U.S. patent application number 15/407183 was filed with the patent office on 2017-08-10 for method and compositions for specifically detecting physiologically acceptable polymer molecules.
The applicant listed for this patent is Baxalta GmbH, Baxalta Incorporated. Invention is credited to Herbert Gritsch, Juergen Siekmann, Peter Turecek, Katalin Varadi, Susanne Vejda, Alfred Weber.
Application Number | 20170227557 15/407183 |
Document ID | / |
Family ID | 40475047 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170227557 |
Kind Code |
A1 |
Turecek; Peter ; et
al. |
August 10, 2017 |
METHOD AND COMPOSITIONS FOR SPECIFICALLY DETECTING PHYSIOLOGICALLY
ACCEPTABLE POLYMER MOLECULES
Abstract
The present invention relates to a method for determining the
amount of a physiologically acceptable polymer molecule bound to a
protein, an antibody or other composition being capable of
specifically binding to a physiologically acceptable polymer
molecule, and a kit containing said antibody or composition.
Inventors: |
Turecek; Peter;
(Klosterneuburg, AT) ; Siekmann; Juergen;
(Austria, AT) ; Weber; Alfred; (Vienna, AT)
; Gritsch; Herbert; (Vienna, AT) ; Varadi;
Katalin; (Vienna, AT) ; Vejda; Susanne;
(Vienna, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baxalta Incorporated
Baxalta GmbH |
Bannockburn
Glattpark (Opfikon) |
IL |
US
CH |
|
|
Family ID: |
40475047 |
Appl. No.: |
15/407183 |
Filed: |
January 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14026146 |
Sep 13, 2013 |
9547016 |
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15407183 |
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12342405 |
Dec 23, 2008 |
8557534 |
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14026146 |
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61009327 |
Dec 27, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2430/60 20130101;
G01N 33/86 20130101; G01N 33/5308 20130101; G01N 33/564
20130101 |
International
Class: |
G01N 33/86 20060101
G01N033/86; G01N 33/564 20060101 G01N033/564; G01N 33/53 20060101
G01N033/53 |
Claims
1. A method for determining the number of water soluble polymer
molecules bound to a protein or protein complex in a
polymer-protein conjugate, comprising the steps of detecting
binding between (i) a polymer:protein conjugate having one or more
polymers bound to the protein and (ii) an antibody that
specifically binds said polymer, said antibody detectable when
bound to said polymer:protein conjugate, wherein the number of
polymers in the polymer:protein conjugate correlates with levels of
antibody detected bound to the polymer:protein conjugate when
compared to a known control.
2. The method of claim 1, wherein the antibody comprises a
detectable label.
3. The method of claim 2, wherein the detectable label is selected
from the group consisting of an enzyme, a radioactive label, a
fluorophore, an electron dense reagent, biotin, digoxigenin,
haptens, and proteins which are made detectable by addition of any
of these labels.
4. The method of claim 1, wherein the polymer:protein conjugate is
bound to a carrier matrix prior to binding with the antibody.
5. The method of claim 4, wherein the matrix is selected from the
group consisting of a microcarrier, a particle, a membrane, a
strip, paper, a film, a bead or a plate.
6. The method of claim 4, wherein the level of antibody detected is
measured as absorbance of the detectable label.
7. The method of claim 1, wherein the polymer:protein conjugate is
isolated using sodium dodecylsulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to a membrane prior to
the detecting.
8. The method of claim 7, wherein the number of polymers in the
polymer:protein conjugate is calculated based on the molecular
weight of the protein-polymer conjugate compared to a known
control.
9. The method of claim 7, wherein the molecular weight of the
polymer-protein complex correlates with the protein subunit
comprising the polymer molecule.
10. The method of claim 1, wherein the protein or protein complex
is a blood clotting factor or a blood clotting factor complex.
11. The method of claim 10 wherein the blood clotting factor or
blood clotting factor complex is human.
12. The method of claim 10 wherein the blood clotting factor is
selected from the group consisting of Factor II, Factor III, Factor
V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor
XII, Facter XIII, von Willebrand Factor, protein C and antithrombin
III.
13. The method of claim 11 wherein the blood clotting factor
complex is FactorVIII:VWF.
14. The method of claim 1, wherein the water soluble polymer is
releasable.
15. The method of claim 1, wherein the water soluble polymer is
hydrolyzable.
16. The method of claim 1, wherein the polymer is selected from the
group consisting of poly(alkylene glycol), poly(propylene glycol),
copolymers of ethylene glycol and propylene glycol,
poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides),
poly(.alpha.-hydroxy acid), poly(vinyl alcohol),
polyphosphasphazene, polyoxazoline, and
poly(N-acryloylmorpholine).
17. The method of claim 1, wherein the water soluble polymer is
polyethylene glycol (PEG) or a derivative thereof.
18. The method of claim 17, wherein the PEG is from 3 to 100
kDa.
19. The method of claim 18, wherein the PEG has a molecular weight
in a range of about 5 kDa to about 60 kDa.
20. The method of claim 18, wherein the PEG has a molecular weight
in a range of about 5 kDa to about 40 kDa.
21. The method of claim 18, wherein the PEG has a molecular weight
in a range of about 5 kDa to about 15 kDa.
22. The method of claim 18, wherein the PEG has a molecular weight
in a range of about 5 kDa to about 10 kDa.
23. A method for determining the number of water soluble polymer
molecules bound to a protein or a protein complex comprising,
contacting said polymer with an antibody that specifically binds
said polymer, said antibody detectable when bound to said polymer,
wherein the number of polymers bound by the antibody correlates
with levels of antibody detected bound when compared to a known
control.
24. The method of claim 1 or 23, wherein the antibody is a
polyclonal antibody.
25. The method of claim 1 or 23, wherein the antibody is a
monoclonal antibody.
Description
[0001] This application is a Continuation of U.S. patent
application Ser. No. 14/026,146, filed Sep. 13, 2013 (now issued as
U.S. Pat. No. 9,547,016), which is a Continuation of U.S. patent
application Ser. No. 12/342,405, filed Dec. 23, 2008 (now issued as
U.S. Pat. No. 8,557,534), which claims the priority benefit of U.S.
Provisional Patent Application No. 61/009,327, filed Dec. 27, 2007,
both of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for determining
the amount of a physiologically acceptable polymer molecule bound
to a protein, an antibody being capable of specifically binding to
a physiologically acceptable polymer molecule, and a kit containing
said antibody.
BACKGROUND OF THE INVENTION
[0003] The in vivo function of a protein is improved by binding it
to a physiologically acceptable polymer molecule. In particular,
binding a physiologically active protein to a physiologically
acceptable polymer molecule has been found to substantially prolong
its in vivo half-life. For example, U.S. Pat. No. 4,970,300
describes that the conjugation of a physiologically acceptable
polymer molecule to factor VIII results in a factor VIII protein
being activable by thrombin and having a substantially decreased
antigenicity and immunoreactivity and a substantially increased in
vivo disappearance time in the bloodstream of a mammal.
[0004] U.S. Pat. No. 4,970,300 describes that the conjugation of a
polymer molecule (dextran) to Factor VIII (FVIII) results in a
FVIII protein activatable by thrombin, and having a substantially
decreased antigenicity and immunoreactivity and a substantially
increased in vivo retention time in the bloodstream of a mammal.
International patent application WO 94/15625 describes that
conjugating factor VIII to a physiologically acceptable polymer
molecule improves the in vivo function of factor VIII (i) by
increasing its resistance to in vivo hydrolysis and thus prolonging
its activity after administration, (ii) by significantly prolonging
its circulating life in vivo over unmodified protein, and (iii) by
increasing its absorption time into the blood stream. U.S. Pat. No.
6,037,452 describes FVIII and Factor IX (FIX) conjugates, where the
protein is covalently bound to a poly(alkylene oxide) through
carbonyl-groups in the protein. Further, improving the in vivo
function of factor IX by binding it to physiologically acceptable
polymer molecules, in particular poly(ethylene glycol) ("PEG"), has
been described in international patent application WO 94/29370. A
PEGylated FVIII that retains specific activity was disclosed in
International Patent Publication WO/2007/126808.The conjugation of
physiologically acceptable polymer to an active agent such as a
protein is performed by preparing stable polymer-protein conjugates
or polymer-protein conjugates in which the physiologically
acceptable polymer is attached to the protein via releasable
covalent bonds (pro-drug concept), i.e. a hydrolyzable or
releaseable linker. For example, a releasable PEG moiety has been
developed using a 9-flourenemethoxycarbonyl (FMOC) conjugation
system containing two PEG chains (Nektar Inc., Huntsville Ala.). In
addition an N-hydroxysuccinimide ester (NHS) group, which is useful
for the chemical modification of lysine residues of the protein,
may be linked to the fluorene ring system via the methoxycarbonyl
group to generate the releasable PEG moiety. International Patent
Publication WO 2008/082669 (incorporated herein by reference)
describes a series of PEGylated recombinant FVIII variants based on
the releasable PEG concept.
