U.S. patent application number 14/988931 was filed with the patent office on 2016-05-05 for blood coagulation protein conjugates.
The applicant listed for this patent is BAXALTA GMBH, BAXALTA INCORPORATED. Invention is credited to Stefan Haider, Hanspeter Rottensteiner, Juergen Siekmann, Peter Turecek.
Application Number | 20160120994 14/988931 |
Document ID | / |
Family ID | 46582462 |
Filed Date | 2016-05-05 |
United States Patent
Application |
20160120994 |
Kind Code |
A1 |
Siekmann; Juergen ; et
al. |
May 5, 2016 |
BLOOD COAGULATION PROTEIN CONJUGATES
Abstract
The invention relates to materials and methods of conjugating a
water soluble polymer to an oxidized carbohydrate moiety of a blood
coagulation 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 linkage is formed between the oxidized carbohydrate moiety
and the active aminooxy group on the water soluble polymer. In one
embodiment of the invention the conjugation is carried out in the
presence of the nucleophilic catalyst aniline. In addition the
generated oxime linkage can be stabilized by reduction with
NaCNBH.sub.3 to form an alkoxyamine linkage.
Inventors: |
Siekmann; Juergen; (Vienna,
AT) ; Haider; Stefan; (Prinzersdorf, AT) ;
Rottensteiner; Hanspeter; (Vienna, AT) ; Turecek;
Peter; (Klosterneuburg, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAXALTA INCORPORATED
BAXALTA GMBH |
Bannockburn
Glattpark (Opfikon) |
IL |
US
CH |
|
|
Family ID: |
46582462 |
Appl. No.: |
14/988931 |
Filed: |
January 6, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14136266 |
Dec 20, 2013 |
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14988931 |
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12843542 |
Jul 26, 2010 |
8637640 |
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14136266 |
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61228828 |
Jul 27, 2009 |
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61347136 |
May 21, 2010 |
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Current U.S.
Class: |
435/188 ;
530/383; 536/53 |
Current CPC
Class: |
A61K 38/4846 20130101;
A61P 7/04 20180101; A61K 38/37 20130101; A61K 47/60 20170801; C12N
9/6437 20130101; C12N 9/644 20130101; A61P 43/00 20180101; C12Y
304/21021 20130101; C12N 9/96 20130101; A61K 47/61 20170801; C12Y
304/21022 20130101; C08B 37/0006 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 38/37 20060101 A61K038/37; C12N 9/96 20060101
C12N009/96; A61K 38/48 20060101 A61K038/48; C12N 9/64 20060101
C12N009/64; C08B 37/00 20060101 C08B037/00 |
Claims
1. A method of conjugating a water soluble polymer to an oxidized
carbohydrate moiety of a blood coagulation protein comprising
contacting the oxidized carbohydrate moiety with an activated water
soluble polymer under conditions that allow conjugation; 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; 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(1-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 (NaIO.sub.4), lead tetraacetate (Pb(OAc).sub.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.
2. The method according to claim 1 wherein the water soluble
polymer is PSA.
3. The method according to claim 2 wherein the PSA is comprised of
about 10-300 sialic acid units.
4. The method according to any one of claims 1-3 wherein the blood
coagulation protein is FIX.
5. The method according to any one of claims 1-3 wherein the blood
coagulation protein is FVIIa.
6. The method according to any one of claims 1-3 wherein the blood
coagulation protein is FVIII.
7. The method according to any one of claims 1-6 wherein the
oxidizing agent is sodium periodate (NaIO.sub.4).
8. The method according to any one of claims 4-7 wherein the
oxidized carbohydrate moiety of the blood coagulation protein is
located in the activation peptide of the blood coagulation
protein.
9. The method according to claim 2 wherein the PSA is prepared by
reacting an activated aminooxy linker with oxidized PSA; wherein
the aminooxy linker is selected from the group consisting of: a) a
3-oxa-pentane-1,5-dioxyamine linker of the formula: ##STR00010##
and b) a 3,6,9-trioxa-undecane-1,11-dioxyamine linker of the
formula: ##STR00011## 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.
10. The method according to claim 7 wherein the aminooxy linker is
3-oxa-pentane-1,5-dioxyamine.
11. The method according to claim 7 wherein the oxidizing agent is
NaIO.sub.4.
12. The method according to any one of claims 1-9 wherein the
contacting of the oxidized carbohydrate moiety with the activated
water soluble polymer occurs in a buffer comprising a nucleophilic
catalyst selected from the group consisting of aniline and aniline
derivatives.
13. The method according to claim 2 further comprising the step of
reducing an oxime linkage in the conjugated blood coagulation
protein by incubating the conjugated blood coagulation protein in a
buffer comprising a reducing compound selected from the group
consisting of sodium cyanoborohydride (NaCNBH.sub.3) and ascorbic
acid (vitamin C).
14. The method according to claim 11 wherein the reducing compound
is sodium cyanoborohydride (NaCNBH.sub.3).
15. A modified blood coagulation protein produced by the method
according to any one of claims 1-12.
16. A modified FIX comprising: (a) a FIX molecule or a biologically
active fragment, derivative or variant thereof; and (b) at least
one aminooxy PSA bound to the FIX molecule of (a), wherein said
aminooxy PSA is attached to the FIX via one or more carbohydrate
moieties.
17. A modified FVIIa comprising: (a) a FVIIa molecule or a
biologically active fragment, derivative or variant thereof; and
(b) at least one aminooxy PSA bound to the FVIIa molecule of (a),
wherein said aminooxy PSA is attached to the FVIIa via one or more
carbohydrate moieties.
18. A modified FVIII comprising: (a) a FVIII molecule or a
biologically active fragment, derivative or variant thereof; and
(b) at least one aminooxy PSA bound to the FVIII molecule of (a),
wherein said aminooxy PSA is attached to the FVIII via one or more
carbohydrate moieties.
19. A modified FIX comprising: (a) a FIX molecule or a biologically
active fragment, derivative or variant thereof; and (b) at least
one aminooxy PEG bound to the FIX molecule of (a), wherein said
aminooxy PEG is attached to the FIX via one or more carbohydrate
moieties.
20. A modified FVIIa comprising: (a) a FVIIa molecule or a
biologically active fragment, derivative or variant thereof; and
(b) at least one aminooxy PEG bound to the FVIIa molecule of (a),
wherein said aminooxy PEG is attached to the FVIIa via one or more
carbohydrate moieties.
21. A modified FVIII comprising: (a) a FVIII molecule or a
biologically active fragment, derivative or variant thereof; and
(b) at least one aminooxy PEG bound to the FVIII molecule of (a),
wherein said aminooxy PEG is attached to the FVIII via one or more
carbohydrate moieties.
22. A water soluble polymer comprising an active aminooxy linker;
said water soluble polymer 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(1-hydroxymethylethylene hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC), said
active aminooxy linker is selected from the group consisting of: a)
a 3-oxa-pentane-1,5-dioxyamine linker of the formula: ##STR00012##
and b) a 3,6,9-trioxa-undecane-1,11-dioxyamine linker of the
formula: ##STR00013##
Description
[0001] This application claims benefit to U.S. Provisional
Application Ser. No. 61/347,136, filed May 21, 2010, and U.S.
Provisional Application Ser. No. 61/228,828 filed Jul. 27, 2009,
all of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to materials and methods for
conjugating a water soluble polymer to a blood coagulation
protein.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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, Biochimica
Biophysica Acta 1341:26-34, 1997).
[0006] 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. For example,
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 et 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.
[0009] 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.
SUMMARY OF THE INVENTION
[0010] 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.
[0011] In one embodiment of the invention, a method of conjugating
a water soluble polymer to an oxidized carbohydrate moiety of a
blood coagulation protein comprising contacting the oxidized
carbohydrate moiety with an activated water soluble polymer under
conditions that allow conjugation; the 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; the 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(1-hydroxymethylethylene hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC); and the
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.
