eluate are
collected and concentrated by ultra-/diafiltration (UF/DF) using a
membrane made of regenerated cellulose (Millipore).
[0407] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 24
Polysialylation of TNF-Alpha Using Aminooxy-PSA and m-Toluidine as
a Nucleophilic Catalyst
[0408] A starting concentration of tumor necrosis factor-alpha
(TNF-alpha) is transferred into a reaction buffer (e.g., 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 is added to give a final concentration of 200
.mu.M. The oxidation is carried at RT for 30 min in the dark under
gentle shaking. The reaction is then quenched with cysteine (final
concentration: 10 mM) for 60 min at RT.
[0409] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized TNF-alpha is eluted
with Buffer B (20 mM Hepes, 5 mM CaCl2, 1M NaCl, pH 7.0). The
TNF-alpha containing fractions are collected. The protein content
is determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M
HCl.
[0410] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently, the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the
PSA-TNF-alpha-containing fractions are collected and subjected to
UF/DF by use of a a membrane made of regenerated cellulose
(Millipore). The preparation is next analytically characterized by
measuring total protein (Coomassie, Bradford) and biological
activity according to methods known in the art.
[0411] In an alternative embodiment, Method 1 is carried out as
follows. Tumor necrosis factor-alpha (TNF-alpha) is transferred
into a reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5
mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give
a final concentration of 200 .mu.M. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at
RT.
[0412] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof. A 50-fold molar excess of aminooxy-PSA reagent
with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (final concentration: 10
mM). The coupling reaction is performed for 2 hours in the dark
under gentle shaking at room temperature. The excess of aminooxy
reagent is removed by means of ion exchange chromatography. The
PSA-TNF-alpha containing fractions of the eluate are collected and
subjected to UF/DF by use of a a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.
Method 2:
[0413] TNF-alpha is transferred or dissolved in reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution. Subsequently, a 40 mM
aqueous sodium periodate solution is added within 10 minutes to
give a concentration of 200 .mu.M. The oxidation reaction is
carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2.degree. C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at
T=+22+/-2.degree. C. to give a final concentration of 10 mM in the
reaction mixture and incubation for 60+/-5 min.
[0414] The oxidized TNF-alpha is further purified by ion exchange
chromatography. The oxidized TNF-alpha containing fractions of the
eluate are collected and used for the conjugation reaction.
[0415] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized TNF-alpha within a maximum time period (t) of 15 minutes
under gentle stirring. Then an aqueous m-toluidine solution (50 mM)
is added within 15 minutes to get a final concentration of 10 mM.
The reaction mixture is incubated for 120+/-10 min. at pH 6.0 in
the dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0416] The obtained PSA-TNF-alpha conjugate is further purified by
ion exchange chromatography. The PSA-TNF-alpha conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
[0417] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0418] Tumor necrosis factor-alpha (TNF-alpha) is transferred into
reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSA
reagent with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (10 mM final concentration)
and NaIO4 (final concentration: 400 .mu.M). The coupling reaction
is performed for 2 hours in the dark under gentle shaking at room
temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM). Then the
conductivity of the reaction mixture is adjusted by adding a buffer
containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5
mM calcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto
a column filled with Phenyl Sepharose FF (GE Healthcare, Fairfield,
Conn.) pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate,
350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH
6.9. Subsequently the conjugate is eluted with 50 mM Hepes, 5 mM
calcium chloride, pH 7.5. Finally the PSA-TNF-alpha-containing
fractions are collected and subjected to UF/DF by use of a membrane
made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0419] In an alternative embodiment, Method 3 is carried out as
follows. Tumor necrosis factor-alpha (TNF-alpha) is transferred
into reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5
mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSA
reagent with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (10 mM final concentration)
and NaIO4 (final concentration: 400 .mu.M). The coupling reaction
is performed for 2 hours in the dark under gentle shaking at room
temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM). and the conjugate
is purified by ion exchange chromatography. The PSA-TNF-alpha
containing fractions of the eluate are collected and subjected to
UF/DF by use of a membrane made of regenerated cellulose
(Millipore). The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity
according to methods known in the art.
Method 4:
[0420] TNF-alpha is dissolved in or transferred to a reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) to get a final protein concentration of
1.0+/-0.25 mg/ml. Then the pH of the solution is corrected to 6.0
by drop wise addition of a 0.5 N aqueous HCl solution.
[0421] Subsequently the aminooxy-polysialic acid (PSA-ONH2) reagent
is added in a 50-fold molar excess to this TNF-alpha-solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0422] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0423] The obtained TNF-alpha conjugate is purified by ion-exchange
chromatography. The PSA-TNF-alpha containing fractions of the
eluate are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose
(Millipore).
[0424] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 25
Polysialylation of Insulin Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0425] As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art. A starting concentration of insulin is
transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to
obtain a protein concentration of 1 mg/ml. To this solution, NaIO4
is added to give a final concentration of 200 .mu.M. The oxidation
is carried at RT for 30 min in the dark under gentle shaking. The
reaction is then quenched with cysteine (final concentration: 10
mM) for 60 min at RT.
[0426] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized insulin is eluted
with Buffer B (20 mM Hepes, 5 mM CaCl2, 1 M NaCl, pH 7.0). The
insulin containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5
M HCl.
[0427] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the PSA-insulin
containing fractions are collected and subjected to UF/DF by use of
a a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
[0428] In an alternative embodiment, Method 1 is carried out as
follows. As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art. Insulin is transferred into a reaction
buffer (e.g., 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and diluted to obtain a protein concentration of
1 mg/ml. To this solution, NaIO4 is added to give a final
concentration of 200 .mu.M. The oxidation is carried at RT for 30
min in the dark under gentle shaking. The reaction is then quenched
with cysteine (final concentration: 10 mM) for 60 min at RT.
[0429] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0430] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of ion exchange chromatography. The PSA-insulin containing
fractions of the eluate are collected and subjected to UF/DF by use
of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
Method 2:
[0431] As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art.
[0432] Insulin is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5 N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0433] The oxidized insulin is further purified by ion exchange
chromatography. The oxidized insulin containing fractions of the
eluate are collected and used for the conjugation reaction.
[0434] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized insulin within a maximum time period (t) of 15 minutes
under gentle stirring. Then an aqueous m-toluidine solution (50 mM)
is added within 15 minutes to get a final concentration of 10 mM.
The reaction mixture is incubated for 120+/-10 min. at pH 6.0 in
the dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0435] The obtained PSA-insulin conjugate is further purified by
ion exchange chromatography. The PSA-insulin conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
[0436] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0437] As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art.
[0438] Insulin is transferred into reaction buffer (50 mM Hepes,
350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted
to obtain a protein concentration of 1 mg/ml. A 50-fold molar
excess of aminooxy-PSA reagent with a MW of 20 kD (described above)
is added followed by m-toluidine as a nucleophilic catalyst (10 mM
final concentration) and NaIO4 (final concentration: 400 .mu.M).
The coupling reaction is performed for 2 hours in the dark under
gentle shaking at room temperature. Subsequently, the reaction is
quenched with cysteine for 60 min at RT (cysteine concentration: 10
mM). Then the conductivity of the reaction mixture is adjusted by
adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with Phenyl Sepharose FF (GE
Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM Hepes,
2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, 0.01% Tween 80, pH 6.9. Subsequently the conjugate is
eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally,
the PSA-insulin containing fractions are collected and subjected to
UF/DF by use of a membrane made of regenerated cellulose
(Millipore). The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity
according to methods known in the art.
[0439] In an alternative embodiment, Method 3 is carried out as
follows. As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art.
[0440] Insulin is transferred into reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. A 50-fold
molar excess of aminooxy-PSA reagent with a MW of 20 kD (described
above) is added followed by m-toluidine as a nucleophilic catalyst
(10 mM final concentration) and NaIO4 (final concentration: 400
.mu.M). The coupling reaction is performed for 2 hours in the dark
under gentle shaking at room temperature. Subsequently, the
reaction is quenched with cysteine for 60 min at RT (cysteine
concentration: 10 mM) and the conjugate is purified by ion exchange
chromatography. PSA-insulin containing fractions of the eluate are
collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
Method 4:
[0441] As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art.
[0442] Insulin is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0443] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this insulin-solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0444] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0445] The obtained insulin conjugate is purified by ion-exchange
chromatography. The PSA-insulin containing fractions of the eluate
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose (Millipore).
[0446] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 26
Polysialylation of Interferon-Alpha Using Aminooxy-PSA and
m-Toluidine as a Nucleophilic Catalyst
Method 1:
[0447] A starting concentration of interferon-alpha is transferred
into a reaction buffer (e.g., 50 mM Hepes, 350 mM sodium chloride,
5 mM calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give
a final concentration of 200 .mu.M. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at
RT.
[0448] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized interferon-alpha is
eluted with Buffer B (20 mM Hepes, 5 mM CaCl2, 1M NaCl, pH 7.0).
The interferon-alpha containing fractions are collected. The
protein content is determined (Coomassie, Bradford) and adjusted to
1 mg/ml with reaction buffer and adjusted to pH 6.0 by dropwise
addition of 0.5 M HCl.
[0449] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently, the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the
PSA-interferon-alpha containing fractions are collected and
subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.
[0450] In an alternative embodiment, Method 1 is carried out as
follows. Interferon-alpha is transferred into a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and diluted to obtain a protein concentration of 1 mg/ml.
To this solution, NaIO4 is added to give a final concentration of
200 .mu.M. The oxidation is carried at RT for 30 min in the dark
under gentle shaking. The reaction is then quenched with cysteine
(final concentration: 10 mM) for 60 min at RT.
[0451] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0452] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of ion-exchange chromatography. The PSA-interferon-alpha
containing fractions of the eluate are collected and subjected to
UF/DF by use of a membrane made of regenerated cellulose
(Millipore). The preparation is next analytically characterized by
measuring total protein (Coomassie, Bradford) and biological
activity according to methods known in the art.
Method 2:
[0453] Interferon-alpha is transferred or dissolved in reaction
buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) to get a final protein concentration of
1.0+/-0.25 mg/ml. Then the pH of the solution is corrected to 6.0
by drop wise addition of a 0.5 N aqueous HCl solution.
Subsequently, a 40 mM aqueous sodium periodate solution is added
within 10 minutes to give a concentration of 200 .mu.M. The
oxidation reaction is carried out for 30+/-5 min at a temperature
(T) of T=+22+/-2.degree. C. Then the reaction is stopped by
addition of an aqueous L-cysteine solution (1 M) within 15 minutes
at T=+22+/-2.degree. C. to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0454] The oxidized interferon-alpha is further purified by ion
exchange chromatography. The oxidized interferon-alpha containing
fractions of the eluate are collected and used for the conjugation
reaction.
[0455] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized interferon-gamma within a maximum time period (t) of 15
minutes under gentle stirring. Then an aqueous m-toluidine solution
(50 mM) is added within 15 minutes to get a final concentration of
10 mM. The reaction mixture is incubated for 120+/-10 min. at pH
6.0 in the dark at a temperature (T) of T=+22+/-2.degree. C. under
gentle shaking (protein concentration: 1 mg/ml).
[0456] The obtained PSA-interferon-alpha conjugate is further
purified by ion exchange chromatography. The PSA-interferon-alpha
conjugate containing fractions are collected and concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with an appropriate molecular weight cut off
(Millipore).
Method 3:
[0457] Interferon-alpha is transferred into reaction buffer (50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. A 50-fold
molar excess of a PSA aminooxy reagent with a MW of 20 kD
(described above) is added followed by m-toluidine as a
nucleophilic catalyst (10 mM final concentration) and NaIO4 (final
concentration: 400 .mu.M). The coupling reaction is performed for 2
hours in the dark under gentle shaking at room temperature.
Subsequently, the reaction is quenched with cysteine for 60 min at
RT (cysteine concentration: 10 mM). Then the conductivity of the
reaction mixture is adjusted by adding a buffer containing ammonium
acetate (50 mM Hepes, 350 mM sodium chloride, 5 mM calcium
chloride, 8 M ammonium acetate, pH 6.9) and loaded onto a column
filled with Phenyl Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate, 350 mM
sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6.9.