[0005] However, at present no reliable method for the quantitative
determination of physiologically acceptable polymer molecules bound
to proteins or nanoparticles is available apart from insensitive
colorimetric methods (Nag et al. 1997, Anal Biochem 250:35-43),
which allow only an estimation of the content of physiologically
acceptable polymer molecules. Moreover, monoclonal antibodies for
the determination of PEG concentrations have been disclosed (U.S.
Pat. No. 6,617,118), but so far no system is available for the
reliable determination of the amount of physiologically acceptable
polymer molecule bound to a protein.
[0006] Therefore, a need exists for a new system to determine the
amount of a physiologically acceptable polymer molecule, in
particular PEG, bound to a protein, particularly a physiologically
active protein.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a method for determining
the amount of a physiologically acceptable polymer molecule bound
to a protein. Additionally, an antibody being capable of
specifically binding to a physiologically acceptable polymer
molecule wherein for example said polymer molecule is present bound
to a protein is provided according to the present invention.
Further, the present invention relates to the use of said antibody
for determining the amount of a physiologically acceptable polymer
molecule bound to a protein.
[0008] In one aspect, the invention provides a method for
determining the amount of a physiologically acceptable polymer
molecule bound to a protein, comprising the steps of: (a) providing
at least one protein bound to at least one physiologically
acceptable polymer molecule; (b) providing at least one antibody
being capable of specifically binding to said physiologically
acceptable polymer molecule; (c) bringing the antibody of step (b)
into contact with the protein of step (a) under conditions suitable
for binding said antibody to the at least one polymer molecule
bound to said protein; and (d) detecting a formation of a complex
between the antibody and the physiologically acceptable polymer
molecule.
[0009] In one embodiment, in step (a) the protein bound to at least
one physiologically acceptable polymer molecule is immobilized on a
substrate or carrier matrix.
[0010] In a further embodiment, the antibody is selected from the
group consisting of a polyclonal antibody and a monoclonal
antibody.
[0011] In another embodiment, the protein is von Willebrand factor
(VWF) or a derivative thereof. In a further embodiment, the protein
is Factor VIII or a derivative thereof.
[0012] In some embodiments, the physiologically acceptable polymer
molecule is selected from the group consisting of poly(alkylene
glycol), poly(propylene glycol), copolymers of ethylene glycol and
propylene glycol, poly(oxyethylated polyol), poly(olefinic
alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides),
poly(.alpha.-hydroxy acid), poly(vinyl alcohol),
polyphosphasphazene, polyoxazoline, and poly(N-acryloylmorpholine).
In a related embodiment, the physiologically acceptable polymer
molecule is poly(ethylene glycol) (PEG) or a derivative
thereof.
[0013] In another aspect, the invention contemplates, an antibody
being capable of specifically binding to a physiologically
acceptable polymer molecule. In one embodiment, the antibody is a
polyclonal antibody.
[0014] In a related embodiment, physiologically acceptable polymer
molecule is bound to a protein. In a further embodiment, the
protein is von Willebrand factor (VWF) or a derivative thereof. In
another embodiment, the physiologically acceptable polymer molecule
is selected from the group consisting of poly(alkylene glycol),
poly(propylene glycol), copolymers of ethylene glycol and propylene
glycol, poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides),
poly(.alpha.-hydroxy acid), poly(vinyl alcohol),
polyphosphasphazene, polyoxazoline, and poly(N-acryloylmorpholine).
In a related embodiment, the physiologically acceptable polymer
molecule is poly(ethylene glycol) (PEG) or a derivative
thereof.
[0015] In a further aspect, the invention provides a kit for
determining the amount of a physiologically acceptable polymer
molecule bound to a protein, comprising an antibody as described
herein.
[0016] In another aspect, the invention provides a method for
determining the number of physiologically acceptable polymer
molecules bound to a protein or protein complex in a
polymer-protein conjugate, comprising the steps of detecting
binding between (i) a polymer:protein conjugate having one or more
polymers bound to the protein and (ii) an antibody that
specifically binds said polymer, said antibody detectable when
bound to said polymer:protein conjugate, wherein the number of
polymers in the polymer:protein conjugate correlates with levels of
antibody detected bound to the polymer:protein conjugate when
compared to a known control.
[0017] In one embodiment, the antibody comprises a detectable
label. In a related embodiment, the detectable label is selected
from the group consisting of an enzyme, a radioactive label, a
fluorophore, an electron dense reagent, biotin, digoxigenin,
haptens, and proteins which are made detectable by addition of any
of these labels.
[0018] In a further embodiment, the polymer:protein conjugate is
bound to a carrier matrix prior to binding with the antibody. In
certain embodiments, the carrier matrix is selected from the group
consisting of a microcarrier, a particle, a membrane, a strip,
paper, a film, a bead or a plate. In a related embodiment, the
polymer:protein conjugate is isolated using sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a
membrane prior to the detecting. In a further embodiment, the
molecular weight of the polymer-protein complex correlates with the
protein subunit comprising the polymer molecule.
[0019] In yet another embodiment, the level of antibody detected is
measured as absorbance of the detectable label. In a related
embodiment, the number of polymers in the polymer:protein conjugate
is calculated based on the molecular weight of the protein-polymer
conjugate compared to a known control. Exemplary methods to measure
polymer molecules for a known control include, but are not limited
to size exclusion chromatography, high performance liquid
chromatography (HPLC) and mass spectrometry.
[0020] In one embodiment of the invention, the protein or protein
complex is a blood clotting factor or a blood clotting factor
complex. In a related embodiment, the blood clotting factor or
blood clotting factor complex is human. In a still further
embodiment, the blood clotting factor is selected from the group
consisting of Factor II, Factor V, Factor VII, Factor VIII, Factor
IX, Factor X, Factor XI, Factor XII, Factor XIII, von Willebrand
Factor, protein C, antithrombin III, and activated forms thereof.
In another embodiment, the blood clotting factor complex is
FactorVIII:VWF.
[0021] In certain embodiments, the polymer is releasable. In a
related embodiment, the polymer is hydrolyzable. In one embodiment,
the physiologically acceptable molecule is attached to the protein
or protein complex via a linker.
[0022] In one embodiment, the polymer is selected from the group
consisting of poly(alkylene glycol), poly(propylene glycol),
copolymers of ethylene glycol and propylene glycol,
poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides), poly(hydroxy
acid), such as poly(.alpha.-hydroxy acid) and poly(.beta.-hydroxy
acid), poly(vinyl alcohol), polyphosphasphazene, polyoxazoline, and
poly(N-acryloylmorpholine).
[0023] In a related embodiment, the polymer is polyethylene glycol
(PEG) or a derivative thereof. In another embodiment, the PEG is
from about 3 to about 200 kDa. In a further embodiment, the PEG has
a molecular weight in a range of about 5 kDa to about 60 kDa. In
another embodiment, the PEG has a molecular weight in a range of
about 5 kDa to about 40 kDa. In still another embodiment, the PEG
has a molecular weight in a range of about 5 kDa to about 15 kDa.
And in a still further embodiment, the PEG has a molecular weight
in a range of about 5 kDa to about 10 kDa. Additional PEG
compositions contemplated for use herein include, but are not
limited to, PEG in the range of from about 5 to about 150 kDa,
about 5 to about 120 kDa, from about 10 to about 100 kDa, from
about 20 to about 50 kDa, and from about 5 to about 25 kDa, as well
as PEG having a molecular weight of about 5 kDa, about 10 kDa,
about 15 kDa, about 20 kDa, about 25 kDa, is about 30 kDa, about 35
kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about
60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa,
about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa, about 110
kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa,
about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, or
about 200 kDa.
[0024] In another aspect, the invention provides a method for
determining the number of physiologically acceptable polymer
molecules bound to a protein or a protein complex or free in
solution comprising, contacting said polymer with an antibody that
specifically binds said polymer, said antibody detectable when
bound to said polymer, wherein the number of polymers bound by the
antibody correlates with levels of antibody detected bound when
compared to a known control.
[0025] In a related aspect, the invention contemplates a method for
determining the number of physiologically acceptable polymer
molecules bound to a protein or a protein complex, contacting said
protein or protein complex with an antibody that specifically binds
said protein or protein complex, said antibody detectable when
bound to said protein or protein complex, wherein the number of
polymers bound by the antibody correlates with levels of antibody
detected bound when compared to a known control.
[0026] In related embodiments, the method of the invention is
carried out using an ELISA technique. It is contemplated that the
ELISA reagents are used as follows, wherein the first antibody
listed is the antibody bound to the substrate and the second
antibody bound in the antibody that is detectable. Exemplary assays
useful to detect the number of polymers bound to a protein or
protein complex include an anti-polymer-anti-protein detection
method, an anti-protein-anti-polymer detection method, or an
anti-polymer-anti-polymer detection method, wherein the
anti-polymer antibody is the same antibody for each binding step,
or is a different polymer-specific antibody for each step. In a
related embodiment, the assay is carried out using only an
anti-polymer specific antibody or an anti-protein-specific
antibody.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIGS. 1A-1B show a direct Enzyme Linked Immunosorbent Assay
(ELISA) on the antigen HSAP-2-SS (PEGylated human serum albumin
(hSA)). Rabbits were inoculated with preparations of the antigen
HSAP-2-h-SS having about 380 .mu.g/ml protein and a PEG
concentration of 250 .mu.g/ml. Serum samples of all animals were
taken before the start and after 3 and 4 weeks and were
subsequently tested for detectable antibody formation against the
antigen HSAP-2-h-SS. The antigen HSAP-2-h-SS is coated on a surface
in 0.1 M carbonate at pH 9.6 at 1 .mu.g/ml. The samples are diluted
in PBS-gelatin buffer and incubated with the wells and subsequently
with a goat anti-rabbit IgG-HRP antibody using Single Incubation
Multilayer Immune Technique (SIMIT). The optical density (OD)
(vertical axis) is shown for the log dilution (horizontal axis) of
the respective samples. , SPF (normal rabbit serum); .diamond. ,
Pool 0 (4 animals before); , Pool 3 weeks (4 animals); , Pool 4
weeks (4 animals).