[0012] In another embodiment of the invention, the water soluble
polymer according to the aforementioned method is PSA. In a related
embodiment, the PSA is comprised of about 5-500 or 10-300 sialic
acid units. In still another embodiment, the blood coagulation
protein according to the aforementioned method is FIX. In another
embodiment, the blood coagulation protein according to the
aforementioned method is FVIIa. In still another embodiment, the
blood coagulation protein according to the aforementioned method is
FVIII. In yet another embodiment, the aforementioned method is
provided wherein the oxidizing agent is sodium periodate (NaIO4).
In another embodiment, the oxidized carbohydrate moiety of the
blood coagulation protein according to the aforementioned method is
located in the activation peptide of the blood coagulation
protein.
[0013] In yet another embodiment of the invention, the
aforementioned method is provided wherein the PSA is prepared by
reacting an activated aminooxy linker with oxidized PSA;
[0014] wherein the aminooxy linker is selected from the group
consisting of:
[0015] a 3-oxa-pentane-1,5-dioxyamine linker of the formula:
##STR00001##
and
[0016] a 3,6,9-trioxa-undecane-1,11-dioxyamine linker of the
formula:
##STR00002##
[0017] 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 still another embodiment, the aforementioned method is
provided wherein the activated aminooxy linker comprises 1-50
ethylene glycol units.
In still another embodiment, an aforementioned method is provided
wherein the aminooxy linker is 3-oxa-pentane-1,5-dioxyamine. In a
related embodiment, the oxidizing agent is NaIO.sub.4.
[0018] In another embodiment of the invention, the aforementioned
method is provided wherein the contacting of the oxidized
carbohydrate moiety with the activated water soluble polymer occurs
in a buffer comprising a nucleophilic catalyst selected from the
group consisting of aniline and aniline derivatives.
[0019] In yet another embodiment of the invention, an
aforementioned method is provided further comprising the step of
reducing an oxime linkage in the conjugated blood coagulation
protein by incubating the conjugated blood coagulation protein in a
buffer comprising a reducing compound selected from the group
consisting of sodium cyanoborohydride (NaCNBH3) and ascorbic acid
(vitamin C). In a related embodiment the reducing compound is
sodium cyanoborohydride (NaCNBH3).
[0020] In another embodiment of the invention, a modified blood
coagulation protein produced by an aforementioned method is
provided.
[0021] In still another embodiment of the invention, a modified FIX
is provided comprising a FIX molecule or a biologically active
fragment, derivative or variant thereof; and at least one aminooxy
PSA bound to the FIX molecule, wherein said aminooxy PSA is
attached to the FIX via one or more carbohydrate moieties.
[0022] In another embodiment of the invention, a modified FVIIa is
provided comprising a FVIIa molecule or a biologically active
fragment, derivative or variant thereof; and at least one aminooxy
PSA bound to the FVIIa molecule, wherein said aminooxy PSA is
attached to the FVIIa via one or more carbohydrate moieties.
[0023] In still another embodiment of the invention, a modified
FVIII is provided comprising a FVIII molecule or a biologically
active fragment, derivative or variant thereof; and at least one
aminooxy PSA bound to the FVIII molecule, wherein said aminooxy PSA
is attached to the FVIII via one or more carbohydrate moieties.
[0024] In still another embodiment of the invention, a modified FIX
is provided comprising a FIX molecule or a biologically active
fragment, derivative or variant thereof; and at least one aminooxy
PEG bound to the FIX molecule, wherein said aminooxy PEG is
attached to the FIX via one or more carbohydrate moieties.
[0025] In another embodiment of the invention, a modified FVIIa is
provided comprising a FVIIa molecule or a biologically active
fragment, derivative or variant thereof; and at least one aminooxy
PEG bound to the FVIIa molecule, wherein said aminooxy PEG is
attached to the FVIIa via one or more carbohydrate moieties.
[0026] In still another embodiment of the invention, a modified
FVIII is provided comprising a FVIII molecule or a biologically
active fragment, derivative or variant thereof; and at least one
aminooxy PEG bound to the FVIII molecule, wherein said aminooxy PEG
is attached to the FVIII via one or more carbohydrate moieties.
[0027] In yet another embodiment, a water soluble polymer is
provided comprising an active aminooxy linker; said water soluble
polymer 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, poly acryloylmorpholine, polyvinyl
alcohol (PVA), polycarboxylate, polyvinylpyrrolidone,
polyphosphazene, polyoxazoline, polyethylene-co-maleic acid
anhydride, polystyrene-co-maleic acid anhydride,
poly(1-hydroxymethylethylene hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC); said
active aminooxy linker is selected from the group consisting of: a
3-oxa-pentane-1,5-dioxyamine linker of the formula:
##STR00003##
and
[0028] a 3,6,9-trioxa-undecane-1,11-dioxyamine linker of the
formula:
##STR00004##
[0029] In still another embodiment, the aforementioned method is
provided wherein activated aminooxy linker comprises 1-50 ethylene
glycol units.
FIGURES
[0030] FIG. 1 shows the primary structure of coagulation Factor
IX.
[0031] FIG. 2 shows the coupling of oxidized rFIX to
aminooxy-PSA.
[0032] 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.
[0033] FIG. 4 shows the preparation of aminooxy-PSA.
[0034] FIG. 5 shows the analytical characterization of the PSA-rFIX
conjugate employing SDS-PAGE and Coomassie staining.
[0035] FIG. 6 shows the analytical characterization of the PSA-rFIX
conjugate employing detection with anti-FIX and anti-PSA
antibodies.
[0036] FIG. 7 shows activity of native rFIX and PSA-rFIX conjugate
relative to time post infusions.
[0037] FIG. 8 shows PSA-rFVIII and Advate levels relative to time
post infusion.
DETAILED DESCRIPTION OF THE INVENTION
[0038] 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), 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(1-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.
[0039] The addition of a soluble polymer, such as through
polysialylation is one approach to improve the properties of a
blood coagulation protein such as FIX, as well as other coagulation
proteins (e.g., VWF, FVIIa (see, e.g., US 2008/0221032A1,
incorporated herein by reference) and FVIII).
Blood Coagulation Proteins
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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/tis sue
factor/FXa product complex.
[0046] A. Polypeptides
[0047] 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. No. 4,757,006; U.S. Pat. No.
5,733,873; U.S. Pat. No. 5,198,349; U.S. Pat. No. 5,250,421; U.S.
Pat. No. 5,919,766; and EP 306 968. As described herein, the term
blood coagulation protein refers to any blood coagulation protein
molecule which exhibits biological activity that is associated with
the native blood coagulation protein. In one embodiment of the
invention, the blood coagulation protein molecule is a full-length
blood coagulation protein.
[0048] Blood coagulation 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 blood coagulation proteins. Thus, blood coagulation
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.
[0049] According to the present invention, the term "recombinant
blood coagulation protein" includes any blood coagulation protein
obtained via recombinant DNA technology. In certain embodiments,
the term encompasses proteins as described herein.
[0050] As used herein, "endogenous blood coagulation protein"
includes a blood coagulation protein which originates from the
mammal intended to receive treatment. The term also includes blood
coagulation protein transcribed from a transgene or any other
foreign DNA present in said mammal. As used herein, "exogenous
blood coagulation protein" includes a blood coagulation protein
which does not originate from the mammal intended to receive
treatment.
[0051] 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.
[0052] 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.
[0053] An "analog," "variant" or "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" refers to a polypeptide sharing the same or
substantially similar structure as a reference polypeptide that has
been modified, e.g., chemically.
[0054] Variant or analog polypeptides include insertion variants,
wherein one or more amino acid residues are added to a blood
coagulation 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 blood
coagulation 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.