Subsequently the conjugate is eluted with 50 mM Hepes, 5 mM calcium
chloride, pH 7.5. Finally, the PSA-interferon-alpha containing
fractions are collected and subjected to UF/DF by use of a membrane
made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0458] In an alternative embodiment, Method 3 is carried out as
follows. Interferon-alpha is transferred into reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
and diluted to obtain a protein concentration of 1 mg/ml. A 50-fold
molar excess of aminooxy-PSA reagent with a MW of 20 kD (described
above) is added followed by m-toluidine as a nucleophilic catalyst
(10 mM final concentration) and NaIO4 (final concentration: 400
.mu.M). The coupling reaction is performed for 2 hours in the dark
under gentle shaking at room temperature. Subsequently, the
reaction is quenched with cysteine for 60 min at RT (cysteine
concentration: 10 mM) and the conjugate is purified by ion exchange
chromatography. The PSA-interferon-alpha containing fractions of
the eluate are collected and subjected to UF/DF by use of a
membrane made of regenerated cellulose (Millipore). The preparation
is analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
Method 4:
[0459] Interferon-alpha is dissolved in or transferred to a
reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) to get a final protein concentration of
1.0+/-0.25 mg/ml. Then the pH of the solution is corrected to 6.0
by drop wise addition of a 0.5 N aqueous HCl solution.
[0460] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this interferon-alpha
solution within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM.
Finally, a 40 mM aqueous sodium periodate solution is added to give
a concentration of 400 .mu.M.
[0461] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0462] The obtained interferon-alpha conjugate is purified by
ion-exchange chromatography. The PSA-interferon-alpha containing
fractions of the eluate are collected and concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose (Millipore).
[0463] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 27
Polysialylation of Interferon-Gamma Using Aminooxy-PSA and
m-Toluidine as a Nucleophilic Catalyst
Method 1:
[0464] 10 mg interferon-gamma is dissolved in 5 ml histidine
buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl). 100 .mu.l of an
aqueous sodium periodate solution (5 mM) is then added and the
reaction mixture is incubated for 1 h in the dark at 4.degree. C.
under gentle stirring and quenched for 15 min at room temperature
by the addition of 50 .mu.l of a 1 M aqueous cysteine solution. The
mixture is subsequently subjected to UF/DF employing Vivaspin 15R
10 kD centrifugal filtrators to remove excess periodate, quencher
and the byproducts thereof.
[0465] The retentate (approx. 7 ml), containing oxidized
interferon-gamma, is mixed with 2 ml of an aqueous m-toluidine
solution (50 mM) and incubated for 30 min at room temperature. Then
aminooxy-PSA reagent with a MW of 20 kD (described above) is added
to give a 5-fold molar reagent excess. This mixture is incubated
for 2.5 h at RT in the dark under gentle stirring.
[0466] The free Interferon-gamma is removed by means of cation
exchange chromatography (CEC). The reaction mixture is diluted with
20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep
SPFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with
Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Free interferon-gamma is
eluted by washing the column with 25% Buffer B and the conjugate at
50% Buffer B. The conductivity of the conjugate containing
fractions is subsequently raised to .about.190 mS/cm with Buffer C
(50 mM Hepes, 5 M NaCl, pH 6.9) and loaded onto a 20 ml HiPrep
Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, pH 6.9).
Free PSA-reagent is washed out within 5 CV Buffer D. Subsequently,
the conjugate is eluted with 100% Buffer E (50 mM Hepes, pH 6.9).
The conjugate containing fractions are concentrated by UF/DF using
a 10 kD membrane made of regenerated cellulose (88 cm2, cut-off 10
kD, Millipore). The final diafiltration step is performed against
histidine buffer, pH 6.9 containing 150 mM NaCl. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art. For
the PSA-Interferon-gamma conjugate a specific activity of >50%
in comparison to native Interferon-gamma is determined. The
conjugate is additionally analytically characterized by Size
Exclusion HPLC using a Agilent 1200 HPLC system equipped with a
Shodex KW 803 column under conditions as previously described
(Kolarich et al, Transfusion 2006; 46:1959-77). It is shown that
the preparation contains no free Interferon gamma.
Method 2:
[0467] 10 mg interferon-gamma is dissolved in 8 ml histidine
buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl). 200 .mu.l of an
aqueous sodium periodate solution (5 mM) and 2 ml of an aqueous
m-toluidine solution (50 mM) are then added. Subsequently the
aminooxy-PSA reagent with a MW of 20 kD (described above) is added
to give a 5-fold molar reagent excess. The mixture is incubated for
2 h in the dark at room temperature under gentle stirring and
quenched for 15 min at room temperature by the addition of 100
.mu.l of 1 M aqueous cysteine solution.
[0468] The free interferon gamma is removed by means of cation
exchange chromatography (CEC). The reaction mixture is diluted with
20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep
SPFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with
Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Free interferon-gamma is
eluted by washing the column with 25% Buffer B and the conjugate at
50% Buffer B. The conductivity of the conjugate containing
fractions is subsequently raised to .about.190 mS/cm with Buffer C
(50 mM Hepes, 5 M NaCl, pH 6.9) and loaded onto a 20 ml HiPrep
Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, pH 6.9).
Free PSA-reagent is washed out within 5 CV Buffer D. Subsequently,
the conjugate is eluted with 100% Buffer E (50 mM Hepes, pH 6.9).
The conjugate containing fractions are concentrated by UF/DF using
a 10 kD membrane made of regenerated cellulose (88 cm2, cut-off 10
kD/Millipore). The final diafiltration step is performed against
histidine buffer, pH 6.9 containing 150 mM NaCl. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art. For
the PSAinterferon-gamma conjugate a specific activity of >50% in
comparison to native interferon-gamma is determined. The conjugate
is additionally analytically characterized by Size Exclusion HPLC
using a Agilent 1200 HPLC system equipped with a Shodex KW 803
column under conditions as previously described (Kolarich et al,
Transfusion 2006; 46:1959-77). It is shown that the preparation
contains no free interferon-gamma.
Method 3:
[0469] 10 mg interferon-gamma is dissolved in 8 ml histidine
buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl). 200 .mu.l of an
aqueous sodium periodate solution (5 mM) and 2 ml of an aqueous
m-toluidine solution (50 mM) are then added. Subsequently the
aminooxy-PSA reagent with a MW of 20 kD (described above) is added
to give a 5-fold molar reagent excess. The mixture is incubated for
2 h in the dark at room temperature under gentle stirring and
quenched for 15 min at room temperature by the addition of 100
.mu.l of 1 M aqueous cysteine solution.
[0470] The free interferon gamma is removed by means of cation
exchange chromatography (CEC). The reaction mixture is diluted with
20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep
SPFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with
Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Free interferon-gamma is
eluted by washing the column with 25% Buffer B and the conjugate at
50% Buffer B. The conductivity of the conjugate containing
fractions is subsequently raised to .about.190 mS/cm with Buffer C
(50 mM Hepes, 5 M NaCl, pH 6.9) and loaded onto a 20 ml HiPrep
Butyl FF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer D (50 mM Hepes, 3 M NaCl, pH 6.9).
Free PSA-reagent is washed out within 5 CV Buffer D. Subsequently
the conjugate is eluted with 100% Buffer E (50 mM Hepes, pH 6.9).
The conjugate containing fractions are concentrated by UF/DF using
a 10 kD membrane made of regenerated cellulose (88 cm2, cut-off 10
kD/Millipore). The final diafiltration step is performed against
histidine buffer, pH 6.9 containing 150 mM NaCl. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art. For
the PSAinterferon-gamma conjugate a specific activity of >50% in
comparison to native interferon-gamma is determined. The conjugate
is additionally analytically characterized by Size Exclusion HPLC
using a Agilent 1200 HPLC system equipped with a Shodex KW 803
column under conditions as previously described (Kolarich et al,
Transfusion 2006; 46:1959-77). It is shown that the preparation
contains no free interferon-gamma.
Method 4:
[0471] Interferon-gamma is dissolved in or transferred to a
reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) to get a final protein concentration of
1.0+/-0.25 mg/ml. Then the pH of the solution is corrected to 6.0
by drop wise addition of a 0.5 N aqueous HCl solution.
[0472] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this interferon-gamma
solution within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM.
Finally, a 40 mM aqueous sodium periodate solution is added to give
a concentration of 400 .mu.M.
[0473] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0474] The obtained interferon-gamma conjugate is purified by
ion-exchange chromatography. The PSA-interferon-gamma containing
fractions of the eluate are collected and concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose (Millipore).
[0475] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 28
Polysialylation of G-CSF Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0476] A starting concentration of granulocyte-colony stimulating
factor (G-CSF) is transferred into a reaction buffer (e.g., 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 is added to give a final concentration of 200
.mu.M. The oxidation is carried at RT for 30 min in the dark under
gentle shaking. The reaction is then quenched with cysteine (final
concentration: 10 mM) for 60 min at RT.
[0477] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized G-CSF is eluted with
Buffer B (20 mM Hepes, 5 mM CaCl2, 1 M NaCl, pH 7.0). The G-CSF
containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5
M HCl.
[0478] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently, the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally the
PSA-G-CSF-containing fractions are collected and subjected to UF/DF
by use of a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
[0479] In an alternative embodiment, Method 1 is carried out as
follows. Granulocyte-colony stimulating factor (G-CSF) is
transferred into a reaction buffer (e.g., 50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to
obtain a protein concentration of 1 mg/ml. To this solution, NaIO4
is added to give a final concentration of 200 .mu.M. The oxidation
is carried at RT for 30 min in the dark under gentle shaking. The
reaction is then quenched with cysteine (final concentration: 10
mM) for 60 min at RT.
[0480] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof. A 50-fold molar excess of aminooxy-PSA reagent
with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (final concentration: 10
mM). The coupling reaction is performed for 2 hours in the dark
under gentle shaking at room temperature. The excess of aminooxy
reagent is removed by means of ion exchange chromatography. The
PSA-G-CSF containing fractions of the eluate are collected and
subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.
Method 2:
[0481] G-CSF is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5 N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0482] The oxidized G-CSF is further purified by ion exchange
chromatography. The oxidized G-CSF containing fractions of the
eluate are collected and used for the conjugation reaction.
[0483] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized G-CSF within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. at pH 6.0 in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0484] The obtained PSA-G-CSF conjugate is further purified by ion
exchange chromatography. The PSA-G-CSF conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
[0485] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0486] Granulocyte-colony stimulating factor (G-CSF) is transferred
into reaction buffer (50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. A 50-fold molar excess of aminooxy-PSA
reagent with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (10 mM final concentration)
and NaIO4 (final concentration: 400 .mu.M). The coupling reaction
is performed for 2 hours in the dark under gentle shaking at room
temperature. Subsequently, the reaction is quenched with cysteine
for 60 min at RT (cysteine concentration: 10 mM). Then the
conductivity of the reaction mixture is adjusted by adding a buffer
containing ammonium acetate (50 mM Hepes, 350 mM sodium chloride, 5
mM calcium chloride, 8 M ammonium acetate, pH 6.9) and loaded onto
a column filled with Phenyl Sepharose FF (GE Healthcare, Fairfield,
Conn.) pre-equilibrated with 50 mM Hepes, 2.5 M ammonium acetate,
350 mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH
6.9. Subsequently the conjugate is eluted with 50 mM Hepes, 5 mM
calcium chloride, pH 7.5. Finally, the PSA-G-CSF-containing
fractions are collected and subjected to UF/DF by use of a membrane
made of regenerated cellulose (Millipore). The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0487] In an alternative embodiment, Method 3 is carried out as
follows. Granulocyte-colony stimulating factor (G-CSF) is
transferred into reaction buffer (e.g. 50 mM Hepes, 350 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and diluted to obtain a
protein concentration of 1 mg/ml. A 50-fold molar excess of
aminooxy-PSA reagent with a MW of 20 kD (described above) is added
followed by m-toluidine as a nucleophilic catalyst (10 mM final
concentration) and NaIO4 (final concentration: 400 .mu.M). The
coupling reaction is performed for 2 hours in the dark under gentle
shaking at room temperature. Subsequently, the reaction is quenched
with cysteine for 60 min at RT (cysteine concentration: 10 mM) and
the conjugate is purified by ion exchange chromatography. The
PSA-G-CSF containing fractions of the eluate are collected and
subjected to UF/DF by use of a membrane made of regenerated
cellulose (Millipore). The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
Method 4:
[0488] G-CSF is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0489] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this G-CSF solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally, a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0490] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0491] The obtained G-CSF conjugate is purified by ion-exchange
chromatography. The PSA-G-CSF containing fractions of the eluate
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose (Millipore).
[0492] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 29
Polysialylation of Humira Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0493] A starting concentration of Humira is transferred into a
reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give
a final concentration of 200 .mu.M. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at
RT.