[0028] FIGS. 2A-2C show the inhibition of the direct ELISA on the
antigen HSAP-2-SS by PEG. Rabbits were immunized with the antigen
HSAP-2-SS and serum samples are prepared as described in FIG. 1.
The antigen HSAP-2-h-SS is coated on a surface in 0.1 M carbonate
at pH 9.6 at 1 .mu.g/ml. The samples were diluted in PBS-gelatin
buffer or PBS-gelatin-1% PEG 5000 buffer (+1% PEG) and incubated
with the wells and subsequently with a goat anti-rabbit IgG-HRP
antibody (SIMIT). The optical density (OD) (vertical axis) is shown
for the log dilution (horizontal axis) of the respective samples.
.quadrature., 3 weeks+1% PEG; .box-solid., 3 weeks; .largecircle.,
4 weeks+1% PEG; , 4 weeks.
[0029] FIG. 3 shows the direct ELISA on a PEG-modified plate.
Rabbits were immunized with the antigen HSAP-2-SS and serum samples
are prepared as described in FIG. 1. A substrate (NUNC Maxisorp
F96) is coated with mPEG-NPC 5000 at 1 mg/ml in 15 mM HEPES 2 hours
at room temperature and then blocked with PBS-gelatin (5 mg/ml).
The samples were diluted in PBS-gelatin buffer and incubated with
the wells and subsequently with a goat anti-rabbit IgG-HRP antibody
(SIMIT). The optical density (OD) (vertical axis) is shown for the
log dilution (horizontal axis) of the respective samples. The
optical density (OD) (vertical axis) is shown for the log dilution
(horizontal axis) of the respective samples. , Pool 3 week; , Pool
SPF (normal rabbit serum).
[0030] FIGS. 4A-4B show the direct ELISA on VWF and PEG-VWF.
Rabbits were immunized with the antigen HSAP-2-SS and serum samples
are prepared as described FIG. 1. A substrate is coated with
PEGylated VWF (PEG-VWF) in 0.1 M carbonate at pH 9.6, another
substrate is coated with recombinant VWF (rVWF-12) in 0.1 M
carbonate at pH 9.6. The samples were diluted in PBS-gelatin buffer
and incubated with the wells and subsequently with a goat
anti-rabbit IgG-HRP antibody (SIMIT). The optical density (OD)
(vertical axis) is shown for the log dilution (horizontal axis) of
the respective samples. , Pool 3 week (Coat: PEG-VWF); , Pool 3
week (Coat: rVWF-12).
[0031] FIGS. 5A-5C show the ELISA for the detection of
VWF-PEGylation. A substrate (NUNC Maxisorp F96) was coated with
anti-VWF antibody and incubated with decreasing amounts of
PEGylated VWF followed by an incubation with an anti-PEG peroxidase
conjugate. The bound peroxidase was detected by a color reaction
with SureBlue and the signal intensity is correlated with the
concentration of PEGylated VWF in the dilution. The optical density
(OD) (vertical axis) is shown for the log mU anti-VWF antibody/ml
dilution (horizontal axis) of the respective samples. , wP-005-1-SS
a (A); .DELTA., wP-005-1-SS e (E); .diamond., wP-005-1-SS f (F); ,
wP-005-1-SS g (G). Sample A represents the native rVWF before
modification whereas the preparations E, F and G were prepared
using the PEGylation reagent PEG-SS-5K in the molar concentrations
of 1 mM, 2.5 mM and 7.5 mM.
[0032] FIG. 6 shows inhibition of the rVWF-PEG detection when free
PEG 5000 is added to the culture.
[0033] FIG. 7 shows dose-response curves of a PEG-PEG ELISA.
[0034] FIG. 8 illustrates the specificity of a PEG-PEG ELISA.
[0035] FIG. 9 shows the strong detection of PEG protein using the
PEG-protein ELISA, as shown with stable PEGylated rVWF.
[0036] FIG. 10 illustrates the strong detection of PEGylated
protein using the PEG-protein ELISA, as shown with releasable
PEGylated rVWF.
[0037] FIG. 11 illustrates the specificity of the PEG-protein ELISA
for protein-bound PEG as shown with PEGylated rVWF.
[0038] FIG. 12 shows the specificity of the PEG-rFVIII ELISA.
[0039] FIG. 13 is a comparison of detection of different anti-FVIII
peroxidase conjugates in the PEG-FVIII ELISA assay.
[0040] FIGS. 14A-14B show the detection of PEG-rFVIII ELISA in the
plasma of FVIII-deficient mice and in rat plasma.
[0041] FIG. 15 is a comparison of the ELISA assay in the detection
of PEGylated rFVIII preparations with different degree of
PEGylation.
[0042] FIG. 16. shows the influence of free PEG on the PEG-rFVIII
ELISA.
[0043] FIG. 17 depicts the ability of the PEG-rFVIII ELISA assay to
measure PEG release from a releasable PEGylated rFVIII preparation
and demonstrates the ELISA is capable to differentiate PEGylated
FVIII molecules with different degrees of PEGylation.
[0044] FIG. 18 shows that PEGylated protein was detectable using
the sensitive ECL method in all applied concentrations.
[0045] FIGS. 19A-19B are a comparison of the levels of detection of
PEGylated protein diluted in buffer (FIG. 2A) or in human plasma
(FIG. 2B).
[0046] FIG. 20 illustrates that the method detects the change in
degree of PEGylation of the PEGylated rFVIIa over time.
[0047] FIGS. 21A-21B show that the method is able to differentiate
between degrees of PEGylation (FIG. 4A, PD=3.7, FIG. 4B, PD=6),
wherein a higher PEGylation degree resulted in a stronger
signal.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention is directed to methods for determining
the amount of a physiologically acceptable polymer molecule bound
to a protein.
[0049] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
following references provide one of skill with a general definition
of many of the terms used in this invention: Singleton, et al.,
DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE
CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988);
THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger, et al. (eds.),
Springer Verlag (1991); and Hale and Marham, THE HARPER COLLINS
DICTIONARY OF BIOLOGY (1991).
[0050] Each publication, patent application, patent, and other
reference cited herein is incorporated by reference in its entirety
to the extent that it is not inconsistent with the present
disclosure.
[0051] It is noted here that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise.
[0052] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0053] The term "sample" as used herein refers to any sample
containing at least one protein bound to at least one
physiologically acceptable polymer molecule, such as any fluid or
solution originating from a process for preparing pharmaceutical
products.
[0054] The term "protein" as used herein refers to any protein,
protein complex or polypeptide, including recombinant proteins,
protein complexes and polypeptides composed of amino acid residues
linked via peptide bonds. Proteins may be obtained by isolation of
a protein from in vivo, by synthetic methods or obtained via
recombinant DNA technology. Synthetic polypeptides are synthesized,
for example, using an automated polypeptide synthesizer. A
recombinant protein used according to the present invention may be
produced by any method known in the art as described herein below.
In one embodiment, the protein is a physiologically active protein,
including a therapeutic protein or a biologically active derivative
thereof. The term "biologically active derivative" refers to a
modification of a protein having substantially the same functional
and/or biological properties of the parent protein. The term
"protein" typically refers to large polypeptides. The term
"peptide" typically refers to short polypeptides. As used herein,
polypeptide, protein and peptide are used interchangeably. A
"protein complex" refers to a molecule that is comprised of at
least one protein bound to at least one other protein. Examples of
protein complexes include, but are not limited to, a protein bound
to a cofactor or chaperone protein, ligand-receptor complexes and
multisubunit proteins such as integrins and other cell surface
receptors comprises of multiple protein subunits.
[0055] As used herein a "fragment" of a polypeptide refers to any
portion of the polypeptide smaller than the full-length polypeptide
or protein expression product. Fragments are typically deletion
analogs of the full-length polypeptide wherein one or more amino
acid residues have been removed from the amino terminus and/or the
carboxy terminus of the full-length polypeptide. Accordingly,
"fragments" are a subset of deletion analogs described below.
[0056] As used herein an "analog" or "derivative" (which may be
used interchangeably) refers to a polypeptide substantially similar
in structure and having the same biological activity, albeit in
certain instances to a differing degree, to a naturally-occurring
molecule. 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, (ii) insertion or
addition of one or more amino acids at one or more termini
(typically an "addition" analog) of the polypeptide and/or one or
more internal regions (typically an "insertion" analog) 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. Substitutions are
conservative or non-conservative based on the physico-chemical or
functional relatedness of the amino acid that is being replaced and
the amino acid replacing it.