[0055] In deletion variants, one or more amino acid residues in a
blood coagulation protein polypeptide as described herein are
removed. Deletions can be effected at one or both termini of the
blood coagulation protein polypeptide, and/or with removal of one
or more residues within the blood coagulation protein amino acid
sequence. Deletion variants, therefore, include fragments of a
blood coagulation protein polypeptide sequence.
[0056] In substitution variants, one or more amino acid residues of
a blood coagulation 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-00001 [0057] 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
[0058] Alternatively, exemplary conservative substitutions are set
out immediately below.
Conservative Substitutions II
TABLE-US-00002 [0059] 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
[0060] B. Polynucleotides
[0061] Nucleic acids encoding a blood coagulation protein of the
invention include, for example and without limitation, genes,
pre-mRNAs, mRNAs, cDNAs, polymorphic variants, alleles, synthetic
and naturally-occurring mutants.
[0062] Polynucleotides encoding a blood coagulation 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 42oC in 50% formamide, 5.times.SSC, 20 mM
Na.cndot.PO4, pH 6.8; and washing in 1.times.SSC at 55oC 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.
[0063] A "naturally-occurring" polynucleotide or polypeptide
sequence is typically 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).
[0064] In certain embodiments of the invention, the aforementioned
polypeptides and polynucleotides are exemplified by the following
blood coagulation proteins.
[0065] Factor VIIa
[0066] 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).
[0067] 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.
[0068] 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 NH.sub.2-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.
[0069] 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.
[0070] 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.
[0071] 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).
[0072] Factor IX
[0073] 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.
[0074] 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.
[0075] 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).
[0076] 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.
[0077] Factor VIII
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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
haemophilia 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.
[0082] 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).
[0083] Von Willebrand Factor
[0084] 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.
[0085] 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).
[0086] 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).
[0087] 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.
[0088] The polynucleotide and amino acid sequences of prepro-VWF
are available at GenBank Accession Nos. NM_000552 and NP_000543,
respectively.
[0089] 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.
[0090] C. Production of Blood Coagulation Proteins
[0091] Production of a blood coagulation 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 blood
coagulation 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 blood coagulation protein.
[0092] In other aspects, the blood coagulation 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.
[0093] A wide variety of vectors are used for the preparation of
the blood coagulation 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 po1 h, 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.
[0094] D. Administration
[0095] In one embodiment a conjugated blood coagulation protein of
the present invention may be administered by injection, such as
intravenous, intramuscular, or intraperitoneal injection.
[0096] To administer compositions comprising a conjugated blood
coagulation 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.
[0097] As used herein, "effective amount" includes a dose suitable
for treating a mammal having a bleeding disorder as described
herein.
[0098] 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.
[0099] 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.
[0100] The present invention also relates to a pharmaceutical
composition comprising an effective amount of a conjugated blood
coagulation 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.
[0101] 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 blood
coagulation 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 blood
coagulation 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
[0102] In one aspect, a blood coagulation protein derivative (i.e.,
a conjugated blood coagulation protein) molecule provided is bound
to a water-soluble polymer including, but 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(1-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.
[0103] In one embodiment, the derivative retains the full
functional activity of native therapeutic blood coagulation protein
products, and provides an extended half-life in vivo, as compared
to native therapeutic blood coagulation 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 blood
coagulation protein.
[0104] A. Sialic Acid and PSA
[0105] 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.
[0106] 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.
[0107] 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.
[0108] B. Polyethylene Glycol (PEG) and Pegylation
[0109] In certain aspects, blood coagulation factor, e.g., FVIII,
FVIIa, FIX, or other blood coagulation factor molecules 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 FVIII, FVIIa, or FIX 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 FVIII,
FVIIa, or FIX after prior oxidation.
[0110] The conjugation is in one aspect performed by direct
coupling (or coupling via linker systems) of the water soluble
polymer to blood coagulation factor, e.g., FVIII, FVIIa, or FIX,
under formation of stable bonds. In addition degradable, releasable
or hydrolys able 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).
[0111] In one embodiment of the invention, a blood coagulation
factor, e.g., FVIII, FVIIa, or FIX, 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 FVIII, FVIIa, or FIX
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.
[0112] 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.
[0113] In one embodiment of the present invention a blood
coagulation factor, e.g., FVIII, FVIIa, FIX, or other blood
coagulation factor, molecule is chemically modified using PEG
derivatives that are commercially available. These PEG derivatives
in alternative aspects have a linear or branched structures.
Examples of PEG-derivatives containing NHS groups are listed
below.
[0114] 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):
##STR00005##
[0115] This reagent with branched structure is described in more
detail by Kozlowski et al. (BioDrugs 2001; 5:419-29).
[0116] Other non-limiting examples of PEG derivatives are
commercially available from NOF Corporation (Tokyo, Japan; see
www.nof.co.jp/english: Catalogue 2005)
##STR00006##
[0117] 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.
[0118] 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.
[0119] Surprisingly, the PEGylated FVIII, FVIIa, FIX, or other
blood coagulation factor 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.
[0120] C. Methods of Attachment
[0121] A blood coagulation 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 blood coagulation 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.
[0122] 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.
[0123] 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 blood coagulation
protein or polysaccharide. Another linkage by which the blood
coagulation 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 NH4Cl 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.
[0124] 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.
[0125] 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 blood coagulation protein
forms a Schiff base with the N-deacetylated 5-amino group of a
sialic acid residue.
[0126] Alternatively, the polysaccharide compound is associated in
a non-covalent manner with a blood coagulation 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.
[0127] In various embodiments, the blood coagulation 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.
[0128] In various embodiments, the blood coagulation 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 blood coagulation 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).
[0129] D. Aminooxy Linkage
[0130] 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
blood coagulation protein according to the present invention) is
first oxidized with a oxidizing agent such as sodium periodate
(NaIO.sub.4) (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).sub.4), manganese acetate
(MnO(Ac).sub.3), cobalt acetate (Co(OAc).sub.2), thallium acetate
(TlOAc), cerium sulfate (Ce(SO.sub.4).sub.2) (U.S. Pat. No.
4,367,309) or potassium perruthenate (KRuO.sub.4) (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.
[0131] 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).
[0132] In one embodiment of the invention, the reaction steps to
conjugate a water soluble polymer to a blood coagulation protein
are carried out separately and sequentially (i.e., starting
materials (e.g., blood coagulation protein, water soluble polymer,
etc), reagents (e.g., oxidizing agents, aniline, etc) and reaction
products (e.g., oxidized carbohydrate on a blood coagulation
protein, activated aminooxy water soluble polymer, etc) are
separated between individual reaction steps).
[0133] 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.
[0134] 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 blood
coagulation 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(1-hydroxymethylethylene
hydroxymethylformal) (PHF),
2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC).
[0135] 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
[0136] The homobifunctional linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.2ONH.sub.2
##STR00007##
(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
[0137] The homobifunctional linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.4ONH.sub.2
##STR00008##
(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 Aminooxy-PSA
[0138] 500 mg of oxidized PSA (MW=18.8 kD) obtained from the Serum
Institute of India (Pune, India) was dissolved in 8 ml 50 mM sodium
acetate buffer, pH 5.5. Next, 100 mg 3-oxa-pentane-1,5-dioxyamine
was added. After shaking for 2 hrs at room temperature, 44 mg
sodium cyanoborohydride was added. After shaking for another 4 hrs
at 4.degree. C., the reaction mix was loaded into a Slide-A-Lyzer
(Pierce, Rockford, Ill.) dialysis cassette (3.5 kD membrane,
regenerated cellulose) and dialyzed against PBS pH 7.2 for 4 days.
The product was frozen at -80.degree. C. The preparation of the
aminooxy-PSA according to this procedure is illustrated in FIG.
4.
Alternative Procedure for Preparation of Aminooxy PSA
[0139] 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
(Millipore).