[0494] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized Humira is eluted with
Buffer B (20 mM Hepes, 5 mM CaCl2, 1M NaCl, pH 7.0). The Humira
containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5M
HCl.
[0495] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the PSA-Humira
containing fractions are collected and subjected to UF/DF by use of
a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
[0496] In an alternative embodiment, Method 1 is carried out as
follows. Humira is transferred into a reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. To this
solution, NaIO4 is added to give a final concentration of 200
.mu.M. The oxidation is carried at RT for 30 min in the dark under
gentle shaking. The reaction is then quenched with cysteine (final
concentration: 10 mM) for 60 min at RT.
[0497] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof. A 50-fold molar excess of aminooxy-PSA reagent
with a MW of 20 kD (described above) is added followed by
m-toluidine as a nucleophilic catalyst (final concentration: 10
mM). The coupling reaction is performed for 2 hours in the dark
under gentle shaking at room temperature. The excess of aminooxy
reagent is removed by means of ion exchange chromatography The
PSA-Humira containing fractions of the elutae are collected and
subjected to UF/DF by use of a a membrane made of regenerated
cellulose (Millipore). The preparation is next analytically
characterized by measuring total protein (Coomassie, Bradford) and
biological activity according to methods known in the art.
Method 2:
[0498] Humira is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5 N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0499] The oxidized Humira is further purified by ion exchange
chromatography. The oxidized Humira containing fractions of the
eluate are collected and used for the conjugation reaction.
[0500] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized Humira within a maximum time period (t) of 15 minutes
under gentle stirring. Then an aqueous m-toluidine solution (50 mM)
is added within 15 minutes to get a final concentration of 10 mM.
The reaction mixture is incubated for 120+/-10 min. at pH 6.0 in
the dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0501] The obtained PSA-Humira conjugate is further purified by ion
exchange chromatography. The PSA-Humira conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
[0502] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0503] Humira is transferred into reaction buffer (50 mM Hepes, 350
mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to
obtain a protein concentration of 1 mg/ml. A 50-fold molar excess
of aminooxy-PSA reagent with a MW of 20 kD (described above) is
added followed by m-toluidine as a nucleophilic catalyst (10 mM
final concentration) and NaIO4 (final concentration: 400 .mu.M).
The coupling reaction is performed for 2 hours in the dark under
gentle shaking at room temperature. Subsequently, the reaction is
quenched with cysteine for 60 min at RT (cysteine concentration: 10
mM). Then the conductivity of the reaction mixture is adjusted by
adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with Phenyl Sepharose FF (GE
Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM Hepes,
2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, 0.01% Tween 80, pH 6.9. Subsequently the conjugate is
eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally the
PSA-Humira containing fractions are collected and subjected to
UF/DF by use of a membrane made of regenerated cellulose
(Millipore). The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity
according to methods known in the art.
[0504] In an alternative embodiment, Method 3 is carried out as
follows. Humira is transferred into reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) and
diluted to obtain a protein concentration of 1 mg/ml. A 50-fold
molar excess of aminooxy-PSA reagent with a MW of 20 kD (described
above) is added followed by m-toluidine as a nucleophilic catalyst
(10 mM final concentration) and NaIO4 (final concentration: 400
.mu.M). The coupling reaction is performed for 2 hours in the dark
under gentle shaking at room temperature. Subsequently, the
reaction is quenched with cysteine for 60 min at RT (cysteine
concentration: 10 mM) and the conjugate is purified by ion exchange
chromatography. The PSA-Humira containing fractions of the eluate
are collected and subjected to UF/DF by use of a membrane made of
regenerated cellulose (Millipore). The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
Method 4:
[0505] Humira is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0506] Subsequently, the aminooxy-polysialic acid (PSA-ONH2)
reagent is added in a 50-fold molar excess to this Humira solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0507] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0508] The obtained Humira-conjugate is purified by ion-exchange
chromatography. The PSA-Humira containing fractions of the eluate
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose (Millipore).
[0509] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 30
Polysialylation of Prolia Using Aminooxy-PSA and m-Toluidine as a
Nucleophilic Catalyst
Method 1:
[0510] A starting concentration of Prolia is transferred into a
reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) and diluted to obtain a protein
concentration of 1 mg/ml. To this solution, NaIO4 is added to give
a final concentration of 200 .mu.M. The oxidation is carried at RT
for 30 min in the dark under gentle shaking. The reaction is then
quenched with cysteine (final concentration: 10 mM) for 60 min at
RT.
[0511] The solution is next subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof or, in the alternative, to an IEX column with a
volume of 20 ml (Merck EMD TMAE (M)) which is equilibrated with
Buffer A (20 mM Hepes, 5 mM CaCl2, pH 7.0). The column is
equilibrated with 5 CV Buffer A. The oxidized Prolia is eluted with
Buffer B (20 mM Hepes, 5 mM CaCl2, 1M NaCl, pH 7.0). The Prolia
containing fractions are collected. The protein content is
determined (Coomassie, Bradford) and adjusted to 1 mg/ml with
reaction buffer and adjusted to pH 6.0 by dropwise addition of 0.5
M HCl.
[0512] A 50-fold molar excess of aminooxy-PSA reagent with a MW of
20 kD (described above) is added followed by m-toluidine as a
nucleophilic catalyst (final concentration: 10 mM). The coupling
reaction is performed for 2 hours in the dark under gentle shaking
at room temperature. The excess of aminooxy reagent is removed by
means of HIC. The conductivity of the reaction mixture is adjusted
by adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with 80 ml Phenyl Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, pH 6.9. Subsequently the conjugate is eluted with 50 mM
Hepes buffer pH 7.5 containing 5 mM CaCl2. Finally, the PSA-Prolia
containing fractions are collected and subjected to UF/DF by use of
a a membrane made of regenerated cellulose (Millipore). The
preparation is next analytically characterized by measuring total
protein (Coomassie, Bradford) and biological activity according to
methods known in the art.
[0513] In an alternative embodiment, Method 1 is carried out as
follows. 10 mg Prolia is dissolved in 5 ml histidine buffer, pH 6.0
(20 mM L-histidine, 150 mM NaCl). 100 .mu.l of an aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 50
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin 15R 10 kD
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0514] The retentate (approx. 7 ml), containing oxidized Prolia, is
mixed with 2 ml of an aqueous m-toluidine solution (50 mM) and
incubated for 30 min at room temperature. Then aminooxy-PSA reagent
with a MW of 20 kD (described above) is added to give a 5-fold
molar reagent excess. This mixture is incubated for 2.5 h at RT in
the dark under gentle stirring.
[0515] The free Prolia is removed by means of cation exchange
chromatography (CEC). The reaction mixture is diluted with 20 ml
Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF
16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated
with Buffer A. Then the column is eluted with Buffer B (50 mM
Hepes, 1 M NaCl, pH 6.5). Free Prolia is eluted by washing the
column with 25% Buffer B and the conjugate at 50% Buffer B. The
conductivity of the conjugate containing fractions is subsequently
raised to .about.190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl, pH
6.9) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GE
Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer D (50 mM
Hepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed out within 5
CV Buffer D. Subsequently, the conjugate is eluted with 100% Buffer
E (50 mM Hepes, pH 6.9). The conjugate containing fractions are
concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm2, cut-off 10 kD, Millipore). The final
diafiltration step is performed against histidine buffer, pH 6.9
containing 150 mM NaCl. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art. For the PSA-Prolia
conjugate a specific activity of >50% in comparison to native
Prolia is determined. The conjugate is additionally analytically
characterized by Size Exclusion HPLC using a Agilent 1200 HPLC
system equipped with a Shodex KW 803 column under conditions as
previously described (Kolarich et al, Transfusion 2006;
46:1959-77). It is shown that the preparation contains no free
Prolia.
Method 2:
[0516] Prolia is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5 N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0517] The oxidized Prolia is further purified by ion exchange
chromatography. The oxidized Prolia containing fractions of the
eluate are collected and used for the conjugation reaction.
[0518] The aminooxy-polysialic acid (PSA-ONH2) reagent is added in
a 50-fold molar excess to the eluate containing the purified
oxidized Prolia within a maximum time period (t) of 15 minutes
under gentle stirring. Then an aqueous m-toluidine solution (50 mM)
is added within 15 minutes to get a final concentration of 10 mM.
The reaction mixture is incubated for 120+/-10 min. at pH 6.0 in
the dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking (protein concentration: 1 mg/ml).
[0519] The obtained Prolia conjugate is further purified by ion
exchange chromatography. The Prolia conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose with an appropriate
molecular weight cut off (Millipore).
[0520] The conjugate prepared by use of this procedure is
analytically characterized by measuring total protein, biological
activity, and determination of the polysialyation degree by
measuring the PSA content (resorcinol assay).
Method 3:
[0521] Prolia is transferred into reaction buffer (50 mM Hepes, 350
mM sodium chloride, 5 mM calcium chloride, pH 6.0) and diluted to
obtain a protein concentration of 1 mg/ml. A 50-fold molar excess
of aminooxy-PSA reagent with a MW of 20 kD (described above) is
added followed by m-toluidine as a nucleophilic catalyst (10 mM
final concentration) and NaIO4 (final concentration: 400 .mu.M).
The coupling reaction is performed for 2 hours in the dark under
gentle shaking at room temperature. Subsequently, the reaction is
quenched with cysteine for 60 min at RT (cysteine concentration: 10
mM). Then the conductivity of the reaction mixture is adjusted by
adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
sodium chloride, 5 mM calcium chloride, 8 M ammonium acetate, pH
6.9) and loaded onto a column filled with Phenyl Sepharose FF (GE
Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM Hepes,
2.5 M ammonium acetate, 350 mM sodium chloride, 5 mM calcium
chloride, 0.01% Tween 80, pH 6.9. Subsequently the conjugate is
eluted with 50 mM Hepes, 5 mM calcium chloride, pH 7.5. Finally the
PSA Prolia-containing fractions are collected and subjected to
UF/DF by use of a membrane made of regenerated cellulose
(Millipore). The preparation is analytically characterized by
measuring total protein (Bradford) and biological activity
according to methods known in the art.
[0522] In an alternative embodiment, Method 3 is carried out as
follows. 10 mg Prolia is dissolved in 8 ml histidine buffer, pH 6.0
(20 mM L-histidine, 150 mM NaCl). 200 .mu.l of an aqueous sodium
periodate solution (5 mM) and 2 ml of an aqueous m-toluidine
solution (50 mM) are then added. Subsequently the aminooxy-PSA
reagent with a MW of 20 kD (described above) is added to give a 5
fold molar reagent excess. The mixture is incubated for 2 h in the
dark at room temperature under gentle stirring and quenched for 15
min at room temperature by the addition of 100 .mu.l of 1 M aqueous
cysteine solution.
[0523] The free Prolia is removed by means of cation exchange
chromatography (CEC). The reaction mixture is diluted with 20 ml
Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml HiPrep SPFF
16/10 column (GE Healthcare, Fairfield, Conn.) pre-equilibrated
with Buffer A. Then the column is eluted with Buffer B (50 mM
Hepes, 1 M NaCl, pH 6.5). Free Prolia is eluted by washing the
column with 25% Buffer B and the conjugate at 50% Buffer B. The
conductivity of the conjugate containing fractions is subsequently
raised to .about.190 mS/cm with Buffer C (50 mM Hepes, 5 M NaCl, pH
6.9) and loaded onto a 20 ml HiPrep Butyl FF 16/10 column (GE
Healthcare, Fairfield, Conn.) pre-equilibrated with Buffer D (50 mM
Hepes, 3 M NaCl, pH 6.9). Free PSA-reagent is washed out within 5
CV Buffer D. Subsequently the conjugate is eluted with 100% Buffer
E (50 mM Hepes, pH 6.9). The conjugate containing fractions are
concentrated by UF/DF using a 10 kD membrane made of regenerated
cellulose (88 cm.sup.2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against histidine buffer, pH 6.9
containing 150 mM NaCl. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art. For the PSA-Prolia
conjugate a specific activity of >50% in comparison to native
Prolia is determined. The conjugate is additionally analytically
characterized by Size Exclusion HPLC using a Agilent 1200 HPLC
system equipped with a Shodex KW 803 column under conditions as
previously described (Kolarich et al, Transfusion 2006;
46:1959-77). It is shown that the preparation contains no free
Prolia.
Method 4:
[0524] Prolia is dissolved in or transferred to a reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution.