[0057] In one aspect, an analog exhibits about 70% sequence
similarity but less than 100% sequence similarity with a given
compound, e.g., a peptide. Such analogs or derivatives are, in one
aspect, comprised of non-naturally occurring amino acid residues,
including by way of example and not limitation, homoarginine,
ornithine, penicillamine, and norvaline, as well as naturally
occurring amino acid residues. Such analogs or derivatives are, in
another aspect, composed of one or a plurality of D-amino acid
residues, or contain non-peptide interlinkages between two or more
amino acid residues. The term "derived from" as used herein refers
to a polypeptide or peptide sequence that is a modification
(including amino acid substitution or deletion) of a wild-type or
naturally-occurring polypeptide or peptide sequence and has one or
more amino acid substitutions, additions or deletions, such that
the derivative sequence shares about 70% but less than 100%
sequence similarity to the wild-type or naturally-occurring
sequence. In one embodiment, the derivative may be a fragment of a
polypeptide, wherein the fragment is substantially homologous
(i.e., at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, or at least 95% homologous) over a length of at least 5,
10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of the wild-type
polypeptide.
[0058] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0059] Optimal alignment of sequences for comparison is conducted,
in certain embodiments, by the local homology algorithm of Smith
& Waterman, Adv. Appl. Math. 2:482 (1981), by the homology
alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970), by the search for similarity method of Pearson &
Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection. One example of a useful algorithm is PILEUP, which uses
a simplification of the progressive alignment method of Feng &
Doolittle, J. Mol. Evol. 35:351-360 (1987) and is similar to the
method described by Higgins & Sharp, CABIOS 5:151-153 (1989).
Another algorithm useful for generating multiple alignments of
sequences is Clustal W (Thompson et al., Nucleic Acids Research 22:
4673-4680 (1994)). An example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm (Altschul et al., J. Mol. Biol. 215:403-410
(1990); Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1989); Karlin & Altschul, Proc. Natl. Acad. Sci. USA
90:5873-5787 (1993)). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information.
[0060] Substitutions are conservative or non-conservative based on
the physico-chemical or functional relatedness of the amino acid
that is being replaced and the amino acid replacing it.
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 set out below.
TABLE-US-00001 CONSERVATIVE SUBSTITUTIONS 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
[0061] Alternatively, exemplary conservative substitutions are set
out immediately below.
TABLE-US-00002 CONSERVATIVE SUBSTITUTIONS II ORIGINAL EXEMPLARY
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
[0062] As used herein a "variant" refers to a protein or analog
thereof that is modified to comprise additional chemical moieties
not normally a part of the molecule. Such moieties improve, in
various aspects, the molecule's solubility, absorption, biological
half-life, etc. The moieties alternatively decrease the toxicity of
the molecule and eliminate or attenuate any undesirable side effect
of the molecule, etc. Moieties capable of mediating such effects
are disclosed in Remington's Pharmaceutical Sciences (1980).
Procedure for coupling such moieties to a molecule are well known
in the art. In certain aspects, without limitation, variants are
polypeptides that are modified by glycosylation, PEGylation, or
polysialylation.
[0063] As used herein, "naturally-occurring," as applied to a
protein or polypeptide, refers to a protein found in nature. For
example, a polypeptide or polynucleotide sequence that is present
in an organism (including viruses) that are isolated from a source
in nature and which has not been intentionally modified by man in
the laboratory is naturally-occurring. The terms
"naturally-occurring" and "wild-type" are used interchangeably
throughout.
[0064] As used herein, "plasma-derived," as applied to a protein or
polypeptide, refers to a naturally-occurring polypeptide or
fragment thereof that is found in blood plasma or serum of a
subject.
[0065] The term "physiologically acceptable polymer molecule" as
used herein refers to polymer molecules which are substantially
soluble in aqueous solution or may be present in form of a
suspension and have substantially no negative impact to mammals
upon administration of a polymer-protein conjugate in a
pharmaceutically effective amount and are regarded as
biocompatible. In one embodiment, physiologically acceptable
molecules comprise from 2 to about 1000, or from about 2 to about
300 repeating units. Exemplary physiologically acceptable polymers
include, but are not limited to, poly(alkylene glycols) such as
polyethylene glycol (PEG), poly(propylene glycol) ("PPG"),
copolymers of ethylene glycol and propylene glycol and the like,
poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides),
poly(.alpha.-hydroxy acid), poly(vinyl alcohol),
polyphosphasphazene, polyoxazoline, poly(N-acryloylmorpholine),
poly(alkylene oxide) polymers, poly(maleic acid), poly(DL-alanine),
polysaccharides, such as carboxymethylcellulose, dextran,
hyaluronic acid and chitin, poly(meth)acrylates, and combinations
of any of the foregoing.
[0066] The physiologically acceptable polymer molecule is not
limited to a particular structure and, in certain aspects, is
linear (e.g. alkoxy PEG or bifunctional PEG), branched or
multi-armed (e.g. forked PEG or PEG attached to a polyol core),
dendritic, or with degradable linkages. Moreover, the internal
structure of the polymer molecule are, in still other
aspects,organized in any number of different patterns and are
selected from the group consisting of, without limitation,
homopolymer, alternating copolymer, random copolymer, block
copolymer, alternating tripolymer, random tripolymer, and block
tripolymer.
[0067] The term "linker" refers to a molecular fragment that links
the physioloigically acceptable polymer to a biologically active
molecule. The fragment typically has two functional groups that can
be coupled to or activated to react with another linker or directly
with the biologically active nucleophile. As an example,
.omega.-aminoalkanoic acid such as lysine is commonly used. In the
present invention, linkers includes stable, releasable and
hydrolyzable linkers.
[0068] The expression "protein bound to at least one
physiologically acceptable polymer molecule" as used herein
includes a protein covalently bound or non-covalently bound by
interactions such as ionic, hydrophobic, affinity, bioaffinity
interactions, to one or more polymer molecules. In various
embodiments, the polymer molecule is coupled to the protein by use
of bifunctional reagents and via a spacer arm. In addition, the
polymer molecule is coupled to the protein by affinity interaction.
For example, the protein, in certain embodiments, is biotinylated
and avidin or streptavidin conjugated polymer molecules can be
bound to the protein. Further, polyclonal or monoclonal antibodies
as well as fragments thereof are bound to a polymer molecule, and
then this complex can be bound to the protein. Polymer molecules
are also bound to the protein also by enzymatic methods such as,
for example, the transfer of saccharides with
polyglycosyltransferase (U.S. Pat. No. 6,379,933) or
glycopegylation (US 2004 0132640). Another approach is the binding
of polymer molecules to the protein on the basis of their
biological function, like for example the binding of PEGylated
collagens or collagen fragments to the A1 and A3 domains of the VWF
protein. For this purpose, in some embodiments, collagens from type
I and III, e.g. from human placenta, showing a strong interaction
with the VWFare used. In certain embodiments, the binding of the
polymer molecule is irreversible or reversible under physiological
conditions after an in vivo-application of the protein.
[0069] The term "PEGylated" as used herein refers to a protein,
protein complex or polypeptide bound to one or more PEG moieties.
The term "PEGylation" as used herein refers to the process of
binding one or more PEGs to a protein. In one embodiment, the
molecular weight of said PEG is in the range of from 3 to 200 kDa,
from 5 to 120 kDa, from 10 to 100 kDa, from 20 to 50 kDa, from 5 to
60 kDA, from 5 to 40 kDa, from 5 to 25 kDa, from 5 to 15 kDa, or
from 5 to 10 kDa.
[0070] The term "specifically binds" or is "specific for" a
physiologically acceptable polymer refers to the ability of a
binding agent to recognize and bind a physiologically acceptable
polymer, but not other compounds (or other antigens). For example,
an antibody "specific for" its cognate antigen indicates that the
variable regions of the antibodies recognize and bind the compound
of interest with a detectable preference (i.e., able to distinguish
the compound of interest from other known compounds of the similar
structure or composition, by virtue of measurable differences in
binding affinity, despite the possible existence of localized
sequence identity or homology if the antibody is specific for a
polypeptide, or similarity between compounds). It will be
understood that specific antibodies may also interact with other
proteins (for example, S. aureus protein A or other antibodies in
ELISA techniques) through interactions with sequences outside the
variable region of the antibodies, and in particular, in the
constant region of the molecule. Screening assays to determine
binding specificity of an antibody for use in the methods of the
invention are well known and routinely practiced in the art. For a
comprehensive discussion of such assays, see Harlow et al. (Eds),
Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold
Spring Harbor, N.Y. (1988), Chapter 6. Antibodies for use in the
invention can be produced using any method known in the art.
[0071] A "detectable label" or a "detectable moiety" is a
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, chemical, or other physical means. For
example, labels suitable for use in the present invention include,
for example, radioactive labels (e.g., .sup.32P), fluorophores
(e.g., fluorescein), electron dense reagents, enzymes (e.g., as
commonly used in an ELISA), biotin, digoxigenin, or haptens and
proteins which are made detectable, e.g., by incorporating a
radiolabel into the hapten or peptide, or used to detect antibodies
specifically reactive with the hapten or peptide.