Example 4
Coupling of Aminooxy-PSA to rFIX and Purification of the
Conjugate
[0140] To 12.6 mg rFIX, dissolved in 6.3 ml 50 mM sodium acetate
buffer, pH 6.0, 289 .mu.l of an aqueous sodium periodate solution
(10 mM) was added. The mixture was shaken in the dark for 1 h at
4.degree. C. and quenched for 15 min at room temperature by the
addition of 6.5 .mu.l 1M glycerol. Low molecular weight
contaminates were removed by ultrafiltration/diafiltration (UF/DF)
employing Vivaspin (Sartorius, Goettingen, Germany) concentrators
(30 kD membrane, regenerated cellulose). Next, 43 mg aminooxy-PSA
was added to the UF/DF retentate and the mixture was shaken for 18
hrs at 4.degree. C. The excess PSA reagent was removed by
hydrophobic interaction chromatography (HIC). The conductivity of
the cooled reaction mixture was raised to 180 mS/cm and loaded onto
a 5 ml HiTrap Butyl FF (GE Healthcare, Fairfield, Conn.) HIC column
(1.6.times.2.5 cm), pre-equilibrated with 50 mM HEPES, 3M sodium
chloride, 6.7 mM calcium chloride, 0.01% Tween 80, pH 6.9. The
conjugate was eluted within 2.4 column volumes (CV) with 50 mM
HEPES, 6.7 mM calcium chloride, 0.005% Tween 80, pH 7.4 at a flow
rate of 5 ml/min. The preparation was analytically characterized by
measuring total protein (BCA) and FIX chromogenic activity. For the
PSA-rFIX conjugate a specific activity of 80.2 IU/mg protein was
determined (56.4% in comparision to native rFIX). The results are
summarized in Table 1.
TABLE-US-00003 TABLE 1 FIX: Specific Activity Specific BCA Chrom
[IU FIX: Activity Item [mg/ml] [IU/ml] Chrom/mg BCA [%] rFIX 8.58
1221 142.3 100 PSA-rFIX 1.15 92.2 80.2 56.4
[0141] The analytical characterization of the PSA-rFIX conjugate by
SDS-PAGE with Coomassie staining is illustrated in FIG. 5. An
SDS-PAGE followed by Western blot employing anti-FIX and anti-PSA
antibodies is shown in FIG. 6.
Example 5
Coupling of Aminooxy-PSA to rFIX in the Presence of aniline as
nucleophilic Catalyst
[0142] To 3.0 mg rFIX, dissolved in 1.4 ml 50 mM sodium acetate
buffer, pH 6.0, 14.1 .mu.l of an aqueous sodium periodate solution
(10 mM) was added. The mixture was shaken in the dark for 1 h at
4.degree. C. and quenched for 15 min at room temperature by the
addition of 1.5 .mu.l 1M glycerol. Low molecular weight
contaminates were removed by means of size exclusion chromatography
(SEC) employing PD-10 desalting columns (GE Healthcare, Fairfield,
Conn.). 1.2 mg oxidized rFIX, dissolved in 1.33 ml 50 mM sodium
acetate buffer, pH 6.0 was mixed with 70 .mu.l of aniline (200 mM
aqueous stock solution) and shaken for 45 min at room temperature.
Next, 4.0 mg aminooxy-PSA was added and the mixture was shaken for
2 hrs at room temperature and another 16 hrs at 4.degree. C.
Samples were drawn after 1 h, after 2 hrs and at the end of the
reaction after 18 hrs. Next, excess PSA reagent and free rFIX were
removed by means of HIC. The conductivity of the cooled reaction
mixture was raised to 180 mS/cm and loaded onto a 5 ml HiTrap Butyl
FF (GE Healthcare, Fairfield, Conn.) HIC column (1.6.times.2.5 cm),
pre-equilibrated with 50 mM HEPES, 3M sodium chloride, 6.7 mM
calcium chloride, 0.01% Tween 80, pH 6.9. The conjugate was eluted
with a linear gradient to 50 mM HEPES, 6.7 mM calcium chloride,
0.005% Tween 80, pH 7.4 in 20CV with at a flow rate of 5
ml/min.
Example 6
Coupling of Aminooxy-PSA to rFIX and Reduction with
NaCNBH.sub.3
[0143] To 10.5 mg rFIX, dissolved in 5.25 ml 50 mM sodium acetate
buffer, pH 6.0, 53 .mu.l of an aqueous sodium periodate solution
(10 mM) was added. The mixture was shaken in the dark for 1 h at
4.degree. C. and quenched for 15 min at room temperature by the
addition of 5.3 .mu.l 1M glycerol. Low molecular weight
contaminates were removed by means of UF/DF employing Vivaspin
(Sartorius, Goettingen, Germany) concentrators (30 kD membrane,
regenerated cellulose). Next, 35.9 mg aminooxy-PSA was added to the
UF/DF retentate and the mixture was shaken for 2 hrs at room
temperature. Then 53 .mu.l of a aqueous sodium cyanoborohydride
solution (5M) was added and the reaction was allowed to proceed for
another 16 hrs. Then the excess PSA reagent was removed by means of
HIC. The conductivity of the cooled reaction mixture was raised to
180 mS/cm and loaded onto a 5 ml HiTrap Butyl FF HIC (GE
Healthcare, Fairfield, Conn.) column (1.6.times.2.5 cm),
pre-equilibrated with 50 mM HEPES, 3M sodium chloride, 6.7 mM
calcium chloride, 0.01% Tween 80, pH 6.9. The conjugate was eluted
within 2.4CV with 50 mM HEPES, 6.7 mM calcium chloride, 0.005%
Tween 80, pH 7.4 at a flow rate of 5 ml/min.
Example 7
Coupling of Aminooxy-PSA (Linker:
NH.sub.2[OCH.sub.2CH.sub.2].sub.4ONH.sub.2) to rFIX and
Purification of the Conjugate
[0144] To 5.6 mg rFIX, dissolved in 2.8 ml 50 mM sodium acetate
buffer, pH 6.0, 102 .mu.l of an aqueous solution of sodium
periodate (10 mM) was added. The mixture was shaken in the dark for
1 h at 4.degree. C. and quenched for 15 min at room temperature by
the addition of 2.9 .mu.l of 1M glycerol. Low molecular weight
contaminates were removed by means of UF/DF employing Vivaspin
(Sartorius, Goettingen, Germany) concentrators (30 kD membrane,
regenerated cellulose). Then 19 mg aminooxy-PSA was added to the
UF/DF retentate and the mixture was shaken for 18 hrs at 4.degree.
C. The excess PSA reagent was removed by means of HIC. The
conductivity of the cooled reaction mixture was raised to 180 mS/cm
and loaded onto a 5 ml HiTrap Butyl FF (GE Healthcare, Fairfield,
Conn.) HIC column (1.6.times.2.5 cm), pre-equilibrated with 50 mM
HEPES, 3M sodium chloride, 6.7 mM calcium chloride, 0.01% Tween 80,
pH 6.9. The conjugate was eluted within 2.4CV with 50 mM HEPES, 6.7
mM calcium chloride, 0.005% Tween 80, pH 7.4 at a flow rate of 5
ml/min.
Example 8
Coupling of Aminooxy-PSA to rFVIII
[0145] To 11 mg rFVIII, dissolved in 11 ml Hepes buffer pH 6 (50 mM
Hepes, 5 mM CaCl.sub.2, 150 mM NaCl, 0.01% Tween) 57 .mu.l 10 mM
sodium periodate was added. The mixture was shaken in the dark for
30 min at 4.degree. C. and quenched for 30 min at 4.degree. C. by
the addition of 107 .mu.l of an aqueous 1M glycerol solution. Then
19.8 mg aminooxy-PSA (18.8 kD) was added and the mixture was shaken
over night at 4.degree. C. The ionic strength was increased by
adding a buffer containing 8M ammonium acetate (8M ammonium
acetate, 50 mM Hepes, 5 mM CaCl.sub.2, 350 mM NaCl, 0.01% Tween 80,
pH 6.9) to get a final concentration of 2.5M ammonium acetate.