[0525] Subsequently the aminooxy-polysialic acid (PSA-ONH.sub.2)
reagent is added in a 50-fold molar excess to this Prolia-solution
within a maximum time period (t) of 15 minutes under gentle
stirring. Then an aqueous m-toluidine solution (50 mM) is added
within 15 minutes to get a final concentration of 10 mM. Finally a
40 mM aqueous sodium periodate solution is added to give a
concentration of 400 .mu.M.
[0526] The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking. Then the reaction is stopped by the addition of an aqueous
L-cysteine solution (1 M) to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0527] The obtained Prolia conjugate is purified by ion-exchange
chromatography. The PSA-Prolia containing fractions of the eluate
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose (Millipore).
[0528] The conjugates prepared by use of this procedure are
analytically characterized by measuring total protein, biological
activity according to methods known in the art, and determination
of the polysialyation degree by measuring the PSA content
(resorcinol assay).
Example 31
Polysialylation of Other Therapeutic Proteins
[0529] Polysialylation reactions performed in the presence of
alternative nucleophilic catalysts like m-toluidine or
o-aminobenzoic acid as described herein may be extended to other
therapeutic proteins. For example, in various aspects of the
invention, the above polysialylation or PEGylation reactions as
described herein with PSA aminooxy or PEG aminooxy reagents is
repeated with therapeutic proteins such as those proteins described
herein.
Example 32
PEGylation of EPO Using an Aminooxy-PEG Reagent and m-Toluidine as
a Nucleophilic Catalyst
Method 1:
[0530] Erythropoietin (EPO) is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). EPO is dissolved in 7.0 ml histidine buffer,
pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous
sodium periodate solution (5 mM) is then added and the reaction
mixture is incubated for 1 h in the dark at 4.degree. C. under
gentle stirring and quenched for 15 min at room temperature by the
addition of 7.5 .mu.l of a 1 M aqueous cysteine solution. The
mixture is subsequently subjected to UF/DF employing Vivaspin
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0531] The retentate containing oxidized EPO is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0532] Finally, the PEG-EPO conjugate is purified by ion-exchange
chromatography (e.g. on Q Sepharose FF). For example, 1.5 mg
protein/ml gel is loaded on the column equilibrated with 50 mM
Hepes buffer, pH 7.4 containing 5 mM CaCl.sub.2). The conjugate is
eluted with 50 mM Hepes buffer containing 5 mM CaCl.sub.2) and 500
mM sodium chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0533] In an alternative embodiment, Method 1 is carried out as
follows. EPO is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). 10 mg EPO is dissolved in 5 ml histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl). 100 .mu.l of an aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 50
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin 15R 10 kD
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0534] The retentate (approx. 7 ml), containing oxidized EPO, is
mixed with 2 ml of an aqueous m-toluidine solution (50 mM) and
incubated for 30 min at room temperature. Then aminooxy-PEG reagent
with a MW of 20 kD (described above) is added to give a 5-fold
molar reagent excess. This mixture is incubated for 2.5 h at RT in
the dark under gentle stirring.
[0535] Finally, the PEG-EPO conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. The reaction mixture is diluted
with 20 ml Buffer A (50 mM Hepes, pH 7.5) and loaded onto a 20 ml
HiPrep QFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with
Buffer B (50 mM Hepes, 1 M NaCl, pH 7.5). Free EPO is eluted by
washing the column with 25% Buffer B and the conjugate at 50%
Buffer B. The conjugate containing fractions are concentrated by
UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,
cut-off 10 kD/Millipore). The final diafiltration step is performed
against histidine buffer, pH 7.2 containing 150 mM NaCl. The
preparation is analytically characterized by measuring total
protein (Bradford) and biological activity biological activity
according to methods known in the art. For the PEG-EPO conjugate a
specific activity of >50% in comparison to native EPO is
determined. The conjugate is additionally analytically
characterized by Size Exclusion HPLC using a Agilent 1200 HPLC
system equipped with a Shodex KW 803 column under conditions as
previously described (Kolarich et al, Transfusion 2006;
46:1959-77). It is shown that the preparation contains no free
EPO.
Method 2:
[0536] EPO is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan).
[0537] EPO is transferred or dissolved in reaction buffer (e.g. 50
mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous HCl solution. Subsequently a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0538] The oxidized EPO is further purified by ion exchange
chromatography. The oxidized EPO containing fractions of the eluate
are collected and used for the conjugation reaction.
[0539] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized EPO within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. in the dark at a
temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0540] The obtained PEG-EPO conjugate is further purified by ion
exchange chromatography. The PEG-EPO conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose with an appropriate
molecular weight cut off (Millipore).
[0541] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0542] EPO is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
EPO is dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and mixed with an aqueous
sodium periodate solution (10 mM), and an aqueous m-toluidine
solution (50 mM). Subsequently, the aminooxy reagent is added to
give a 20-fold molar reagent excess. The mixture is incubated for 2
h in the dark at room temperature under gentle stirring and
quenched for 15 min at room temperature by the addition of 8 .mu.l
of aqueous cysteine solution (1 M).
[0543] Finally, the PEG-EPO conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded
on the column pre equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes
buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and
is then subjected to UF/DF using a membrane. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0544] In an alternative embodiment, Method 3 is carried out as
follows. EPO is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). 10 mg EPO is dissolved in .about.8 ml histidine buffer, pH
6.0 (20 mM L-histidine, 150 mM NaCl). 200 .mu.l of an aqueous
sodium periodate solution (5 mM) and 2 ml of an aqueous m-toluidine
solution (50 mM) are then added. Subsequently, the aminooxy-PEG
reagent with a MW of 20 kD (described above) is added to give a
5-fold molar reagent excess. The mixture is incubated for 2 h in
the dark at room temperature under gentle stirring and quenched for
15 min at room temperature by the addition of 100 .mu.l of 1 M
aqueous cysteine solution.
[0545] Finally, the PEG-EPO conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. The reaction mixture is diluted
with 20 ml Buffer A (50 mM Hepes, pH 7.5) and loaded onto a 20 ml
HiPrep QFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with
Buffer B (50 mM Hepes, 1 M NaCl, pH 7.5). Free EPO is eluted by
washing the column with 25% Buffer B and the conjugate at 50%
Buffer B. The conjugate containing fractions are concentrated by
UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,
cut-off 10 kD/Millipore). The final diafiltration step is performed
against histidine buffer, pH 7.2 containing 150 mM NaCl. The
preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art. For the PEG-EPO conjugate a specific activity of
>50% in comparison to native EPO is determined. The conjugate is
additionally analytically characterized by Size Exclusion HPLC
using a Agilent 1200 HPLC system equipped with a Shodex KW 803
column under conditions as previously described (Kolarich et al,
Transfusion 2006; 46:1959-77). It is shown that the preparation
contains no free EPO.
Method 4:
[0546] EPO is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan). An
initial concentration or weight of EPO is transferred or dissolved
in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) to get a final protein concentration of 2 mg
EPO/ml. Subsequently an 5 mM aqueous sodium periodate solution is
added within 15 minutes to give a final concentration of 100 .mu.M,
followed by addition of an 50 mM aqueous m-toluidine solution to
get a final concentration of 10 mM within a time period of 30
minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of a 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0547] The PEG-EPO conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm.sup.2, cut-off 10 kD/Millipore). The
final diafiltration step is performed against Hepes buffer (50 mM
Hepes, 5 mM CaCl.sub.2), pH 7.5).
[0548] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 33
PEGylation of Ang-2 Using an Aminooxy-PEG Reagent and m-Toluidine
as a Nucleophilic Catalyst
Method 1:
[0549] Ang-2 is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Ang-2 is dissolved in 7.0 ml histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0550] The retentate containing oxidized Ang-2 is next mixed with
an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0551] Finally, the PEG-Ang-2 conjugate is purified by ion-exchange
chromatography (e.g. on Q Sepharose FF). For example, 1.5 mg
protein/ml gel is loaded on the column equilibrated with 50 mM
Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted
with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0552] In an alternative embodiment, Method 1 is carried out as
follows. Ang-2 is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Ang-2 is dissolved in 7.0 ml histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0553] The retentate containing oxidized Ang-2 is next mixed with
an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0554] Finally, the PEG-Ang-2 conjugate is purified by ion-exchange
chromatography. The conjugate containing fraction of the eluate are
collected and then subjected to UF/DF using an appropriate MW
cutoff membrane. The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological
activity according to methods known in the art.
Method 2:
[0555] Ang-2 is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan).
[0556] Ang-2 is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5N aqueous HCl solution. Subsequently a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0557] The oxidized Ang-2 is further purified by ion exchange
chromatography. The oxidized Ang-2 containing fractions of the
eluate are collected and used for the conjugation reaction.
[0558] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized Ang-2 within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. in the dark at a
temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0559] The obtained PEG-Ang-2 conjugate is further purified by ion
exchange chromatography. The PEG-Ang-2 conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
[0560] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0561] Ang-2 is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Ang-2 is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0562] Finally, the PEG-Ang-2 conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded
on the column pre equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes
buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and
is then subjected to UF/DF using a membrane. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0563] In an alternative embodiment, Method 3 is carried out as
follows. Ang-2 is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Ang-2 is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0564] Finally the PEG-Ang-2 conjugate is purified by ion-exchange
chromatography The conjugate containing reactions of the eluate are
collected and then subjected to UF/DF. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
Method 4:
[0565] Ang-2 is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of Ang-2 is transferred
or dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride,
5 mM calcium chloride, pH 6.0) to get a final protein concentration
of 2 mg Ang-2/ml. Subsequently an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration
of 100 .mu.M, followed by addition of an 50 mM aqueous m-toluidine
solution to get a final concentration of 10 mM within a time period
of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of an 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0566] The PEG-Ang-2 conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes,
5 mM CaCl2, pH 7.5).
[0567] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
[0568] Subsequently, the free Ang-2 is removed by means of ion
exchange chromatography (IEC). The conjugate containing fractions
of the eluate are concentrated by UF/DF.
Example 34
PEGylation of VEGF Using an Aminooxy-PEG Reagent and m-Toluidine as
a Nucleophilic Catalyst
Method 1:
[0569] VEGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). VEGF is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated
for 1 h in the dark at 4.degree. C. under gentle stirring and
quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0570] The retentate containing oxidized VEGF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0571] Finally, the PEG-VEGF conjugate is purified by ion-exchange
chromatography (e.g., on Q Sepharose FF). For example, 1.5 mg
protein/ml gel is loaded on the column equilibrated with 50 mM
Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted
with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0572] In an alternative embodiment, Method 1 is carried out as
follows. VEGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). VEGF is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated
for 1 h in the dark at 4.degree. C. under gentle stirring and
quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0573] The retentate containing oxidized VEGF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0574] Finally, the PEG-VEGF conjugate is purified by ion-exchange
chromatography The conjugate containing fractions of the eluate are
collected and then subjected to UF/DF using an appropriate MW
cutoff membrane. The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological
activity according to methods known in the art.
Method 2:
[0575] VEGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). VEGF is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5 N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0576] The oxidized VEGF is further purified by ion exchange
chromatography. The oxidized VEGF containing fractions of the
eluate are collected and used for the conjugation reaction.
[0577] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized VEGF within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. in the dark at a
temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0578] The obtained PEG-VEGF conjugate is further purified by ion
exchange chromatography. The PEG-VEGF conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
[0579] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0580] VEGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). VEGF is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently, the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0581] Finally, the PEG-VEGF conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded
on the column pre equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes
buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and
is then subjected to UF/DF using a membrane. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0582] In an alternative embodiment, Method 3 is carried out as
follows. VEGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). VEGF is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently, the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0583] Finally, the PEG-VEGF conjugate is purified by ion-exchange
chromatography. The conjugate conjugate fractions of the eluate are
collected and then subjected to UF/DF. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
Method 4:
[0584] VEGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of VEGF is transferred
or dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride,
5 mM calcium chloride, pH 6.0) to get a final protein concentration
of 2 mg VEGF/ml. Subsequently, an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration
of 100 .mu.M, followed by addition of an 50 mM aqueous m-toluidine
solution to get a final concentration of 10 mM within a time period
of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of an 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0585] The PEG-VEGF conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes,
5 mM CaCl2, pH 7.5).