[0072] The term "substrate" or "carrier matrix" does not mean any
specific limitations, and relates, for example, to an insoluble
polymer material, which can be an organic polymer, such as
polyamide or a vinyl polymer (e.g. poly(meth)acrylate, polystyrene
and polyvinyl alcohol, or derivatives thereof), a natural polymer
such as cellulose, dextrane, agarose, chitin and polyamino acids,
or an inorganic polymer, such as glass or metallohydroxide. In
certain embodiments, the substrate is in the form of a
microcarrier, particles, membranes, strips, paper, film, pearls,
beads or plates, such as microtiter plates. In one aspect, the
protein bound to at least one physiologically acceptable polymer
molecule is immobilized on the substrate directly by covalent
coupling or via a carrier such as a linker molecule or an antibody
immobilized on the substrate.
[0073] "Pharmaceutical composition" refers to a composition
suitable for pharmaceutical use in subject animal, including humans
and mammals. A pharmaceutical composition comprises a
pharmacologically effective amount of a polymer-polypeptide
conjugate and also comprises a pharmaceutically acceptable carrier.
A pharmaceutical composition encompasses a composition comprising
the active ingredient(s), and the inert ingredient(s) that make up
the carrier, as well as any product which results, directly or
indirectly, from combination, complexation or aggregation of any
two or more of the ingredients, or from dissociation of one or more
of the ingredients, or from other types of reactions or
interactions of one or more of the ingredients. Accordingly, the
pharmaceutical compositions of the present invention encompass any
composition made by admixing a compound or conjugate of the present
invention and a pharmaceutically acceptable carrier.
[0074] "Pharmaceutically acceptable carrier" refers to any of the
standard pharmaceutical carriers, buffers, and excipients, such as
a phosphate buffered saline solution, 5% aqueous solution of
dextrose, and emulsions, such as an oil/water or water/oil
emulsion, and various types of wetting agents and/or adjuvants.
Suitable pharmaceutical carriers and formulations are described in
Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co.,
Easton, 1995). Preferred pharmaceutical carriers depend upon the
intended mode of administration of the active agent. Typical modes
of administration include enteral (e.g., oral) or parenteral (e.g.,
subcutaneous, intramuscular, intravenous or intraperitoneal
injection; or topical, transdermal, or transmucosal
administration). A "pharmaceutically acceptable salt" is a salt
that is formulated into a compound or conjugate for pharmaceutical
use including, e.g., metal salts (sodium, potassium, magnesium,
calcium, etc.) and salts of ammonia or organic amines.
[0075] "Pharmaceutically acceptable" refers to a material which is
not biologically or otherwise undesirable, i.e., the material may
be administered to an individual without causing any undesirable
biological effects or interacting in a deleterious manner with any
of the components of the composition in which it is contained.
[0076] One aspect of the present invention relates to a method for
determining the amount of a physiologically acceptable polymer
molecule bound to a protein, comprising the steps of:
[0077] (a) providing at least one protein bound to at least one
physiologically acceptable polymer molecule;
[0078] (b) providing at least one antibody being capable of
specifically binding to said physiologically acceptable polymer
molecule;
[0079] (c) bringing the antibody of step (b) into contact with the
protein of step (a) under conditions suitable for binding said
antibody to the at least one polymer molecule bound to said
protein; and
[0080] (d) detecting a formation of a complex between the antibody
and the physiologically acceptable polymer molecule.
[0081] The complex between the antibody and the polymer molecule is
detected by methods well known in the art. Examples for the
detection of the above mentioned complex include, but are not
limited to, the use of a labelled antibody directed against the
antibody being capable of specifically binding to the
physiologically acceptable polymer molecule or the antibody being
capable of specifically binding to a physiologically acceptable
polymer molecule is covalently linked to a detectable label which
is any suitable detectable label known in the art. The detection
method for measuring the detectable label is, for example, and
without limitation, selected from the group consisting of an enzyme
assay, a chromogenic assay, a lumino assay, a fluorogenic assay,
and a radioimmune assay. The reaction conditions to perform
detection of the detectable label depend upon the detection method
selected. It is within the knowledge of the person skilled in the
art to choose the optimal parameters, such as buffer system,
temperature and pH for the respective detection system to be
used.
[0082] The quantification of the detectable label resulting in the
determination of the amount of the physiologically acceptable
polymer molecules bound to the protein is carried out by standard
methods. For example, in one aspect, the antibody being capable of
specifically binding to the physiologically acceptable polymer
molecule is conjugated to an enzyme (e.g., a peroxidase), and for
detection, an enzymatic substrate reaction is carried out. The
amount of physiologically acceptable polymer molecules is
calculated from a calibration curve obtained by a protein of
interest bound to the physiologically acceptable polymer molecules
defined amounts. The amounts of physiologically acceptable polymer
molecules bound to the protein of interest can are obtained, for
example, by evaluating data from SDS-gel electrophoresis and
determining the mass increase after binding of the physiologically
acceptable polymer molecules.
[0083] In one aspect, the antibody according to the present
invention is selected from the group consisting of a polyclonal
antibody, a chimeric antibody, a monoclonal antibody derived by
conventional hybridoma techniques, and an antibody or antibody
fragment obtained by recombinant techniques, e.g. phage display or
ribosome display. In one embodiment of the present invention, the
antibody is a polyclonal antibody.
[0084] According to the present invention, the term "protein" does
not underlie a specific restriction and may include any protein,
protein complex or polypeptide, including recombinant proteins,
protein complexes and polypeptides obtained via recombinant DNA
technology. The recombinant protein used according to the present
invention may be produced by any method known in the art. This may
include any method known in the art for (i) the production of
recombinant DNA by genetic engineering, e.g. via reverse
transcription of RNA and/or amplification of DNA, (ii) the
introduction of recombinant DNA into prokaryotic or eukaryotic
cells by transfection, e.g. via electroporation or microinjection,
(iii) the cultivation of said transformed cells, e.g. in a
continuous or batchwise manner, (iv) the expression of the protein,
e.g. constitutive or upon induction, and (v) the isolation of the
protein, e.g. from the culture medium or by harvesting the
transformed cells, in order to (vi) obtain purified recombinant
protein, e.g. via anion exchange chromatography or affinity
chromatography.
Proteins and Protein Complexes
[0085] Proteins contemplated for use in the compositions include
physiologically active proteins useful for administration to a
subject. In one embodiment, the physiologically active protein is a
therapeutic protein. The physiologically active protein, is in one
aspect, a protein or any fragment of such that still retains some,
substantially all, or all of the therapeutic or biological activity
of the protein. In some embodiments, the protein is one that, if
not expressed or produced or if substantially reduced in expression
or production, would give rise to a disease. Preferably, the
protein is derived or obtained from a mammal.
[0086] In various embodiments of the invention, when the
physiologically active protein conjugated to a physiologically
acceptable polymer is a protein or fragment thereof possessing a
biological activity of the protein, the physiologically active
protein has an amino acid sequence identical to the amino acid
sequence to the corresponding portion of the unconjugated human or
mammalian protein. In other embodiments, the physiologically active
protein of the conjugate is a protein native to the species of the
human or mammal. In other embodiments, the protein or fragment
thereof, is substantially homologous (i.e., at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical in amino acid sequence over a
length of at least 10, 25, 50, 100, 150, or 200 amino acids, or the
entire length of the active agent) to a native sequence of the
corresponding human or mammalian protein.
Methods of Making a Protein
[0087] Methods for making recombinant proteins are well-known in
the art. Methods of producing cells, including mammalian cells,
which express DNA or RNA encoding a recombinant protein are
described in U.S. Pat. Nos. 6,048,729, 5,994,129, and 6,063,630.
The teachings of each of these applications are incorporated herein
by reference in their entirety.
[0088] In one embodiment, a nucleic acid construct used to express
a polypeptide or fragment, or analog thereof is one which is
expressed extrachromosomally (episomally) in the transfected
mammalian cell or one which integrates, either randomly or at a
pre-selected targeted site through homologous recombination, into
the recipient cell's genome. A construct which is expressed
extrachromosomally comprises, in addition to polypeptide-encoding
sequences, sequences sufficient for expression of the protein in
the cells and, optionally, for replication of the construct. It
typically includes a promoter, a polypeptide-encoding DNA sequence
and a polyadenylation site. The DNA encoding the protein is
positioned in the construct in such a manner that its expression is
under the control of the promoter. Optionally, the construct may
contain additional components such as one or more of the following:
a splice site, an enhancer sequence, a selectable marker gene under
the control of an appropriate promoter, and an amplifiable marker
gene under the control of an appropriate promoter.
[0089] In those embodiments in which the DNA construct integrates
into the cell's genome, it includes the polypeptide-encoding
nucleic acid sequences. Optionally, it can include a promoter and
an enhancer sequence, a polyadenylation site or sites, a splice
site or sites, nucleic acid sequences which encode a selectable
marker or markers, nucleic acid sequences which encode an
amplifiable marker and/or DNA homologous to genomic DNA in the
recipient cell to target integration of the DNA to a selected site
in the genome (targeting DNA or DNA sequences).