Next, the reaction mixture was loaded on a HiTrap Butyl FF (GE
Healthcare, Fairfield, Conn.) column which was equilibrated with
equilibration buffer (2.5M ammonium acetate, 50 mM Hepes, 5 mM
CaCl.sub.2, 350 mM NaCl, 0.01% Tween 80, pH 6.9). The product was
eluted with elution buffer (50 mM Hepes, 5 mM CaCl.sub.2, 0.01%
Tween 80, pH 7.4), and the eluate was concentrated by centrifugal
filtration using Vivaspin (Sartorius, Goettingen, Germany) devices
with 30,000 MWCO.
Example 9
PK Studies in Hemophilic Mice
[0146] FIX-deficient mice were injected with either rFIX or
PSA-rFIX (prepared according to Example 4) in formulation buffer
(10 mM histidine, 260 mM glycine, 29 mM sucrose, 0.005% Tween 80,
pH 6.8) in a volume dose of 10 ml/kg bodyweight. Groups of 6 mice
were sacrificed 5 minutes, 3 hours, 9, 16, 24 and 48 hours after
substance injection and blood was collected by heart puncture.
Citrated plasma was prepared and stored frozen until analysis of
FIX activity.
[0147] FIX activity was determined with a chromogenic FIX assay
(Biophen FIX assay, Hyphen Biomed, Neuville-sur-Oise, France) and
elimination curves were constructed (FIG. 7). Actual FIX activity
doses were 123 IU FIX/kg for PSA-rFIX and 143 IU FIX/kg for rFIX.
Pharmacokinetic parameters were calculated with program R (The R
Foundation for Statistical Computing, 2008). In vivo recovery was
13% for rFIX and 29% for PSA-rFIX. Dose adjusted AUC for PSA-rFIX
increased 6.4-fold relative to rFIX, terminal half life increased
by a factor of 1.2 and MRT was 1.7-times longer for PSA-rFIX
compared to rFIX (Table 2).
TABLE-US-00004 TABLE 2 In vivo Terminal recovery AUC Increase HL
Increase MRT Increase Item [%] [(IU/ml)/(IU/kg)] factor [h] factor
[h] factor rFIX 13 0.0100 =1 8.0 =1 7.3 =1 PSA-rFIX 29 0.0650 6.4x
9.6 1.2x 12.3 1.7x
Example 10
Polysialylation of Blood Coagulation Proteins
[0148] Polysialylation as described herein may be extended to other
coagulation proteins. For example, in various aspects of the
invention, the above polysialylation as described in Examples 5, 6
and 9 with aminooxy-PSA is repeated with coagulation proteins such
as FVIII, FVIIa and VWF.
Example 11
Preparation of the Homobifunctional Linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.6ONH.sub.2
[0149] The homobifunctional linker
NH.sub.2[OCH.sub.2CH.sub.2].sub.6ONH.sub.2
##STR00009##
(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 12
Polysialylation of rFIX Employing a Maleimido/Aminooxy Linker
System
[0150] A. Preparation of the Modification Reagent
[0151] An Aminooxy-PSA reagent is prepared by use of a
maleimido/aminooxy linker system (Toyokuni et al., Bioconjugate
Chem 2003; 14, 1253-9). PSA-SH (20 kD) containing a free terminal
SH-group is prepared using a two step procedure: a) Preparation of
PSA-NH.sub.2 by reductive amination of oxidized PSA with NH.sub.4Cl
according to WO05016973A1 and b) introduction of a sulfhydryl group
by reaction of the terminal primary amino group with
2-iminothiolane (Traut's reagent/Pierce, Rockford, Ill.) as
described in U.S. Pat. No. 7,645,860. PSA-SH is coupled to the
maleimido-group of the linker at pH 7.5 in PBS-buffer using a 10
fold molar excess of the linker and a PSA-SH concentration of 50
mg/ml. The reaction mixture is incubated for 2 hours under gentle
shaking at room temperature. Then the excess linker reagent is
removed and the aminooxy-PSA is buffer exchanged into oxidation
buffer (50 mM sodium phosphate, pH 6.0) by diafiltration. The
buffer is exchanged 25 times employing a Pellicon XL5 kD
regenerated cellulose membrane (Millipore, Billerica, Mass.).
[0152] B. Modification of rFIX After Prior Oxidation with
NaIO.sub.4
[0153] rFIX is oxidized in 50 mM sodium phosphate buffer, pH 6.0
employing 100 .mu.M sodium periodate in the buffer. The mixture was
shaken in the dark for 1 h at 4.degree. C. and quenched for 15 min
at room temperature by the addition of glycerol to a final
concentration of 5 mM. Low molecular weight contaminates were
removed by means of size exclusion chromatography (SEC) employing
PD-10 desalting columns (GE Healthcare, Fairfield, Conn.). Oxidized
rFIX is then spiked with aniline to obtain a final concentration of
10 mM and mixed with the aminooxy-PSA reagent to achieve a 5 fold
molar excess of PSA. The reaction mixture was incubated for 2 hours
under gentle shaking in the dark at room temperature.
[0154] C. Purification of the Conjugates
[0155] The excess of PSA reagent and free rFIX is removed by means
of HIC. The conductivity of the reaction mixture is raised to 180
mS/cm and loaded onto a column filled with 48 ml Butyl-Sepharose FF
(GE Healthcare, Fairfield, Conn.) pre-equilibrated with 50 mM
Hepes, 3 M sodium chloride, 6.7 mM calcium chloride, 0.01% Tween
80, pH 6.9. Subsequently the conjugate is eluted with a linear
gradient of 60% elution buffer (50 mM Hepes, 6.7 mM calcium
chloride, pH 7.4) in 40 CV. Finally the PSA-rFIX containing
fractions are collected and subjected to UF/DF by use of a 30 kD
membrane made of regenerated cellulose (Millipore). The preparation
is analytically characterized by measuring total protein (BCA) and
FIX chromogenic activity. For the PSA-rFIX conjugates prepared with
both variants a specific activity of >50% in comparison to
native rFIX is determined.
Example 13
Preparation of Aminooxy-PSA Reagent
[0156] An Aminooxy-PSA reagent was prepared according to Example 3.
The final product was diafiltrated against buffer, pH 7.2 (50 mM
Hepes) using a 5 kD membrane (regenerated cellulose, Millipore),
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 14
Pharmacokinetics of Polysialylated rFVIII in a FVIII Deficient
Knock Out Mouse Model
[0157] A PSA-FVIII conjugate was prepared according Example 8. The
conjugate showed a specific activity of 6237 IU/mg (FVIII activity
determined by the chromogenic assay; total protein determined by
the Bradford assay) and had a polysialylation degree of 6.7 (mole
PSA per mole FVIII) as measured by the Resorcinol assay
(Svennerholm L, Biochim Biophys Acta 1957; 24: 604-11).
[0158] FVIII deficient mice described in detail by Bi et al. (Nat
Genet 1995; 10:119-21) were used as a model of severe human
hemophilia A. Groups of 6 mice received a bolus injection (200 IU
FVIII/kg) via the tail vein with either PSA-rFVIII prepared
according to Example 8 or native rFVIII (ADVATE, Baxter Healthcare
Corporation) in a dose of 200 IU FVIII/kg bodyweight. Citrate
plasma by heart puncture after anesthesia was prepared from the
respective groups 5 minutes, 3, 6, 9, 16, 24, 32 and 42 hours after
injection. FVIII activity levels were measured in plasma samples by
use of the chromogenic assay. The results of this experiment are
summarized in Table 3 and illustrated in FIG. 8. All calculations
were performed with R version 2.10.1 (A language and environment
for statistical computing. R Foundation for Statistical Computing,
Vienna, Austria. http://www.R-project.org.). As a result the mean
residence time (MRT) increased from 5.4 h (Advate control) to 11.1
h for the PSA-rFVIII conjugate.