[0586] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 35
PEGylation of EGF Using an Aminooxy-PEG Reagent and m-Toluidine as
a Nucleophilic Catalyst
Method 1:
[0587] EGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
EGF is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated
for 1 h in the dark at 4.degree. C. under gentle stirring and
quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0588] The retentate containing oxidized EGF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0589] Finally, the PEG-EGF conjugate is purified by ion-exchange
chromatography (e.g., on Q Sepharose FF). For example, 1.5 mg
protein/ml gel is loaded on the column equilibrated with 50 mM
Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted
with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0590] In an alternative embodiment, Method 1 is carried out as
follows. EGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). EGF is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated
for 1 h in the dark at 4.degree. C. under gentle stirring and
quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0591] The retentate containing oxidized EGF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0592] Finally, the PEG-EGF conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions of the eluate
are collected and then subjected to UF/DF using an appropriate MW
cutoff membrane. The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological
activity according to methods known in the art.
Method 2:
[0593] EGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
EGF is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5
N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0594] The oxidized EGF is further purified by ion exchange
chromatography. The oxidized EGF containing fractions of the eluate
are collected and used for the conjugation reaction.
[0595] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized NGF within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. in the dark at a
temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0596] The obtained PEG-EGF conjugate is further purified by ion
exchange chromatography. The PEG-EGF conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose with an appropriate
molecular weight cut off (Millipore).
[0597] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0598] EGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
EGF is dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and mixed with an aqueous
sodium periodate solution (10 mM), and an aqueous m-toluidine
solution (50 mM). Subsequently the aminooxy reagent is added to
give a 20-fold molar reagent excess. The mixture is incubated for 2
h in the dark at room temperature under gentle stirring and
quenched for 15 min at room temperature by the addition of 8 .mu.l
of aqueous cysteine solution (1 M).
[0599] Finally, the PEG-EGF conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded
on the column pre equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes
buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and
is then subjected to UF/DF using a membrane. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0600] In an alternative embodiment, Method 3 is carried out as
follows. EGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). EGF is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0601] Finally, the PEG-EGF conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions of the eluate
are collected and then subjected to UF/DF. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
Method 4:
[0602] EGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan). An
initial concentration or weight of EGF is transferred or dissolved
in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) to get a final protein concentration of 2 mg
EGF/ml. Subsequently an 5 mM aqueous sodium periodate solution is
added within 15 minutes to give a final concentration of 100 .mu.M,
followed by addition of an 50 mM aqueous m-toluidine solution to
get a final concentration of 10 mM within a time period of 30
minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of an 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0603] The PEG-EGF conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes,
5 mM CaCl2, pH 7.5).
[0604] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 36
PEGylation of NGF Using an Aminooxy-PEG Reagent and m-Toluidine as
a Nucleophilic Catalyst
Method 1:
[0605] NGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
NGF is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated
for 1 h in the dark at 4.degree. C. under gentle stirring and
quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0606] The retentate containing oxidized NGF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0607] Finally, the PEG-NGF conjugate is purified by ion-exchange
chromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg
protein/ml gel is loaded on the column equilibrated with 50 mM
Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted
with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0608] In an alternative embodiment, Method 1 is carried out as
follows. NGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). NGF is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated
for 1 h in the dark at 4.degree. C. under gentle stirring and
quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0609] The retentate containing oxidized NGF is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0610] Finally, the PEG-NGF conjugate is purified by ion-exchange
chromatography (The conjugate containing fractions of the eluate
are collected and then subjected to UF/DF using an appropriate MW
cutoff membrane. The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological
activity according to methods known in the art.
Method 2:
[0611] NGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
NGF is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5N
aqueous HCl solution. Subsequently a 40 mM aqueous sodium periodate
solution is added within 10 minutes to give a concentration of 200
.mu.M. The oxidation reaction is carried out for 30+/-5 min at a
temperature (T) of T=+22+/-2.degree. C. Then the reaction is
stopped by addition of an aqueous L-cysteine solution (1 M) within
15 minutes at T=+22+/-2.degree. C. to give a final concentration of
10 mM in the reaction mixture and incubation for 60+/-5 min.
[0612] The oxidized NGF is further purified by ion exchange
chromatography. The oxidized NGF containing fractions of the eluate
are collected and used for the conjugation reaction.
[0613] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized NGF within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. in the dark at a
temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0614] The obtained PEG-NGF conjugate is further purified by ion
exchange chromatography. The PEG-NGF conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose with an appropriate
molecular weight cut off (Millipore).
[0615] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0616] NGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
NGF is dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and mixed with an aqueous
sodium periodate solution (10 mM), and an aqueous m-toluidine
solution (50 mM). Subsequently the aminooxy reagent is added to
give a 20-fold molar reagent excess. The mixture is incubated for 2
h in the dark at room temperature under gentle stirring and
quenched for 15 min at room temperature by the addition of 8 .mu.l
of aqueous cysteine solution (1 M).
[0617] Finally, the PEG-NGF conjugate is purified by ion-exchange
chromatography on Q Sepharose FF. 1.5 mg protein/ml gel is loaded
on the column pre equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes
buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and
is then subjected to UF/DF using a membrane. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0618] In an alternative embodiment, Method 3 is carried out as
follows. NGF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). NGF is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0619] Finally, the PEG-NGF conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions are collected
and then subjected to UF/DF. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
Method 4:
[0620] NGF is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan). An
initial concentration or weight of NGF is transferred or dissolved
in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) to get a final protein concentration of 2 mg
NGF/ml. Subsequently an 5 mM aqueous sodium periodate solution is
added within 15 minutes to give a final concentration of 100 .mu.M,
followed by addition of an 50 mM aqueous m-toluidine solution to
get a final concentration of 10 mM within a time period of 30
minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of an 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0621] The PEG-NGF conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes,
5 mM CaCl2, pH 7.5).
[0622] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 37
PEGylation of HGH Using an Aminooxy-PEG Reagent and m-Toluidine as
a Nucleophilic Catalyst
Method 1:
[0623] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art.
[0624] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
HGH is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated
for 1 h in the dark at 4.degree. C. under gentle stirring and
quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0625] The retentate containing oxidized HGH is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0626] Finally, the PEG-HGH conjugate is purified by ion-exchange
chromatography (e.g., on Q Sepharose FF). For example, 1.5 mg
protein/ml gel is loaded on the column equilibrated with 50 mM
Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted
with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0627] In an alternative embodiment, Method 1 is carried out as
follows. As described herein, the amino acid sequence of human
growth hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art.
[0628] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
HGH is dissolved in 7.0 ml histidine buffer, pH 6.0 (20 mM
L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium periodate
solution (5 mM) is then added and the reaction mixture is incubated
for 1 h in the dark at 4.degree. C. under gentle stirring and
quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0629] The retentate containing oxidized HGH is next mixed with an
aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0630] Finally, the PEG-HGH conjugate is purified by ion-exchange
chromatography (The conjugate containing fractions of the eluate
are collected and then subjected to UF/DF using an appropriate MW
cutoff membrane. The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological
activity according to methods known in the art.
Method 2:
[0631] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art.
[0632] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
HGH is transferred or dissolved in reaction buffer (e.g. 50 mM
Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 1.0+/-0.25 mg/ml. Then the pH
of the solution is corrected to 6.0 by drop wise addition of a 0.5N
aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0633] The oxidized HGH is further purified by ion exchange
chromatography. The oxidized HGH containing fractions of the eluate
are collected and used for the conjugation reaction.
[0634] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized HGH within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. in the dark at a
temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0635] The obtained PEG-HGH conjugate is further purified by ion
exchange chromatography. The PEG-NGF conjugate containing fractions
are collected and concentrated by ultra-/diafiltration (UF/DF)
using a membrane made of regenerated cellulose with an appropriate
molecular weight cut off (Millipore).
[0636] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0637] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art.
[0638] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan).
HGH is dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium
chloride, 5 mM calcium chloride, pH 6.0) and mixed with an aqueous
sodium periodate solution (10 mM), and an aqueous m-toluidine
solution (50 mM). Subsequently the aminooxy reagent is added to
give a 20-fold molar reagent excess. The mixture is incubated for 2
h in the dark at room temperature under gentle stirring and
quenched for 15 min at room temperature by the addition of 8 .mu.l
of aqueous cysteine solution (1 M).
[0639] Finally, the PEG-HGH conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded
on the column pre equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes
buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and
is then subjected to UF/DF using a membrane. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0640] In an alternative embodiment, Method 3 is carried out as
follows. As described herein, the amino acid sequence of human
growth hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art. HGH is PEGylated by
use of a linear 20 kD PEGylation reagent containing an aminooxy
group. An example of this type of reagent is the Sunbright.RTM. CA
series from NOF (NOF Corp., Tokyo, Japan). HGH is dissolved in
Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) and mixed with an aqueous sodium periodate
solution (10 mM), and an aqueous m-toluidine solution (50 mM).
Subsequently the aminooxy reagent is added to give a 20-fold molar
reagent excess. The mixture is incubated for 2 h in the dark at
room temperature under gentle stirring and quenched for 15 min at
room temperature by the addition of 8 .mu.l of aqueous cysteine
solution (1 M).
[0641] Finally, the PEG-HGH conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions are collected
and then subjected to UF/DF. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
Method 4:
[0642] As described herein, the amino acid sequence of human growth
hormone (HGH) is first modified to incorporate at least one
glycosylation site. Following purification, HGH is glycosylated in
vitro according to methods known in the art.
[0643] HGH is PEGylated by use of a linear 20 kD PEGylation reagent
containing an aminooxy group. An example of this type of reagent is
the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo, Japan). An
initial concentration or weight of HGH is transferred or dissolved
in Hepes buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium
chloride, pH 6.0) to get a final protein concentration of 2 mg
HGH/ml. Subsequently an 5 mM aqueous sodium periodate solution is
added within 15 minutes to give a final concentration of 100 .mu.M,
followed by addition of an 50 mM aqueous m-toluidine solution to
get a final concentration of 10 mM within a time period of 30
minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of a 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0644] The PEG-HGH conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes,
5 mM CaCl2, pH 7.5).
[0645] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 38
PEGylation of TNF-Alpha Using an Aminooxy-PEG Reagent and
m-Toluidine as a Nucleophilic Catalyst
Method 1:
[0646] TNF-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). TNF-alpha is dissolved in 7.0 ml histidine buffer, pH 6.0
(20 mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0647] The retentate containing oxidized TNF-alpha is next mixed
with an aqueous m-toluidine solution (50 mM) and incubated for 30
min at room temperature. Aminooxy-PEG reagent with a MW of 20 kD is
then added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0648] Finally, the PEG-TNF-alpha conjugate is purified by
ion-exchange chromatography (e.g., on Q-Sepharose FF). For example,
1.5 mg protein/ml gel is loaded on the column equilibrated with 50
mM Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is
eluted with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM
sodium chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0649] In an alternative embodiment, Method 1 is carried out as
follows. TNF-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). TNF-alpha is dissolved in 7.0 ml histidine buffer, pH 6.0
(20 mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0650] The retentate containing oxidized TNF-alpha is next mixed
with an aqueous m-toluidine solution (50 mM) and incubated for 30
mm at room temperature. Aminooxy-PEG reagent with a MW of 20 kD is
then added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0651] Finally, the PEG-TNF-alpha conjugate is purified by
ion-exchange chromatography. The conjugate containing fractions of
the eluate are collected and then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
Method 2:
[0652] TNF-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). TNF-alpha is transferred or dissolved in reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5N aqueous HCl solution. Subsequently a 40 mM
aqueous sodium periodate solution is added within 10 minutes to
give a concentration of 200 .mu.M. The oxidation reaction is
carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2.degree. C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at
T=+22+/-2.degree. C. to give a final concentration of 10 mM in the
reaction mixture and incubation for 60+/-5 min.
[0653] The oxidized TNF-alpha is further purified by ion exchange
chromatography. The oxidized TNF-alpha containing fractions of the
eluate are collected and used for the conjugation reaction.
[0654] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized TNF alpha within a maximum time period (t) of 15 minutes
under gentle stirring. Then an aqueous m-toluidine solution (50 mM)
is added within 15 minutes to get a final concentration of 10 mM.
The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0655] The obtained PEG-TNF-alpha conjugate is further purified by
ion exchange chromatography. The PEG-TNF-alpha conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
[0656] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0657] TNF-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). TNF-alpha is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0658] Finally, the PEG-TNF-alpha conjugate is purified by
ion-exchange chromatography on Q-Sepharose FF. 1.5 mg protein/ml
gel is loaded on the column pre equilibrated with 50 mM Hepes
buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted with
50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using a membrane.
The preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
[0659] In an alternative embodiment, Method 3 is carried out as
follows. TNF-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). TNF-alpha is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently, the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0660] Finally, the PEG-TNF-alpha conjugate is purified by
ion-exchange chromatography. The conjugate containing fractions are
collected and then subjected to UF/DF. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
Method 4:
[0661] TNF-alpha is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of TNF-alpha is
transferred or dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final
protein concentration of 2 mg TNF-alpha/ml. Subsequently, an 5 mM
aqueous sodium periodate solution is added within 15 minutes to
give a final concentration of 100 .mu.M, followed by addition of an
50 mM aqueous m-toluidine solution to get a final concentration of
10 mM within a time period of 30 minutes. Then the aminooxy-PEG
reagent with a MW of 20 kD (described above) is added to give a
20-fold molar reagent excess. After correction of the pH to 6.0 the
mixture is incubated for 2 h in the dark at room temperature under
gentle stirring and quenched for 15 min at room temperature by the
addition of an 1 M aqueous L-cysteine solution to give a final
concentration of 10 mM.
[0662] The PEG-TNF-alpha conjugate is purified by means of ion
exchange chromatography (IEC). The conjugate containing fractions
of the eluate are concentrated by UF/DF using a 10 kD membrane made
of regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The
final diafiltration step is performed against Hepes buffer (50 mM
Hepes, 5 mM CaCl2, pH 7.5).
[0663] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 39
PEGylation of Insulin Using an Aminooxy-PEG Reagent and m-Toluidine
as a Nucleophilic Catalyst
Method 1:
[0664] As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art. Insulin is PEGylated by use of a
linear 20 kD PEGylation reagent containing an aminooxy group. An
example of this type of reagent is the Sunbright.RTM. CA series
from NOF (NOF Corp., Tokyo, Japan). Insulin is dissolved in 7.0 ml
histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM
CaCl2). An aqueous sodium periodate solution (5 mM) is then added
and the reaction mixture is incubated for 1 h in the dark at
4.degree. C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 .mu.l of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.
[0665] The retentate containing oxidized insulin is next mixed with
an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0666] Finally, the PEG-insulin conjugate is purified by
ion-exchange chromatography (e.g., on Q-Sepharose FF). For example,
1.5 mg protein/ml gel is loaded on the column equilibrated with 50
mM Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is
eluted with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM
sodium chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0667] In an alternative embodiment, Method 1 is carried out as
follows. As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art. Insulin is PEGylated by use of a
linear 20 kD PEGylation reagent containing an aminooxy group. An
example of this type of reagent is the Sunbright.RTM. CA series
from NOF (NOF Corp., Tokyo, Japan). Insulin is dissolved in 7.0 ml
histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM
CaCl2). An aqueous sodium periodate solution (5 mM) is then added
and the reaction mixture is incubated for 1 h in the dark at
4.degree. C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 .mu.l of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.
[0668] The retentate containing oxidized insulin is next mixed with
an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0669] Finally, the PEG-insulin conjugate is purified by
ion-exchange chromatography. The conjugate containing fractions of
the eluate are collected and then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
Method 2:
[0670] As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art.
[0671] Insulin is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Insulin is transferred or dissolved in reaction buffer
(e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) to get a final protein concentration of 1.0+/-0.25 mg/ml.
Then the pH of the solution is corrected to 6.0 by drop wise
addition of a 0.5 N aqueous HCl solution. Subsequently, a 40 mM
aqueous sodium periodate solution is added within 10 minutes to
give a concentration of 200 .mu.M. The oxidation reaction is
carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2.degree. C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at
T=+22+/-2.degree. C. to give a final concentration of 10 mM in the
reaction mixture and incubation for 60+/-5 min.
[0672] The oxidized insulin is further purified by ion exchange
chromatography. The oxidized insulin containing fractions of the
eluate are collected and used for the conjugation reaction.
[0673] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized insulin within a maximum time period (t) of 15 minutes
under gentle stirring. Then an aqueous m-toluidine solution (50 mM)
is added within 15 minutes to get a final concentration of 10 mM.
The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0674] The obtained PEG-insulin conjugate is further purified by
ion exchange chromatography. The PEG-insulin conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
[0675] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0676] As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art.
[0677] Insulin is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Insulin is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently, the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0678] Finally, the PEG-insulin conjugate is purified by
ion-exchange chromatography on Q Sepharose FF. 1.5 mg protein/ml
gel is loaded on the column pre equilibrated with 50 mM Hepes
buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted with
50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using a membrane.
The preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
[0679] In an alternative embodiment, Method 3 is carried out as
follows. As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art. Insulin is PEGylated by use of a
linear 20 kD PEGylation reagent containing an aminooxy group. An
example of this type of reagent is the Sunbright.RTM. CA series
from NOF (NOF Corp., Tokyo, Japan). Insulin is dissolved in Hepes
buffer (50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride,
pH 6.0) and mixed with an aqueous sodium periodate solution (10
mM), and an aqueous m-toluidine solution (50 mM). Subsequently the
aminooxy reagent is added to give a 20-fold molar reagent excess.
The mixture is incubated for 2 h in the dark at room temperature
under gentle stirring and quenched for 15 min at room temperature
by the addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0680] Finally, the insulin-conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions are collected
and then subjected to UF/DF. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art.
Method 4:
[0681] As described herein, the amino acid sequence of insulin is
first modified to incorporate at least one glycosylation site.
Following purification, insulin is glycosylated in vitro according
to methods known in the art.
[0682] Insulin is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of insulin is
transferred or dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) to get a final
protein concentration of 2 mg insulin/ml. Subsequently an 5 mM
aqueous sodium periodate solution is added within 15 minutes to
give a final concentration of 100 .mu.M, followed by addition of an
50 mM aqueous m-toluidine solution to get a final concentration of
10 mM within a time period of 30 minutes. Then the aminooxy-PEG
reagent with a MW of 20 kD (described above) is added to give a
20-fold molar reagent excess. After correction of the pH to 6.0 the
mixture is incubated for 2 h in the dark at room temperature under
gentle stirring and quenched for 15 min at room temperature by the
addition of a 1 M aqueous L-cysteine solution to give a final
concentration of 10 mM.
[0683] The PEG-insulin conjugate is purified by means of ion
exchange chromatography (IEC). The conjugate containing fractions
of the eluate are concentrated by UF/DF using a 10 kD membrane made
of regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The
final diafiltration step is performed against Hepes buffer (50 mM
Hepes, 5 mM CaCl2, pH 7.5).
[0684] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 40
PEGylation of Interferon-Alpha Using an Aminooxy-PEG Reagent and
m-Toluidine as a Nucleophilic Catalyst
Method 1:
[0685] Interferon-alpha is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). Interferon-alpha is dissolved in 7.0 ml
histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM
CaCl2). An aqueous sodium periodate solution (5 mM) is then added
and the reaction mixture is incubated for 1 h in the dark at
4.degree. C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 .mu.l of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.
[0686] The retentate containing oxidized interferon-alpha is next
mixed with an aqueous m-toluidine solution (50 mM) and incubated
for 30 min at room temperature. Aminooxy-PEG reagent with a MW of
20 kD is then added to give a 5-fold molar reagent excess. This
mixture is incubated for 2.5 h at room temperature in the dark
under gentle stirring.
[0687] Finally, the PEG-interferon-alpha conjugate is purified by
ion-exchange chromatography (e.g., on Q-Sepharose FF). For example,
1.5 mg protein/ml gel is loaded on the column equilibrated with 50
mM Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is
eluted with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM
sodium chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0688] In an alternative embodiment, Method 1 is carried out as
follows. Interferon-alpha is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). Interferon-alpha is dissolved in 7.0 ml
histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl, 5 mM
CaCl2). An aqueous sodium periodate solution (5 mM) is then added
and the reaction mixture is incubated for 1 h in the dark at
4.degree. C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 7.5 .mu.l of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin centrifugal filtrators to remove excess periodate,
quencher and the byproducts thereof.
[0689] The retentate containing oxidized interferon-alpha is next
mixed with an aqueous m-toluidine solution (50 mM) and incubated
for 30 min at room temperature. Aminooxy-PEG reagent with a MW of
20 kD is then added to give a 5-fold molar reagent excess. This
mixture is incubated for 2.5 h at room temperature in the dark
under gentle stirring.
[0690] Finally, the PEG-interferon-alpha conjugate is purified by
ion-exchange chromatography The conjugate containing freactions are
collected and then subjected to UF/DF using an appropriate MW
cutoff membrane. The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological
activity according to methods known in the art.
Method 2:
[0691] Interferon-alpha is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). Interferon-alpha is transferred or dissolved
in reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) to get a final protein concentration of
1.0+/-0.25 mg/ml. Then the pH of the solution is corrected to 6.0
by drop wise addition of a 0.5 N aqueous HCl solution.
Subsequently, a 40 mM aqueous sodium periodate solution is added
within 10 minutes to give a concentration of 200 .mu.M. The
oxidation reaction is carried out for 30+/-5 min at a temperature
(T) of T=+22+/-2.degree. C. Then the reaction is stopped by
addition of an aqueous L-cysteine solution (1 M) within 15 minutes
at T=+22+/-2.degree. C. to give a final concentration of 10 mM in
the reaction mixture and incubation for 60+/-5 min.
[0692] The oxidized interferon-alpha is further purified by ion
exchange chromatography. The oxidized interferon-alpha containing
fractions of the eluate are collected and used for the conjugation
reaction.
[0693] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized interferon-alpha within a maximum time period (t) of 15
minutes under gentle stirring. Then an aqueous m-toluidine solution
(50 mM) is added within 15 minutes to get a final concentration of
10 mM. The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking.
[0694] The obtained PEG-interferon-alpha conjugate is further
purified by ion exchange chromatography. The PEG-interferon alpha
conjugate containing fractions are collected and concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with an appropriate molecular weight cut off
(Millipore).
[0695] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0696] Interferon-alpha is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). Interferon-alpha is dissolved in Hepes buffer
(50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) and mixed with an aqueous sodium periodate solution (10 mM),
and an aqueous m-toluidine solution (50 mM). Subsequently the
aminooxy reagent is added to give a 20-fold molar reagent excess.
The mixture is incubated for 2 h in the dark at room temperature
under gentle stirring and quenched for 15 min at room temperature
by the addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0697] Finally, the PEG-interferon-alpha conjugate is purified by
ion-exchange chromatography on Q-Sepharose FF. 1.5 mg protein/ml
gel is loaded on the column pre equilibrated with 50 mM Hepes
buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted with
50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using a membrane.
The preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
[0698] In an alternative embodiment, Method 3 is carried out as
follows. Interferon-alpha is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). Interferon-alpha is dissolved in Hepes buffer
(50 mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH
6.0) and mixed with an aqueous sodium periodate solution (10 mM),
and an aqueous m-toluidine solution (50 mM). Subsequently the
aminooxy reagent is added to give a 20-fold molar reagent excess.
The mixture is incubated for 2 h in the dark at room temperature
under gentle stirring and quenched for 15 min at room temperature
by the addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0699] Finally, the PEG-interferon-alpha conjugate is purified by
ion-exchange chromatography. The conjugate containing fractions are
collected and then subjected to UF/DF using a membrane. The
preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
Method 4:
[0700] Interferon-alpha is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). An initial concentration or weight of
interferon-alpha is transferred or dissolved in Hepes buffer (50 mM
Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 2 mg interferon-alpha/ml.
Subsequently, an 5 mM aqueous sodium periodate solution is added
within 15 minutes to give a final concentration of 100 .mu.M,
followed by addition of an 50 mM aqueous m-toluidine solution to
get a final concentration of 10 mM within a time period of 30
minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of an 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0701] The PEG-interferon-alpha conjugate is purified by means of
ion exchange chromatography (IEC). The conjugate containing
fractions of the eluate are concentrated by UF/DF using a 10 kD
membrane made of regenerated cellulose (88 cm2, cut-off 10
kD/Millipore). The final diafiltration step is performed against
Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).
[0702] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 41
PEGylation of Interferon-Gamma Using an Aminooxy-PEG Reagent and
m-Toluidine as a Nucleophilic Catalyst
Method 1:
[0703] Interferon-gamma is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). 10 mg Interferon-gamma is dissolved in 5 ml
histidine buffer, pH 6.0 (20 mM L-histidine, 150 mM NaCl). 100
.mu.l of an aqueous sodium periodate solution (5 mM) is then added
and the reaction mixture is incubated for 1 h in the dark at
4.degree. C. under gentle stirring and quenched for 15 min at room
temperature by the addition of 50 .mu.l of a 1 M aqueous cysteine
solution. The mixture is subsequently subjected to UF/DF employing
Vivaspin 15R 10 kD centrifugal filtrators to remove excess
periodate, quencher and the byproducts thereof.