Host Cells
[0090] Host cells used to produce recombinant proteins are
bacterial, yeast, insect, non-mammalian vertebrate, or mammalian
cells; the mammalian cells include, but are not limited to,
hamster, monkey, chimpanzee, dog, cat, bovine, porcine, mouse, rat,
rabbit, sheep and human cells. The host cells include immortalized
cells (a cell line) or non-immortalized (primary or secondary)
cells and include any of a wide variety of cell types, such as, but
not limited to, fibroblasts, keratinocytes, epithelial cells (e.g.,
mammary epithelial cells, intestinal epithelial cells), ovary cells
(e.g., Chinese hamster ovary or CHO cells), endothelial cells,
glial cells, neural cells, formed elements of the blood (e.g.,
lymphocytes, bone marrow cells), muscle cells, hepatocytes and
precursors of these somatic cell types.
[0091] Commonly used host cells include prokaryotic cells such as
gram negative or gram positive bacteria, i.e., any strain of E.
coli, Bacillus, Streptomyces, Saccharomyces, Salmonella, and the
like; eukaryotic cells such as CHO (Chinese hamster ovary) cells;
baby hamster kidney (BHK) cells; human kidney 293 cells; COS-7
cells; insect cells such as D. Mel-2, Sf4, Sf5, Sf9, and Sf21 and
High 5; plant cells and various yeast cells such as Saccharomyces
and Pichia.
[0092] Host cells containing the polypeptide-encoding DNA or RNA
are cultured under conditions appropriate for growth of the cells
and expression of the DNA or RNA. Those cells which express the
polypeptide are identified, using known methods, and the
recombinant protein isolated and purified, using known methods;
either with or without amplification of polypeptide production.
Identification is carried out, for example and without limitation,
through screening genetically modified mammalian cells displaying a
phenotype indicative of the presence of DNA or RNA encoding the
protein, such as PCR screening, screening by Southern blot
analysis, or screening for the expression of the protein. Selection
of cells having incorporated protein-encoding DNA may be
accomplished by including a selectable marker in the DNA construct
and culturing transfected or infected cells containing a selectable
marker gene under conditions appropriate for survival of only those
cells that express the selectable marker gene. Further
amplification of the introduced DNA construct is affected, in
certain aspects, by culturing genetically modified cells under
conditions appropriate for amplification (e.g., culturing
genetically modified cells containing an amplifiable marker gene in
the presence of a concentration of a drug at which only cells
containing multiple copies of the amplifiable marker gene can
survive).
[0093] In one example of the present invention, the protein is a
physiologically active protein, protein complex or polypeptide,
particularly a therapeutic protein, or a biologically active
derivative thereof. As used herein, the term "biologically active
derivative" includes any derivative of a protein, protein complex
or polypeptide having substantially the same functional and/or
biological properties of said protein, protein complex or
polypeptide, such as binding properties, and/or the same structural
basis, such as a peptidic backbone or a basic polymeric unit.
[0094] Recombinant proteins which are physiologically active
proteins or therapeutic proteins include, but are not limited to,
cytokines, growth factors, therapeutic coagulation proteins or
blood clotting factors, enzymes, chemokines, soluble cell-surface
receptors, cell adhesion molecules, antibodies, hormones,
cytoskeletal proteins, matrix proteins, chaperone proteins,
structural proteins, metabolic proteins, and other therapeutic
proteins known to those of skill in the art. Exemplary recombinant
proteins which are used as therapeutics include, but are not
limited to, Factor VIII, Factor VIII:C, Antihemophilic Factor,
Factor VII, Factor IX and von Willebrand factor, erythropoietin,
interferons, insulin, CTLA4-Ig, alpha-glucocerebrosidase,
alpha-glucosidase, follicle stimulating hormone, anti-CD20
antibody, anti-HER2 antibody, anti-CD52 antibody, TNF receptor, and
others known in the art. See, for example, Physicians Desk
Reference, 62.sup.nd Edition, 2008, Thomson Healthcare, Montvale,
N.J.
[0095] In one embodiment, the protein is a therapeutic coagulation
factor or blood (clotting) factor, including but not limited to,
Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X,
Factor XI, Factor XII, Factor XIII, von Willebrand Factor, protein
C, antithrombin III, and activated forms of any one of these
proteins. In a related embodiment, the protein complex comprises
one or more blood factors. Exemplary protein complexes of blood
factos include a complex between FVIII and VWF.
Blood Factors
[0096] In one specific example of the present invention, the
protein is a plasma-derived (plasmatic) and/or recombinant von
Willebrand factor (VWF) or a biologically active derivative
thereof. The term "plasma-derived VWF (pVWF)" includes mature VWF
obtained from a mammal. One biologically active derivative of said
pVWF is pro-VWF which contains the pro-peptide. In one example of
the present invention the protein is selected from the group
consisting of immature VWF including the precursor VWF molecule
(pre-pro-VWF) synthesized by endothelial cells and megakaryocytes,
the VWF propeptide (pro-VWF), and mature plasma-derived VWF
obtained upon cleavage of the signal peptide and pro-peptide,
respectively, of the precursor molecule. Further examples of
biologically active derivatives of plasmatic VWF include pro-drugs
which are processed or converted into the biologically active form,
or are biologically active as such, truncated forms, forms having
deletions, forms having substitutions, forms having additions other
than pro-forms, fragments of the mature form, chimeric forms, and
forms having post-translational modifications as compared to the
natural form. The term "recombinant VWF (rVWF)" includes VWF
obtained via recombinant DNA technology having optionally a
glycosylation pattern which is pharmacologically acceptable.
Specific examples thereof include VWF without A2 domain thus
resistant to proteolysis (Lankhof et al., Thromb
Haemost.;77:1008-1013,1997) and the VWF fragment from Val 449 to
Asn 730 including the glycoprotein Ib-binding domain and binding
sites for collagen and heparin (Pietu et al., Biochem Biophys Res
Commun.;164:1339-1347, 1989).
[0097] von Willebrand Factor exists in plasma in a series of
multimer forms of a molecular weight of from 1.times.10.sup.6 to
20.times.10.sup.6 Dalton. VWF (Genbank Accession No. NP_000543) is
a glycoprotein primarily formed in the endothelial cells of mammals
and subsequently secreted into circulation. In this connection,
starting from a polypeptide chain having a molecular weight of
approximately 220 kD, a VWF dimer having a molecular weight of 550
kD is produced in the cells by the formation of several sulfur
bonds. Further polymers of the VWF with increasing molecular
weights, up to 20 million Dalton, are formed by the linking of VWF
dimers. It is presumed that particularly the high-molecular VWF
multimers have an essential importance in blood coagulation.
[0098] VWF syndrome manifests clinically when there is either an
underproduction or an overproduction of VWF. Overproduction of VWF
causes increased thrombosis (formation of a clot or thrombus inside
a blood vessel, obstructing the flow of blood) while reduced levels
of, or lack of, high-molecular forms of VWF causes increased
bleeding and an increased bleeding time due to inhibition of
platelet aggregation and wound closure.
[0099] A VWF deficiency may also cause a phenotypic hemophilia A
since VWF is an essential component of functional Factor VIII. In
these instances, the half-life of Factor VIII is reduced to such an
extent that its function in the blood coagulation cascade is
impaired. Patients suffering from von Willebrand disease (VWD) or
VWF syndrome frequently exhibit a Factor VIII deficiency. In these
patients, the reduced Factor VIII activity is not the consequence
of a defect of the X chromosomal gene, but an indirect consequence
of the quantitative and qualitative change of VWF in plasma. The
differentiation between hemophilia A and vWD may normally be
effected by measuring the VWF antigen or by determining the
ristocetin-cofactor activity. Both the VWF antigen content and the
ristocetin cofactor activity are lowered in most vWD patients,
whereas they are normal in hemophilia A patients. VWF products for
the treatment of VWF syndrome include, but are not limited to:
HUMATE-P; and, IMMUNATE.RTM., INNOBRAND.RTM., and 8Y.RTM., which
therapies comprising FVIII/VWF concentrate from plasma.
[0100] In a related embodiment, the protein is Factor VIII. Factor
VIII (FVIII) is a blood plasma glycoprotein of about 260 kDa
molecular mass produced in the liver of mammals (Genbank Accesion
No. NP_000123). It is a critical component of the cascade of
coagulation reactions that lead to blood clotting. Within this
cascade is a step in which Factor IXa, in conjunction with FVIII,
converts Factor X (Genbank Accession No. NP_000495) to an activated
form, Factor Xa. FVIII acts as a cofactor at this step, being
required with calcium ions and phospholipid for the activity of
Factor IXa. The two most common hemophilic disorders are caused by
a deficiency of functional FVIII (Hemophilia A, about 80% of all
cases) or functional Factor IXa (Hemophilia B or Christmas Factor
disease). 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 Factor X (FX) activation by
enhancing the rate of activation in the presence of calcium and
phospholipids or cellular membranes.
[0101] 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, 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.
[0102] 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 assay. 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.