TABLE-US-00005 TABLE 3 In vivo Mean recov- AUC 0-24 Terminal
residence Clearance ery (IU/ml h)/ half- time CL Item IVR % IU/kg
life (h) MRT (h) (ml/h/kg) PSA-rFVIII 71 0.161 7.2 11.1 6.0 rFVIII
58 0.054 4.4 5.4 17.1 control (Advate)
Example 15
Detailed Synthesis of the Aminooxy-PSA Reagent
[0159] 3-oxa-pentane-1,5dioxyamine was synthesized according to
Botyryn et al (Tetrahedron 1997; 53:5485-92) in a two step organic
synthesis as outlined in Example 1.
[0160] Step 1:
[0161] 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).
[0162] Step2:
[0163] 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 16
Polysialylation of rFIX Using PSA Hydrazide
[0164] rFIX is polysialylated by use of a PSA hydrazide reagent,
which was prepared by reaction of oxidized PSA with adipic acid
dihydrazide (ADH).
[0165] Step 1: Preparation of PSA Hydrazide
[0166] 500 mg of oxidized PSA (MW=20 kD) obtained from the Serum
Institute of India (Pune, India) was dissolved in 8 ml 50 mM sodium
acetate buffer, pH 5.5. 100 mg adipic acid dihydrazide (ADH) was
then added. The solution was gently shaken for 2 hrs. 44 mg sodium
cyanoborohydride were then added. After the reaction was incubated
for an additional 4 hrs at 4.degree. C., the reaction mix was
loaded into a Slide-A-Lyzer (Pierce, Rockford, Ill.) dialysis
cassette (3.5 kD membrane, regenerated cellulose) and dialyzed
against PBS pH 7.2 for 4 days. The product was frozen at
-80.degree. C.
[0167] Step 2: Reaction of PSA Hydrazide with rFIX and Purification
of the Conjugate
[0168] rFIX is polysialylated by use of a PSA hydrazide reagent as
described in Step 1. rFIX (concentration 1 mg/ml) is oxidized with
NaIO.sub.4 (concentration: 80 .mu.M) for 1 h at 4.degree. C. in the
dark under gentle shaking. The reaction is stopped by addition of
glycerol and the oxidized FIX is subjected to UF/DF by use of a 30
kD membrane made of regenerated cellulose (Vivaspin). The oxidized
rFIX is then polysialylated at pH 6.5 using a 200-fold molar excess
of reagent and a protein concentration of 1 mg/ml. rFIX and the
polysialyation reagent are incubated for 2 hours under gentle
shaking in the dark at room temperature. Finally, the PSA-rFIX
conjugate is purified by HIC. The conductivity of the reaction
mixture is raised to 130 mS/cm by adding a buffer containing
ammonium acetate (50 mM Hepes, 350 mM NaCl, 5 mM Calcium chloride,
8M ammonium acetate, 0.01% Tween 80, pH 6.9) and loaded onto a
HiTrap Butyl FF column (5 ml, GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 2.5M 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, 0.01% Tween 80, pH 7.4. Finally the PSA-rFIX
containing fractions are collected and subjected to UF/DF by use of
a 30 kD membrane made of regenerated cellulose (Vivaspin). For the
PEG-rFIX conjugate, a specific activity of >50% in comparison to
native rFIX is determined (chromogenic assay).
Example 17
Polysialylation of rFIX using PSA Hydrazide in the Presence of
Aniline as a Nucleophilic Catalyst
[0169] 123 mg rFIX are dissolved in 60 ml phosphate buffer (50 mM
NaPO.sub.4, pH 6.5) buffer. Then 1.2 ml of an aqueous sodium
periodate solution (10 mM) is added and the mixture is incubated
for 1 h in the dark at 4.degree. C. under gentle stirring.
Subsequently the reaction is quenched for 15 min at RT by the
addition of 600 .mu.l of 1M aqueous glycerol solution. The mixture
is subsequently subjected to UF/DF employing a Pellicon XL Ultracel
30 kD membrane.
[0170] The UF/D F retentate (63.4 ml), containing oxidized rFIX, is
further diluted with 59.6 ml phosphate buffer (50 mM NaPO.sub.4, pH
6.0) and mixed with 6.5 ml of an aqueous aniline solution (200 mM)
and incubated for 30 min at RT. Then 12.3 ml of the PSA-hydrazide
reagent (prepared according Example 16) is added to give a 5 fold
molar reagent excess. This mixture is incubated for 2 h at RT in
the dark under gentle stirring.
[0171] The excess of the PSA-hydrazide reagent and free rFIX is
removed by means of HIC. The conductivity of the reaction mixture
is raised to 180 mS/cm and loaded onto a column filled with 48 ml
Butyl-Sepharose FF (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with 50 mM Hepes, 3M sodium chloride, 6.7 mM
calcium chloride, 0.01% Tween 80, pH 6.9. Subsequently the
conjugate is eluted with 50 mM Hepes, 5 mM calcium chloride, 0.01%
Tween 80, pH 7.4. Finally the PSA-rFIX containing fractions are
collected and subjected to UF/DF by use of a 30 kD membrane made of
regenerated cellulose (Millipore). The preparation is analytically
characterized by measuring total protein (BCA) and FIX chromogenic
activity. For the PSA-rFIX conjugate a specific activity of >50%
in comparison to native rFIX is determined.
Example 18
Polysialylation of rFIX and Purification Using a Two Step
Procedure
[0172] 140 mg rFIX was dissolved in 62 ml phosphate buffer (50 mM
NaPO.sub.4, pH 6.0) buffer. Then 1.92 ml of an aqueous sodium
periodate solution (10 mM) were added and the mixture was incubated
for 1 h in the dark at 4.degree. C. under gentle stirring and
quenched for 15 min at RT by the addition of 64 .mu.l of an 1M
aqueous glycerol solution. Subsequently the mixture was subjected
to UF/DF employing a Pellicon XL Ultracel 30 kD membrane.
[0173] The UF/DF retentate (69.4 ml), containing oxidized rFIX, was
further diluted with 73.8 ml phosphate buffer (50 mM NaPO.sub.4, pH
6.0), mixed with 8.2 ml of an aqueous aniline solution (200 mM) and
incubated for 30 min at RT. Then 12.3 ml of the aminooxy reagent
(prepared according to Example 3) were added to give a 2.5 fold
molar reagent excess. This mixture was incubated for 2.5 h at RT in
the dark under gentle stirring.
[0174] The free rFIX is removed by means of anion exchange
chromatography (AIEC). The reaction mixture is diluted with 20 ml
Buffer A (50 mM Hepes, 5 mM CaCl.sub.2, pH 7.5) and loaded onto a
Q-Sepharose FF 26/10 column (GE Healthcare, Fairfield, Conn.)
pre-equilibrated with Buffer A. Then the column is eluted with
Buffer B (50 mM Hepes, 1M NaCl, 5 mM CaCl.sub.2, 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 are subsequently raised to 190 mS/cm by addition of
Buffer C (50 mM Hepes, 5M NaCl, 5 mM CaCl.sub.2, pH 6.9) and loaded
onto a Butyl Sepharose FF 26/10 column (GE Healthcare, Fairfield,
Conn.) pre-equilibrated with Buffer D (50 mM Hepes, 3M NaCl, 5 mM
CaCl.sub.2, pH 6.9). Free PSA-reagent is washed out within 5CV
Buffer D. Subsequently the conjugate is eluted with 100% Buffer E
(50 mM Hepes, 5 mM CaCl.sub.2, pH 7.4). 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
7.2 containing 150 mM NaCl and 5 mM CaCl.sub.2. The preparation is
analytically characterized by measuring total protein (BCA) and FIX
chromogenic activity. For the PSA-rFIX conjugate a specific
activity of >50% in comparison to native rFIX is determined.