[0704] The retentate (approx. 7 ml), containing oxidized
interferon-gamma, is mixed with 2 ml of an aqueous m-toluidine
solution (50 mM) and incubated for 30 min at room temperature. Then
aminooxy-PEG reagent with a MW of 20 kD (described above) is added
to give a 5-fold molar reagent excess. This mixture is incubated
for 2.5 h at RT in the dark under gentle stirring.
[0705] Finally, the PEG-interferon-gamma conjugate is purified by
ion-exchange chromatography on SP Sepharose FF. The reaction
mixture is diluted with 20 ml Buffer A (50 mM Hepes, pH 6.5) and
loaded onto a 20 ml HiPrep SPFF 16/10 column (GE Healthcare,
Fairfield, Conn.) pre-equilibrated with Buffer A. Then the column
is eluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Free
interferon-gamma is eluted by washing the column with 25% Buffer B
and the conjugate at 50% Buffer B. The conjugate containing
fractions are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against histidine buffer, pH 6.9
containing 150 mM NaCl. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according to methods known in the art. For the
PEG-interferon-gamma conjugate a specific activity of >50% in
comparison to native Interferon gamma is determined. The conjugate
is additionally analytically characterized by Size Exclusion HPLC
using a Agilent 1200 HPLC system equipped with a Shodex KW 803
column under conditions as previously described (Kolarich et al,
Transfusion 2006; 46:1959-77). It is shown that the preparation
contains no free Interferon-gamma.
Method 2:
[0706] Interferon-gamma is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). Interferon-gamma is transferred or dissolved
in reaction buffer (e.g. 50 mM Hepes, 350 mM sodium chloride, 5 mM
calcium chloride, pH 6.0) to get a final protein concentration of
1.0+/-0.25 mg/ml. Then the pH of the solution is corrected to 6.0
by drop wise addition of a 0.5 N aqueous HCl solution. Subsequently
a 40 mM aqueous sodium periodate solution is added within 10
minutes to give a concentration of 200 .mu.M. The oxidation
reaction is carried out for 30+/-5 min at a temperature (T) of
T=+22+/-2.degree. C. Then the reaction is stopped by addition of an
aqueous L-cysteine solution (1 M) within 15 minutes at
T=+22+/-2.degree. C. to give a final concentration of 10 mM in the
reaction mixture and incubation for 60+/-5 min.
[0707] The oxidized interferon-gamma is further purified by ion
exchange chromatography. The oxidized interferon-gamma containing
fractions of the eluate are collected and used for the conjugation
reaction.
[0708] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized interferon-gamma within a maximum time period (t) of 15
minutes under gentle stirring. Then an aqueous m-toluidine solution
(50 mM) is added within 15 minutes to get a final concentration of
10 mM. The reaction mixture is incubated for 120+/-10 min. in the
dark at a temperature (T) of T=+22+/-2.degree. C. under gentle
shaking.
[0709] The obtained PEG-interferon-gamma conjugate is further
purified by ion exchange chromatography. The PEG-interferon-gamma
conjugate containing fractions are collected and concentrated by
ultra-/diafiltration (UF/DF) using a membrane made of regenerated
cellulose with an appropriate molecular weight cut off
(Millipore).
[0710] The conjugate prepared by use of this procedure are
analytically characterized by measuring total protein and
biological activity according to methods known in the art.
Method 3:
[0711] Interferon-gamma is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). 10 mg interferon-gamma is dissolved in
.about.8 ml histidine-buffer, pH 6.0 (20 mM L-histidine, 150 mM
NaCl). 200 .mu.l of an aqueous sodium periodate solution (5 mM) and
2 ml of an aqueous m-toluidine solution (50 mM) are then added.
Subsequently the aminooxy-PEG reagent with a MW of 20 kD (described
above) is added to give a 5-fold molar reagent excess. The mixture
is incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 100 .mu.l of 1 M aqueous cysteine solution.
[0712] Finally the PEG-interferon-gamma conjugate is purified by
ion-exchange chromatography on SP-Sepharose FF. The reaction
mixture is diluted with 20 ml Buffer A (50 mM Hepes, pH 6.5) and
loaded onto a 20 ml HiPrep SP FF 16/10 column (GE Healthcare,
Fairfield, Conn.) pre-equilibrated with Buffer A. Then the column
is eluted with Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Free
intergferon-gamma is eluted by washing the column with 25% Buffer B
and the conjugate at 50% Buffer B. The conjugate containing
fractions are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against histidine buffer, pH 6.9
containing 150 mM NaCl. The preparation is analytically
characterized by measuring total protein (Bradford) and biological
activity according according to methods known in the art. For the
PEG-interferon-gamma conjugate a specific activity of >50% in
comparison to native interferon-gamma is determined. The conjugate
is additionally analytically characterized by Size Exclusion HPLC
using a Agilent 1200 HPLC system equipped with a Shodex KW 803
column under conditions as previously described (Kolarich et al,
Transfusion 2006; 46:1959-77). It is shown that the preparation
contains no free interferon-gamma.
Method 4:
[0713] Interferon-gamma is PEGylated by use of a linear 20 kD
PEGylation reagent containing an aminooxy group. An example of this
type of reagent is the Sunbright.RTM. CA series from NOF (NOF
Corp., Tokyo, Japan). An initial concentration or weight of
interferon-gamma is transferred or dissolved in Hepes buffer (50 mM
Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) to
get a final protein concentration of 2 mg interferon-gamma/ml.
Subsequently an 5 mM aqueous sodium periodate solution is added
within 15 minutes to give a final concentration of 100 .mu.M,
followed by addition of an 50 mM aqueous m-toluidine solution to
get a final concentration of 10 mM within a time period of 30
minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of an 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0714] The PEG-interferon-gamma conjugate is purified by means of
ion exchange chromatography (IEC). The conjugate containing
fractions of the eluate are concentrated by UF/DF using a 10 kD
membrane made of regenerated cellulose (88 cm2, cut-off 10
kD/Millipore). The final diafiltration step is performed against
Hepes buffer (50 mM Hepes, 5 mM CaCl2, pH 7.5).
[0715] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 42
PEGylation of G-CSF Using an Aminooxy-PEG Reagent and m-Toluidine
as a Nucleophilic Catalyst
Method 1:
[0716] G-CSF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). G-CSF is dissolved in 7.0 ml histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0717] The retentate containing oxidized G-CSF is next mixed with
an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0718] Finally, the PEG-G-CSF conjugate is purified by ion-exchange
chromatography (e.g., on Q-Sepharose FF). For example, 1.5 mg
protein/ml gel is loaded on the column equilibrated with 50 mM
Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted
with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0719] In an alternative embodiment, Method 1 is carried out as
follows. G-CSF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). G-CSF is dissolved in 7.0 ml histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0720] The retentate containing oxidized G-CSF is next mixed with
an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0721] Finally, the PEG-G-CSF conjugate is purified by ion-exchange
chromatography (The conjugate containing fractions of the eluate
are collected and then subjected to UF/DF using an appropriate MW
cutoff membrane. The preparation is next analytically characterized
by measuring total protein (Coomassie, Bradford) and biological
activity according to methods known in the art.
Method 2:
[0722] G-CSF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). G-CSF is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5N aqueous HCl solution. Subsequently a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0723] The oxidized G-CSF is further purified by ion exchange
chromatography. The oxidized G-CSF containing fractions of the
eluate are collected and used for the conjugation reaction.
[0724] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized G-CSF within a maximum time period (t) of 15 minutes under
gentle stirring. Then an aqueous m-toluidine solution (50 mM) is
added within 15 minutes to get a final concentration of 10 mM. The
reaction mixture is incubated for 120+/-10 min. in the dark at a
temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0725] The obtained PEG-G-CSF conjugate is further purified by ion
exchange chromatography. The PEG-G-CSF conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
Method 3:
[0726] G-CSF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). G-CSF is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently, the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0727] Finally, the PEG-G-CSF conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. 1.5 mg protein/ml gel is loaded
on the column pre equilibrated with 50 mM Hepes buffer, pH 7.4
containing 5 mM CaCl2. The conjugate is eluted with 50 mM Hepes
buffer containing 5 mM CaCl2 and 500 mM sodium chloride, pH 7.4 and
is then subjected to UF/DF using a membrane. The preparation is
analytically characterized by measuring total protein (Bradford)
and biological activity according to methods known in the art.
[0728] In an alternative embodiment, Method 3 is carried out as
follows. G-CSF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). G-CSF is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently, the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0729] Finally, the PEG-G-CSF conjugate is purified by ion-exchange
chromatography. The conjugate containing fractions of the eluate
are collected and then subjected to UF/DF using a membrane. The
preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
Method 4:
[0730] G-CSF is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of G-CSF is transferred
or dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride,
5 mM calcium chloride, pH 6.0) to get a final protein concentration
of 2 mg G-CSF/ml. Subsequently, an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration
of 100 .mu.M, followed by addition of an 50 mM aqueous m-toluidine
solution to get a final concentration of 10 mM within a time period
of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of an 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0731] The G-CSF conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes,
5 mM CaCl2, pH 7.5).
[0732] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 43
PEGylation of Humira Using an Aminooxy-PEG Reagent and m-Toluidine
as a Nucleophilic Catalyst
Method 1:
[0733] Humira is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Humira is dissolved in 7.0 ml histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0734] The retentate containing oxidized Humira is next mixed with
an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0735] Finally, the PEG-Humira conjugate is purified by
ion-exchange chromatography (e.g., on Q-Sepharose FF). For example,
1.5 mg protein/ml gel is loaded on the column equilibrated with 50
mM Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is
eluted with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM
sodium chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0736] In an alternative embodiment, Method 1 is carried out as
follows. Humira is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Humira is dissolved in 7.0 ml histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0737] The retentate containing oxidized Humira is next mixed with
an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0738] Finally, the PEG-Humira conjugate is purified by
ion-exchange chromatography. The conjugate containing fractions of
the eluate are collected and then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
Method 2:
[0739] Humira is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Humira is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5N aqueous HCl solution. Subsequently a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0740] The oxidized Humira is further purified by ion exchange
chromatography. The oxidized Humira containing fractions of the
eluate are collected and used for the conjugation reaction.
[0741] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized Humira within a maximum time period (t) of 15 minutes
under gentle stirring. Then an aqueous m-toluidine solution (50 mM)
is added within 15 minutes to get a final concentration of 10 mM.
The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0742] The obtained PEG-Humira conjugate is further purified by ion
exchange chromatography. The PEG-Humira conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
Method 3:
[0743] Humira is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Humira is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently, the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0744] Finally, the PEG-Humira conjugate is purified by
ion-exchange chromatography on Q-Sepharose FF. 1.5 mg protein/ml
gel is loaded on the column pre equilibrated with 50 mM Hepes
buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted with
50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using a membrane.
The preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
[0745] In an alternative embodiment, Method 3 is carried out as
follows. Humira is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Humira is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0746] Finally, the PEG-Humira conjugate is purified by
ion-exchange chromatography. The conjugate containing fractions are
collected and then subjected to UF/DF using a membrane. The
preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
Method 4:
[0747] Humira is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of Humira is transferred
or dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride,
5 mM calcium chloride, pH 6.0) to get a final protein concentration
of 2 mg Humira/ml. Subsequently an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration
of 100 .mu.M, followed by addition of an 50 mM aqueous m-toluidine
solution to get a final concentration of 10 mM within a time period
of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of a 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0748] The Humira conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes,
5 mM CaCl2, pH 7.5).
[0749] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 44
PEGylation of Prolia Using an Aminooxy-PEG Reagent and m-Toluidine
as a Nucleophilic Catalyst
Method 1:
[0750] Prolia is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Prolia is dissolved in 7.0 ml histidine buffer, pH 6.0 (20
mM L-histidine, 150 mM NaCl, 5 mM CaCl2). An aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 7.5
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin centrifugal
filtrators to remove excess periodate, quencher and the byproducts
thereof.
[0751] The retentate containing oxidized Prolia is next mixed with
an aqueous m-toluidine solution (50 mM) and incubated for 30 min at
room temperature. Aminooxy-PEG reagent with a MW of 20 kD is then
added to give a 5-fold molar reagent excess. This mixture is
incubated for 2.5 h at room temperature in the dark under gentle
stirring.