[0103] Until recently, the standard treatment of Hemophilia A
involved frequent infusion of preparations of FVIII concentrates
derived from the plasmas of human donors. While this replacement
therapy is generally effective, such treatment puts patients at
risk for virus-transmissible diseases such as hepatitis and AIDS.
Although this risk has been reduced by further purification of
FVIII from plasma by immunopurification using monoclonal
antibodies, and by inactivating viruses by treatment with either an
organic solvent or heat, such preparations have greatly increased
the cost of treatment and are not without risk. For these reasons,
patients have been treated episodically, rather than
prophylactically. A further complication is that about 15% of
patients develop inhibitory antibodies to plasma-derived FVIII.
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 can usually
be achieved by giving FVIII two to three times a week.
[0104] An important advance in the treatment of Hemophilia A was
the isolation of cDNA clones encoding the complete 2,351 amino acid
sequence of human FVIII (see, Wood et al, Nature, 312: 330 (1984)
and U.S. Pat. No. 4,757,006) and the provision of the human FVIII
gene DNA sequence and recombinant methods for its production. FVIII
products for the treatment of hemophilia include, but are not
limited to: ADVATE.RTM. (Antihemophilic Factor (Recombinant),
Plasma/Albumin-Free Method, rAHF-PFM), recombinant Antihemophilic
Factor (BIOCLATE.TM., GENARC.RTM., HELIXATE FS.RTM., KOATE.RTM.,
KOGENATE FS.RTM., RECOMBINATE.RTM.): MONOCLATE-P.RTM., purified
preparation of Factor VIII:C, Antihemophilic Factor/von Willebrand
Factor Complex (Human) HUMATE-P.RTM. and ALPHANATE.RTM.,
Anti-hemophilic Factor/von Willebrand Factor Complex (Human); and
HYATE C.RTM., purified pig Factor VIII. ADVATE.RTM., is produced in
CHO-cells and manufactured by Baxter Healthcare Corporation. No
human or animal plasma proteins or albumin are added in the cell
culture process, purification, or final formulation of
ADVATE.RTM..
[0105] Factor VII (proconvertin), a serine protease enzyme, is one
of the central proteins in the blood coagulation cascade (Genbank
Accession No. NP_000122). The main role of Factor VII (FVII) is to
initiate the process of coagulation in conjunction with tissue
factor (TF). Upon vessel injury, TF is exposed to the blood and
circulating Factor VII. Once bound to TF, FVII is activated to
FVIIa by different proteases, among which are thrombin (Factor
IIa), activated Factor X and the FVIIa-TF complex itself.
Recombinant human Factor VIIa (NOVOSEVEN.RTM.) has been introduced
for use in uncontrollable bleeding in hemophilia patients who have
developed inhibitors against replacement coagulation factor.
[0106] Factor IX (FIX, Christmas Factor) (Genbank Accession No.
NP_000124) is a serine protease that is inactive unless activated
by Factor XIa or Factor VIIa (of the tissue factor pathway). When
activated into Factor IXa, it acts by hydrolyzing an
arginine-isoleucine bond in Factor X to form Factor Xa. Factor VIII
is a required cofactor for FIX protease activity (Lowe G D, Br. J.
Haematol. 115: 507-13, 2002). Deficiency of Factor IX causes
hemophilia B or Christmas disease.
[0107] Additional blood factors include Factor II (thrombin)
(Genbank Accession No. NP_000497), deficiencies of which cause
thrombosis and dysprothrombinemia; Factor V, (Genbank Accession No.
NP_000121), deficiencies of which cause hemorrhagic diathesis or a
form of thrombophilia, which is known as activated protein C
resistance, Factor XI (Genbank Accession No. NP_000119),
deficiencies of which cause Rosenthal's syndrome (hemophilia C),
and Factor XIII subunit A (Genbank Accession No. NP_000120) and
subunit B (Genbank Accession No. NP_001985), deficiencies of which
are characterized as a type I deficiency (deficiency in both the A
and B subunits) and type II deficiency (deficiency in the A subunit
alone), either of which can result in a lifelong bleeding tendency,
defective wound healing, and habitual abortion; Factor XII (Genbank
Accession No. NP_000496); protein C (Genbank Accession No.
NP_000303); antithrombin III (Genbank Accession No. NP_000479), and
activated forms thereof.
Polypeptide Variants and Analogs
[0108] Methods of the invention are useful to rapidly detect
recombinant proteins in a sample, as well as fragments, analogs or
variants of the recombinant protein, and further may be useful to
detect naturally-occurring protein which may exist as fragments or
allelic variants in vivo wherein glycosylation differences are
detected.
[0109] Methods for preparing polypeptide fragments, analogs or
variants are well-known in the art. Fragments of a polypeptide are
prepared using methods well known in the art, including enzymatic
cleavage (e.g., trypsin, chymotrypsin) and also using recombinant
means to generate a polypeptide fragment having a specific amino
acid sequence. Fragments may be generated to comprise a
ligand-binding domain, a receptor-binding domain, a dimerization or
multimerization domain, or any other identifiable domain known in
the art.
[0110] Methods of making polypeptide analogs are also well-known.
Analogs are, in certain aspects, substantially homologous or
substantially identical to the naturally-occurring polypeptide from
which the analog is derived, and analogs contemplated by the
invention are those which retain at least some of the biological
activity of the naturally-occurring polypeptide.
[0111] Substitution analogs typically exchange one amino acid of
the wild-type for another at one or more sites within the protein,
and are, in certain aspects, designed to modulate one or more
properties of the polypeptide, such as stability against
proteolytic cleavage, without the loss of other functions or
properties. Substitutions of this kind are generally conservative.
By "conservative amino acid substitution" is meant substitution of
an amino acid with an amino acid having a side chain of a similar
chemical character. Similar amino acids for making conservative
substitutions include those having an acidic side chain (glutamic
acid, aspartic acid); a basic side chain (arginine, lysine,
histidine); a polar amide side chain (glutamine, asparagine); a
hydrophobic, aliphatic side chain (leucine, isoleucine, valine,
alanine, glycine); an aromatic side chain (phenylalanine,
tryptophan, tyrosine); a small side chain (glycine, alanine,
serine, threonine, methionine); or an aliphatic hydroxyl side chain
(serine, threonine).
[0112] Polynucleotide analogs and fragments may be readily
generated by a worker of skill to encode biologically active
fragments, variants, or mutants of the naturally occurring molecule
that possess the same or similar biological activity to the
naturally occurring molecule. Routinely practiced methods include
PCR techniques, enzymatic digestion of DNA encoding the protein
molecule and ligation to heterologous polynucleotide sequences, and
the like. For example, point mutagenesis, using PCR and other
techniques well-known in the art, may be employed to identify with
particularity which amino acid residues are important in particular
activities associated with protein activity. Thus, one of skill in
the art will be able to generate single base changes in the DNA
strand to result in an altered codon and a missense mutation.
[0113] It is further contemplated that the protein or polypeptide
is modified to make an analog which is a fusion protein comprising
a second agent which is a polypeptide. In one embodiment, the
second agent which is a polypeptide is an enzyme, a growth factor,
a cytokine, a chemokine, a cell-surface receptor, the extracellular
domain of a cell surface receptor, a cell adhesion molecule, or
fragment or active domain of a protein described above or of any
other type of protein known in the art. In a related embodiment,
the second agent is a blood clotting factor such as Factor II,
Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI,
Factor XII, Facter XIII, von Willebrand Factor, protein C,
antithrombin III, and activated forms thereof. The fusion protein
contemplated is made by chemical or recombinant techniques
well-known in the art.
[0114] Protein variants contemplated include polypeptides
chemically modified by such techniques as ubiquitination,
glycosylation, conjugation to therapeutic or diagnostic agents,
labeling (e.g., with radionuclides or various enzymes), covalent
polymer attachment such as PEGylation (derivatization with
polyethylene glycol), introduction of non-hydrolyzable bonds, and
insertion or substitution by chemical synthesis of amino acids such
as ornithine, which do not normally occur in human proteins.
Variants retain the binding properties of non-modified molecules of
the invention.
[0115] Additional polypeptide variants useful in the methods of the
present invention include polypeptides comprising polysialylate
(PSA) moieties. Methods for preparing polysialylated polypeptide
are described in U.S. Patent Publication 20060160948 and Saenko et
al., Haemophilia 12:42-51, 2006.
Physiologically Acceptable Polymers
[0116] In one embodiment, the invention contemplates chemically
modified proteins or polypeptides, which have been linked to a
chemical moiety that provides advantageous effects to production,
viability of the protein or polypeptide. For example, nonspecific
or site-specific conjugation of physiologically acceptable polymers
to polypeptides is known in the art to improve half-life by
potentially reducing immunogenicity, renal clearance, and/or
improving protease resistance.
[0117] A physiologically acceptable polymer molecule includes
polymer molecules which, for example, are substantially soluble in
an aqueous solution or may be present in form of a suspension and
have substantially no negative impact, such as side effects, to
mammals upon administration of a polymer molecule-protein-conjugate
in a pharmaceutically effective amount and are regarded as
biocompatible. There is no particular limitation to the
physiologically acceptable polymer molecule used according to the
present invention.