Example 19
Coupling of Aminooxy-PSA to rFVIIa and Purification of the
Conjugate
[0175] A solution of 10 mg rFVIIa in 5 ml reaction buffer (50 mM
Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0) is
mixed with an aqueous solution of NaIO.sub.4 (final concentration:
100 .mu.M) and incubated for 1 h at 4.degree. C. under gentle
stirring in the dark and quenched by the addition of an aqueous
solution of cysteine (final concentration: 1 mM) for 15 min. The
reaction mixture is subsequently subjected to UF/DF. To the
retentate (10 ml) a 30 fold molar excess of Aminooxy reagent
(prepared according to Example 1) is added. The coupling reaction
is performed for 2 hours at room temperature in the dark under
gentle shaking. The excess of aminooxy reagent is removed by HIC.
The conductivity of the reaction mixture is raised to 130 mS/cm by
adding a buffer containing ammonium acetate (50 mM Hepes, 350 mM
NaCl, 5 mM Calcium chloride, 8 M ammonium acetate, 0.01% Tween 80,
pH 6.9) and loaded onto a HiTrap Butyl FF column (5 ml, 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, 0.01% Tween 80, pH
7.4 by a linear gradient of 100% elution buffer in 20 CV. Finally
the PSA-rFVIIa containing fractions are collected and subjected to
UF/DF by use of a 30 kD membrane made of regenerated cellulose
(Vivaspin). The preparation is analytically characterized by
measuring total protein (BCA) and FVIIa chromogenic activity
(Staclot assay, Diagnostica Stago, Asnieres, France) and shows a
specific activity of >20% compared to the rFVIIa starting
material.
Example 20
Coupling of Aminooxy-PSA to rFVIIa in the Presence of Aniline as
Nucleophilic Catalyst
[0176] To 3.0 mg rFVIIa, dissolved in 1.4 ml 50 mM sodium acetate
buffer, pH 6.0, 14.1 .mu.l of an aqueous sodium periodate solution
(10 mM) is added. The mixture is shaken in the dark for 1 h at
4.degree. C. and quenched for 15 min at room temperature by the
addition of 1.5 .mu.l 1M glycerol. Low molecular weight
contaminates are removed by means of size exclusion chromatography
(SEC) employing PD-10 desalting columns (GE Healthcare, Fairfield,
Conn.). 3 mg oxidized rFVIIa, dissolved in 3 ml 50 mM sodium
acetate buffer, pH 6.0 is mixed with aniline (a nucleophilic
catalyst, final concentration: 10 mM) and shaken for 30 min at room
temperature. Next, aminooxy-PSA is added to give a 5 fold molar
excess and the mixture is shaken for 2 hrs at room temperature.
Subsequently the excess PSA reagent and free rFIX are removed by
means of HIC. The conductivity of the cooled reaction mixture is
raised to 180 mS/cm and loaded onto a 5 ml HiTrap Butyl FF (GE
Healthcare, Fairfield, Conn.) HIC column (1.6.times.2.5 cm),
pre-equilibrated with 50 mM Hepes, 3M sodium chloride, 6.7 mM
calcium chloride, 0.01% Tween 80, pH 6.9. The conjugate is eluted
with a linear gradient to 50 mM HEPES, 6.7 mM calcium chloride,
0.005% Tween 80, pH 7.4 in 20 CV with at a flow rate of 5
ml/min.
Example 21
Preparation of an Aminooxy-PEG Reagent
[0177] A branched PEG-aldehyde (MW 40 kD) is used for coupling to
the diaminooxy linker, which is prepared as described in Example 1.
This PEG-aldehyde reagent is available from NOF (NOF Corp., Tokyo,
Japan). 500 mg of PEG-aldehyde is dissolved in 8 ml 50 mM sodium
acetate buffer, pH 5.5. Then 100 mg 3-oxa-pentane-1,5-dioxyamine is
added. After shaking for 2 hrs at room temperature, 44 mg sodium
cyanoborohydride is added. After shaking for another 4 hrs at
4.degree. C., the reaction mix is loaded into a Slide-A-Lyzer
(Pierce, Rockford, Ill.) dialysis cassette (3.5 kD membrane,
regenerated cellulose) and dialyzed against PBS pH 7.2 for 4 days.
The product is frozen at -80.degree. C.
Example 22
PEGylation of rFIX with an Aminooxy PEG-Reagent
[0178] 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). rFIX is oxidized at a protein concentration of 2 mg/ml with
NaIO.sub.4 (final: concentration: 100 .mu.M) for 1 hour under
gentle shaking in the dark at 4.degree. C. in reaction buffer (50
mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
and quenched by the addition of an aqueous solution of glycerol
(final concentration: 1 mM) for 15 min. The reaction mixture is
subsequently subjected to UF/DF. To the retentate a 3 fold molar
excess of Aminooxy reagent and aniline (a nucleophilic catalyst,
final concentration: 10 mM) are added. The coupling reaction is
performed for 2 hours at room temperature in the dark under gentle
shaking. Finally the PEG-rFIX 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 Tris, pH 8.0. The
conjugate is eluted with 50 mM Tris and 1 M sodium chloride, pH 8.0
in 20 CV and is then subjected to UF/DF using a 30 kD membrane. The
preparation is analytically characterized by measuring total
protein (BCA) and FIX chromogenic activity. For the PEG-rFIX
conjugate a specific activity of >75% in comparison to native
rFIX is determined.
Example 23
PEGylation of rFVIII with an Aminooxy PEG-Reagent
[0179] 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). rFVIII is oxidized at a protein concentration of 1 mg/ml
with NaIO.sub.4 (final: concentration: 100 .mu.M) for 1 hour under
gentle shaking in the dark at 4.degree. C. in reaction buffer (50
mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
and quenched by the addition of an aqueous solution of cysteine
(final concentration: 1 mM) for 15 min. The reaction mixture is
subsequently subjected to UF/DF. To the retentate a 20 fold molar
excess of Aminooxy reagent and aniline (a nucleophilic catalyst,
final concentration: 10 mM) are added. The coupling reaction is
performed for 2 hours at room temperature in the dark under gentle
shaking. 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. The analytical characterization of the conjugate by
FVIII chromogenic assay and determination of total protein (BCA
assay) shows a specific activity of >60% compared to the rFVIII
starting material.
Example 24
PEGylation of rFVIIa with an Aminooxy PEG-Reagent
[0180] 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). rFVIIa is oxidized at a protein concentration of 2 mg/ml
with NaIO.sub.4 (final: concentration: 100 .mu.M) for 1 hour under
gentle shaking in the dark at 4.degree. C. in reaction buffer (50
mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
and quenched by the addition of an aqueous solution of glycerol
(final concentration: 1 mM) for 15 min. The reaction mixture is
subsequently subjected to UF/DF. To the retentate a 5 fold molar
excess of Aminooxy reagent and aniline (a nucleophilic catalyst,
final concentration: 10 mM) are added. The coupling reaction is
performed for 2 hours at room temperature in the dark under gentle
shaking. Finally the PEG-rFVIIa 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 20 mM Hepes
buffer containing 1 mM CaCl.sub.2, pH 7.4. The conjugate is eluted
with 20 mM Hepes buffer containing 1 mM CaCl.sub.2 and 500 mM
sodium chloride, pH 7.4 and is then subjected to UF/DF using a 30
kD membrane. The analytical characterization of the conjugate by
measuring FVIIa activity (Staclot assay, Diagnostica Stago,
Asnieres, France) and total protein (BCA assay) shows a specific
activity of >25% compared to the rFVIIa starting material.