[0752] Finally, the PEG-Prolia conjugate is purified by
ion-exchange chromatography (e.g., on Q-Sepharose FF). For example,
1.5 mg protein/ml gel is loaded on the column equilibrated with 50
mM Hepes buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is
eluted with 50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM
sodium chloride, pH 7.4 and is then subjected to UF/DF using an
appropriate MW cutoff membrane. The preparation is next
analytically characterized by measuring total protein (Coomassie,
Bradford) and biological activity according to methods known in the
art.
[0753] In an alternative embodiment, Method 1 is carried out as
follows. Prolia is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). 10 mg rFIX is dissolved in 5 ml histidine-buffer, pH 6.0
(20 mM L-histidine, 150 mM NaCl). 100 .mu.l of an aqueous sodium
periodate solution (5 mM) is then added and the reaction mixture is
incubated for 1 h in the dark at 4.degree. C. under gentle stirring
and quenched for 15 min at room temperature by the addition of 50
.mu.l of a 1 M aqueous cysteine solution. The mixture is
subsequently subjected to UF/DF employing Vivaspin 15R 10 kD
centrifugal filtrators to remove excess periodate, quencher and the
byproducts thereof.
[0754] The retentate (approx. 7 ml), containing oxidized Prolia, is
mixed with 2 ml of an aqueous m-toluidine solution (50 mM) and
incubated for 30 min at room temperature. Then aminooxy-PEG reagent
with a MW of 20 kD (described above) is added to give a 5-fold
molar reagent excess. This mixture is incubated for 2.5 h at RT in
the dark under gentle stirring.
[0755] Finally the PEG-Prolia conjugate is purified by ion-exchange
chromatography on SP Sepharose FF. The reaction mixture is diluted
with 20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml
HiPrep SP FF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with
Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Free Prolia is eluted by
washing the column with 25% Buffer B and the conjugate at 50%
Buffer B. The conjugate containing fractions are concentrated by
UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,
cut-off 10 kD/Millipore). The final diafiltration step is performed
against histidine buffer, pH 6.9 containing 150 mM NaCl. The
preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art. For the PEG-Prolia conjugate a specific activity
of >50% in comparison to native Prolia is determined. The
conjugate is additionally analytically characterized by Size
Exclusion HPLC using a Agilent 1200 HPLC system equipped with a
Shodex KW 803 column under conditions as previously described
(Kolarich et al, Transfusion 2006; 46:1959-77). It is shown that
the preparation contains no free Prolia.
Method 2:
[0756] Prolia is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). Prolia is transferred or dissolved in reaction buffer (e.g.
50 mM Hepes, 350 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
to get a final protein concentration of 1.0+/-0.25 mg/ml. Then the
pH of the solution is corrected to 6.0 by drop wise addition of a
0.5N aqueous HCl solution. Subsequently, a 40 mM aqueous sodium
periodate solution is added within 10 minutes to give a
concentration of 200 .mu.M. The oxidation reaction is carried out
for 30+/-5 min at a temperature (T) of T=+22+/-2.degree. C. Then
the reaction is stopped by addition of an aqueous L-cysteine
solution (1 M) within 15 minutes at T=+22+/-2.degree. C. to give a
final concentration of 10 mM in the reaction mixture and incubation
for 60+/-5 min.
[0757] The oxidized Prolia is further purified by ion exchange
chromatography. The oxidized Humira containing fractions of the
eluate are collected and used for the conjugation reaction.
[0758] The aminooxy-PEG reagent with a MW of 20 kD reagent is added
in a 50-fold molar excess to the eluate containing the purified
oxidized Prolia within a maximum time period (t) of 15 minutes
under gentle stirring. Then an aqueous m-toluidine solution (50 mM)
is added within 15 minutes to get a final concentration of 10 mM.
The reaction mixture is incubated for 120+/-10 min. in the dark at
a temperature (T) of T=+22+/-2.degree. C. under gentle shaking.
[0759] The obtained PEG-Prolia conjugate is further purified by ion
exchange chromatography. The PEG-Prolia conjugate containing
fractions are collected and concentrated by ultra-/diafiltration
(UF/DF) using a membrane made of regenerated cellulose with an
appropriate molecular weight cut off (Millipore).
Method 3:
[0760] Prolia is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). EPO is dissolved in Hepes buffer (50 mM Hepes, 150 mM
sodium chloride, 5 mM calcium chloride, pH 6.0) and mixed with an
aqueous sodium periodate solution (10 mM), and an aqueous
m-toluidine solution (50 mM). Subsequently the aminooxy reagent is
added to give a 20-fold molar reagent excess. The mixture is
incubated for 2 h in the dark at room temperature under gentle
stirring and quenched for 15 min at room temperature by the
addition of 8 .mu.l of aqueous cysteine solution (1 M).
[0761] Finally, the PEG-Prolia conjugate is purified by
ion-exchange chromatography on Q-Sepharose FF. 1.5 mg protein/ml
gel is loaded on the column pre equilibrated with 50 mM Hepes
buffer, pH 7.4 containing 5 mM CaCl2. The conjugate is eluted with
50 mM Hepes buffer containing 5 mM CaCl2 and 500 mM sodium
chloride, pH 7.4 and is then subjected to UF/DF using a membrane.
The preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art.
[0762] In an alternative embodiment, Method 3 is carried out as
follows.
[0763] Prolia is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). 10 mg Prolia is dissolved in .about.8 ml histidine buffer,
pH 6.0 (20 mM L-histidine, 150 mM NaCl). 200 .mu.l of an aqueous
sodium periodate solution (5 mM) and 2 ml of an aqueous m-toluidine
solution (50 mM) are then added. Subsequently, the aminooxy-PEG
reagent with a MW of 20 kD (described above) is added to give a
5-fold molar reagent excess. The mixture is incubated for 2 h in
the dark at room temperature under gentle stirring and quenched for
15 min at room temperature by the addition of 100 .mu.l of 1 M
aqueous cysteine solution.
[0764] Finally the PEG-Prolia conjugate is purified by ion-exchange
chromatography on SP-Sepharose FF. The reaction mixture is diluted
with 20 ml Buffer A (50 mM Hepes, pH 6.5) and loaded onto a 20 ml
HiPrep SPFF 16/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with
Buffer B (50 mM Hepes, 1 M NaCl, pH 6.5). Free Prolia is eluted by
washing the column with 25% Buffer B and the conjugate at 50%
Buffer B. The conjugate containing fractions are concentrated by
UF/DF using a 10 kD membrane made of regenerated cellulose (88 cm2,
cut-off 10 kD/Millipore). The final diafiltration step is performed
against histidine buffer, pH 6.9 containing 150 mM NaCl. The
preparation is analytically characterized by measuring total
protein (Bradford) and biological activity according to methods
known in the art. For the PEG-Prolia conjugate a specific activity
of >50% in comparison to native Prolia is determined. The
conjugate is additionally analytically characterized by Size
Exclusion HPLC using a Agilent 1200 HPLC system equipped with a
Shodex KW 803 column under conditions as previously described
(Kolarich et al, Transfusion 2006; 46:1959-77). It is shown that
the preparation contains no free Prolia.
Method 4:
[0765] Prolia is PEGylated by use of a linear 20 kD PEGylation
reagent containing an aminooxy group. An example of this type of
reagent is the Sunbright.RTM. CA series from NOF (NOF Corp., Tokyo,
Japan). An initial concentration or weight of Humira is transferred
or dissolved in Hepes buffer (50 mM Hepes, 150 mM sodium chloride,
5 mM calcium chloride, pH 6.0) to get a final protein concentration
of 2 mg Prolia/ml. Subsequently an 5 mM aqueous sodium periodate
solution is added within 15 minutes to give a final concentration
of 100 .mu.M, followed by addition of an 50 mM aqueous m-toluidine
solution to get a final concentration of 10 mM within a time period
of 30 minutes. Then the aminooxy-PEG reagent with a MW of 20 kD
(described above) is added to give a 20-fold molar reagent excess.
After correction of the pH to 6.0 the mixture is incubated for 2 h
in the dark at room temperature under gentle stirring and quenched
for 15 min at room temperature by the addition of an 1 M aqueous
L-cysteine solution to give a final concentration of 10 mM.
[0766] The Prolia conjugate is purified by means of ion exchange
chromatography (IEC). The conjugate containing fractions of the
eluate are concentrated by UF/DF using a 10 kD membrane made of
regenerated cellulose (88 cm2, cut-off 10 kD/Millipore). The final
diafiltration step is performed against Hepes buffer (50 mM Hepes,
5 mM CaCl2, pH 7.5).
[0767] The preparation is analytically characterized by measuring
total protein (Bradford and BCA procedure) and biological activity
according to known methods.
Example 45
PEGylation of a Therapeutic Protein Using Branched PEG
[0768] PEGylation of a therapeutic protein of the invention may be
extended to a branched or linear PEGylation reagent, which is made
of an aldehyde and a suitable linker containing an active aminooxy
group.
Sequence CWU 1
1
11422PRTHomo sapiens 1Leu Asn Arg Pro Lys Arg Tyr Asn Ser Gly Lys
Leu Glu Glu Phe Val1 5 10 15Gln Gly Asn Leu Glu Arg Glu Cys Met Glu
Glu Lys Cys Ser Phe Glu 20 25 30Glu Pro Arg Glu Val Phe Glu Asn Thr
Glu Lys Thr Thr Glu Phe Trp 35 40 45Lys Gln Tyr Val Asp Gly Asp Gln
Cys Glu Ser Asn Pro Cys Leu Asn 50 55 60Gly Gly Ser Cys Lys Asp Asp
Ile Asn Ser Tyr Glu Cys Trp Cys Pro65 70 75 80Phe Gly Phe Glu Gly
Lys Asn Cys Glu Leu Asp Val Thr Cys Asn Ile 85 90 95Lys Asn Gly Arg
Cys Glu Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys 100 105 110Val Val
Cys Ser Cys Thr Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys 115 120
125Ser Cys Glu Pro Ala Val Pro Phe Pro Cys Gly Arg Val Ser Val Ser
130 135 140Gln Thr Ser Lys Leu Thr Arg Ala Glu Ala Val Phe Pro Asp
Val Asp145 150 155 160Tyr Val Asn Pro Thr Glu Ala Glu Thr Ile Leu
Asp Asn Ile Thr Gln 165 170 175Gly Thr Gln Ser Phe Asn Asp Phe Thr
Arg Val Val Gly Gly Glu Asp 180 185 190Ala Lys Pro Gly Gln Phe Pro
Trp Gln Val Val Leu Asn Gly Lys Val 195 200 205Asp Ala Phe Cys Gly
Gly Ser Ile Val Asn Glu Lys Trp Ile Val Thr 210 215 220Ala Ala His
Cys Val Glu Thr Gly Val Lys Ile Thr Val Val Ala Gly225 230 235
240Glu His Asn Ile Glu Glu Thr Glu His Thr Glu Gln Lys Arg Asn Val
245 250 255Ile Arg Ala Ile Ile Pro His His Asn Tyr Asn Ala Ala Ile
Asn Lys 260 265 270Tyr Asn His Asp Ile Ala Leu Leu Glu Leu Asp Glu
Pro Leu Val Leu 275 280 285Asn Ser Tyr Val Thr Pro Ile Cys Ile Ala
Asp Lys Glu Tyr Thr Asn 290 295 300Ile Phe Leu Lys Phe Gly Ser Gly
Tyr Val Ser Gly Trp Ala Arg Val305 310 315 320Phe His Lys Gly Arg
Ser Ala Leu Val Leu Gln Tyr Leu Arg Val Pro 325 330 335Leu Val Asp
Arg Ala Thr Cys Leu Arg Ser Thr Lys Phe Thr Ile Tyr 340 345 350Asn
Asn Met Phe Cys Ala Gly Phe His Glu Gly Gly Arg Asp Ser Cys 355 360
365Gln Gly Asp Ser Gly Gly Pro His Val Thr Glu Val Glu Gly Thr Ser
370 375 380Phe Leu Thr Gly Ile Ile Ser Trp Gly Glu Glu Cys Ala Met
Lys Gly385 390 395 400Lys Tyr Gly Ile Tyr Thr Lys Val Ser Arg Tyr
Val Asn Trp Ile Lys 405 410 415Glu Lys Thr Lys Leu Thr 420
* * * * *
References