Example 25
PEGylation of rFIX with an PEG-Hydrazide Reagent
[0181] rFIX is PEGylated by use of a linear 20 kD PEGylation
reagent containing a hydrazide group. An example of this type of
reagent is the Sunbright.RTM. HZ series from NOF (NOF Corp., Tokyo,
Japan). rFIX is oxidized at a protein concentration of 2 mg/ml with
NaIO.sub.4 (final: concentration: 100 .mu.M) for 1 hour under
gentle shaking in the dark at 4.degree. C. in reaction buffer (50
mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
and quenched by the addition of an aqueous solution of glycerol
(final concentration: 1 mM) for 15 min. The reaction mixture is
subsequently subjected to UF/DF. To the retentate a 50 fold molar
excess of Hydrazide reagent and aniline (a nucleophilic catalyst,
final concentration: 10 mM) are added. The coupling reaction is
performed for 2 hours at room temperature in the dark under gentle
shaking. Finally the PEG-rFIX conjugate is purified by ion-exchange
chromatography on Q-Sepharose FF. The reaction mixture is loaded
onto the column (1.5 mg protein/ml gel), which is preequilibrated
with 50 mM Tris-buffer, pH 8.0. The conjugate is eluted with 20 CV
Tris-buffer, pH 8.0 (50 mM Tris, 1 M NaCl) and is then subjected to
UF/DF using a 30 kD membrane. The preparation is analytically
characterized by measuring total protein (BCA) and FIX chromogenic
activity. For the PEG-rFIX conjugate a specific activity of >50%
in comparison to native rFIX is determined (chromogenic assay).
Example 26
Polysialylation of rFVIII in the Presence of 2 mM Aniline
[0182] rFVIII is transferred into reaction buffer (50 mM Hepes, 350
mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6),
diluted to a protein concentration of 1 mg/ml and oxidized with
NaIO.sub.4 (final: concentration: 100 .mu.M) for 1 hour under
gentle shaking in the dark at 4.degree. C. in reaction buffer (50
mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
and quenched by the addition of an aqueous solution of cysteine
(final concentration: 1 mM) for 15 min. The reaction mixture is
subsequently subjected to UF/DF. To the retentate a 20 fold molar
excess of Aminooxy reagent and aniline (a nucleophilic catalyst,
final concentration: 2 mM) are added. The coupling reaction is
performed for 2 hours at room temperature in the dark under gentle
shaking. The excess of aminooxy reagent is removed by means of HIC.
The conductivity of the reaction mixture is 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, 0.01%
Tween 80, pH 6.9) and loaded onto a column filled with 53 ml
Butyl-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, 0.01% Tween 80, pH 7.4. Finally the PSA-rFIX containing
fractions are collected and subjected to UF/DF by use of a 30 kD
membrane made of regenerated cellulose (Millipore, Billerica,
Mass.). The preparation is analytically characterized by measuring
total protein (BCA) and FVIII chromogenic activity. For the
PSA-rFVIII conjugate a specific activity of 80% in comparison to
native rFVIII is determined.
Example 27
Polysialylation of rFVIII in the Presence of 10 mM Aniline
[0183] rFVIII is transferred into reaction buffer (50 mM Hepes, 350
mM sodium chloride, 5 mM calcium chloride, 0.01% Tween 80, pH 6),
diluted to a protein concentration of 1 mg/ml and oxidized with
NaIO.sub.4 (final: concentration: 100 .mu.M) for 1 hour under
gentle shaking in the dark at 4.degree. C. in reaction buffer (50
mM Hepes, 150 mM sodium chloride, 5 mM calcium chloride, pH 6.0)
and quenched by the addition of an aqueous solution of cysteine
(final concentration: 1 mM) for 15 min. The reaction mixture is
subsequently subjected to UF/DF. To the retentate a 20 fold molar
excess of Aminooxy reagent and aniline (a nucleophilic catalyst,
final concentration: 10 mM) are added. The coupling reaction is
performed for 2 hours at room temperature in the dark under gentle
shaking. The excess of aminooxy reagent is removed by means of HIC.
The conductivity of the reaction mixture is 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, 0.01%
Tween 80, pH 6.9) and loaded onto a column filled with 53 ml
Butyl-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, 0.01% Tween 80, pH 7.4. Finally the PSA-rFIX containing
fractions are collected and subjected to UF/DF by use of a 30 kD
membrane made of regenerated cellulose (Millipore, Billerica,
Mass.). The preparation is analytically characterized by measuring
total protein (BCA) and FVIII chromogenic activity. For the
PSA-rFVIII conjugate a specific activity of 80% in comparison to
native rFVIII is determined.
Example 28
PEGylation of a Blood Coagulation Protein Using Branched PEG
[0184] PEGylation of a blood coagulation proteins (such as FIX,
FVIII and FVIIa as described in Examples 22-25) may be extended to
a branched or linear PEGylation reagent as described in Example 21,
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 Val 1 5 10 15 Gln Gly Asn Leu Glu Arg Glu Cys Met
Glu Glu Lys Cys Ser Phe Glu 20 25 30 Glu Pro Arg Glu Val Phe Glu
Asn Thr Glu Lys Thr Thr Glu Phe Trp 35 40 45 Lys Gln Tyr Val Asp
Gly Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn 50 55 60 Gly Gly Ser
Cys Lys Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro 65 70 75 80 Phe
Gly Phe Glu Gly Lys Asn Cys Glu Leu Asp Val Thr Cys Asn Ile 85 90
95 Lys Asn Gly Arg Cys Glu Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys
100 105 110 Val Val Cys Ser Cys Thr Glu Gly Tyr Arg Leu Ala Glu Asn
Gln Lys 115 120 125 Ser Cys Glu Pro Ala Val Pro Phe Pro Cys Gly Arg
Val Ser Val Ser 130 135 140 Gln Thr Ser Lys Leu Thr Arg Ala Glu Ala
Val Phe Pro Asp Val Asp 145 150 155 160 Tyr Val Asn Pro Thr Glu Ala
Glu Thr Ile Leu Asp Asn Ile Thr Gln 165 170 175 Gly Thr Gln Ser Phe
Asn Asp Phe Thr Arg Val Val Gly Gly Glu Asp 180 185 190 Ala Lys Pro
Gly Gln Phe Pro Trp Gln Val Val Leu Asn Gly Lys Val 195 200 205 Asp
Ala Phe Cys Gly Gly Ser Ile Val Asn Glu Lys Trp Ile Val Thr 210 215
220 Ala Ala His Cys Val Glu Thr Gly Val Lys Ile Thr Val Val Ala Gly
225 230 235 240 Glu His Asn Ile Glu Glu Thr Glu His Thr Glu Gln Lys
Arg Asn Val 245 250 255 Ile Arg Ala Ile Ile Pro His His Asn Tyr Asn
Ala Ala Ile Asn Lys 260 265 270 Tyr Asn His Asp Ile Ala Leu Leu Glu
Leu Asp Glu Pro Leu Val Leu 275 280 285 Asn Ser Tyr Val Thr Pro Ile
Cys Ile Ala Asp Lys Glu Tyr Thr Asn 290 295 300 Ile Phe Leu Lys Phe
Gly Ser Gly Tyr Val Ser Gly Trp Ala Arg Val 305 310 315 320 Phe His
Lys Gly Arg Ser Ala Leu Val Leu Gln Tyr Leu Arg Val Pro 325 330 335
Leu Val Asp Arg Ala Thr Cys Leu Arg Ser Thr Lys Phe Thr Ile Tyr 340
345 350 Asn Asn Met Phe Cys Ala Gly Phe His Glu Gly Gly Arg Asp Ser
Cys 355 360 365 Gln Gly Asp Ser Gly Gly Pro His Val Thr Glu Val Glu
Gly Thr Ser 370 375 380 Phe Leu Thr Gly Ile Ile Ser Trp Gly Glu Glu
Cys Ala Met Lys Gly 385 390 395 400 Lys Tyr Gly Ile Tyr Thr Lys Val
Ser Arg Tyr Val Asn Trp Ile Lys 405 410 415 Glu Lys Thr Lys Leu Thr
420
* * * * *
References