[0118] The polymer molecules are typically characterized as having
for example from about 2 to about 1000, or from about 2 to about
300 repeating units. Examples of such polymer molecules include,
but are not limited to, poly(alkylene glycols) such as polyethylene
glycol (PEG), poly(propylene glycol) ("PPG"), copolymers of
ethylene glycol and propylene glycol and the like,
poly(oxyethylated polyol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides),
poly(.alpha.-hydroxy acid), poly(vinyl alcohol),
polyphosphasphazene, polyoxazoline, poly(N-acryloylmorpholine),
poly(alkylene oxide) polymers, poly(maleic acid), poly(DL-alanine),
polysaccharides, such as carboxymethylcellulose, dextran,
hyaluronic acid and chitin, poly(meth)acrylates, and combinations
of any of the foregoing.
[0119] For example water-soluble polymers, including but not
limited to, poly(ethylene glycol) (PEG), poly(ethylene oxide)
(PEO), polyoxyethylene (POE), polyvinyl alcohols, hydroxyethyl
celluloses, or dextrans, are commonly conjugated to proteins or
peptides to increase stability or size, etc., of a protein or
peptide.
[0120] PEG, PEO or POE refers to an oligomer or polymer of ethylene
oxide. PEGs and PEOs include molecules with a distribution of
molecular weights, i.e., polydisperse. The size distribution is
characterized statistically by its weight average molecular weight
(Mw) and its number average molecular weight (Mn), the ratio of
which is called the polydispersity index (Mw/Mn). Mw and Mn are
measured, in certain aspects, by mass spectroscopy. Most of the
PEG-protein conjugates, particularly those conjugated to PEG larger
than 1 KD, exhibit a range of molecular weights due to a
polydisperse nature of the parent PEG molecule. For example, in
case of mPEG2K (Sunbright ME-020HS, NOF), actual molecular masses
are distributed over a range of 1.5.about.3.0 KD with a
polydispersity index of 1.036. Exceptions are proteins conjugated
to MS(PEG)n (N=4, 8, 12 or 24, e.g., PEO4, PEO12)-based reagents
(Pierce), which are specially prepared as monodisperse mixtures
with discrete chain length and defined molecular weight.
[0121] The physiologically acceptable polymer molecule is not
limited to a particular structure and is, in various aspects,
linear (e.g. alkoxy PEG or bifunctional PEG), branched or
multi-armed (e.g. forked PEG or PEG attached to a polyol core),
dentritic, or with degradable linkages. Moreover, the internal
structure of the polymer molecule is organized in any number of
different patterns and is selected from the group consisting of
homopolymer, alternating copolymer, random copolymer, block
copolymer, alternating tripolymer, random tripolymer, and block
tripolymer.
[0122] In one specific example of the present invention, the
physiologically acceptable polymer molecule is PEG and derivatives
thereof. There is no specific limitation of the PEG used according
to the present invention. For example, PEG-protein conjugates
include but are not limited to linear or branched conjugates,
polymer:proteins conjugated by NHS (N-hydroxysuccinimide)- or
aldehyde-based chemistry, variants with a different chemical
linkage between the PEG chain and conjugation site, and variants
differing in lengths. The average molecular weight of the PEG will
range from about 3 kiloDalton ("kDa") to about 200 kDa, from about
5 to about 120 kDa, from about 10 to about 100 kDa, from about 20
to about 50 kDa,from about 5 kDa to about 60 kDa, from about 5 kDa
to about 40 kDa, from about 3 to about 30 kDa, from about 5 kDa to
about 25 kDa, from about 5 kDa to about 15 kDa, or from about 5 kDa
to about 10 kDa. In certain embodiments, the PEG is about 5 kDa,
about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, is about 30
kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about
55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa,
about 80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100
kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa,
about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about
190 kDa, or about 200 kDa.
[0123] The invention contemplates PEG-protein conjugates selected
from the group consisting of linear PEG-protein conjugates that are
NHS-conjugated and range in length from --(CH2-CH2-O)n-, where n=1
to 2000, linear PEG-protein conjugates that are aldehyde-conjugated
and range in length from --(CH2-CH2-O)n-, where n=1 to 2000,
two-arm branched PEG-protein conjugates that are NHS-conjugated and
range in length, from 3 to 100 kDa in mass, and three-arm branched
PEG-protein conjugates that are NHS-conjugated. The invention also
contemplates PEG-protein conjugates that contain different chemical
linkages (--CO(CH2)n-, and --(CH2)n- where n=1 to 5) between its
conjugation site and the PEG chain. The invention further
contemplates charged, anionic PEG-protein conjugates to reduce
renal clearance, including but not limited to carboxylated,
sulfated and phosphorylated compounds (anionic) (Caliceti &
Veronese, Adv Drug Deliv Rev 2003 55(10):1261-77; Perlman et al., J
Clin Endo Metab 2003 88(7):3227-35; Pitkin et al., Antimicrob
Agents Chemother 1986 29(3): 440-44; Vehaskari et al., Kidney Intl
1982 22 127-135). In a further embodiment, the peptide is
optionally conjugated to a moiety including a bisphosphonate, a
water-soluble polymer such as PEG or PEO, carbohydrates, fatty
acids, or further amino acids.
[0124] Macromolecule chemical modification is, in one aspect,
performed in a non-specific fashion (leading to mixtures of
modified species) or in a site-specific fashion (based on wild-type
macromolecule reactivity-directed modification and/or
site-selective modification using a combination of site-directed
mutagenesis and chemical modification) or, alternatively, using
expressed protein ligation methods (Curr Opin Biotechnol.
13(4):297-303 (2002)).
[0125] To discover if the in vivo therapeutic half-life of a
peptide would benefit from PEGylation, a variety of different
PEG-protein conjugates are synthesized, characterized in vitro and
in-vivo for pharmacokinetics. In order to both optimize the
potential effects of PEGylation a design strategy is employed
wherein polymer length, conformation, and charge of PEG is
varied.
[0126] Methods for preparing the PEGylated protein of the present
invention generally comprise the steps of (a) reacting the protein
of interest with polyethylene glycol under conditions whereby PEG
becomes attached to the N-terminus/C-terminus of the protein, and
(b) obtaining the reaction product(s). Because PEGylating a protein
might significantly alter the intrinsic activity of the protein,
different types of PEG are explored. The chemistry used for
PEGylation of protein includes, but is not limited to, the
acylation of the primary amines of the protein using the NHS-ester
of methoxy-PEG
(O-[(N-Succinimidyloxycarbonyl)-methyl]-O'-methylpolyethylene
glycol). Acylation with methoxy-PEG-NHS or methoxy-PEG-SPA results
in an amide linkage that eliminates the charge from the original
primary amine (also, Boc-PEG for C-terminus). Unlike ribosome
protein synthesis, synthetic peptide synthesis proceeds from the
C-terminus to the N-terminus. Therefore, Boc-PEG is one method
(i.e. using tert-(B)utyl (o)xy (c)arbonyl (Boc, t-Boc) synthesis)
to attach PEG to the C-terminus of the peptide (R. B. Merrifield
(1963). "Solid Phase Peptide Synthesis. I. The Synthesis of a
Tetrapeptide". J. Am. Chem. Soc. 85 (14): 2149-2154).
(F)luorenyl-(m)eth(o)xy-(c)arbonyl (FMOC) chemistry (Atherton, E.;
Sheppard, R.C. (1989). Solid Phase peptide synthesis: a practical
approach. Oxford, England: IRL Press.) is favored because it does
not require the hazardous use of hydrofluoric acid to remove
side-chain protecting groups. The present methods provide for a
substantially homogenous mixture of polymer:protein conjugate.
"Substantially homogenous" as used herein means that only
polymer:protein conjugate molecules are observed. The
polymer:protein conjugate has biological activity and the present
"substantially homogenous" PEGylated protein preparations are those
which are homogenous enough to display the advantages of a
homogenous preparation, e.g., ease in clinical application in
predictability of lot to lot pharmacokinetics.
[0127] Exemplary stable linkers that can facilitate conjugation of
the physiologically acceptable polymer to the polypeptide of
interest include, but are not limited to, amide, amine, ether,
carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether,
thioester, and dithiocarbamate linkages, such as w,w-aminoalkane,
N-carboxyalkylmaleimide, or aminoalkanoic acids, maleimidobenzoyl
sulfosuccinimide ester, glutaraldehyde, or succinic anhydride,
N-carboxymethylmaleimide N,N'-disuccinimidyl oxalate and
1,1'-bis[6-(trifluoromethy)benzo-triazolyl] oxalate.
[0128] In other embodiments, the physiologically acceptable polymer
is conjugated to the polypeptide using a releasable linker. In one
embodiment, the releasable linker is a hydrolyzable linkers A
hydrolyzable or degradable bond is a relatively weak bond that
reacts with water (i.e., is hydrolyzed) under physiological
conditions. The tendency of a bond to hydrolyze in water will
depend not only on the general type of linkage connecting two
central atoms but also on the substituents attached to these
central atoms. Methods of making conjugates comprising water
soluble polymers having hydrolyzable linkers are described in U.S.
Pat. No. 7,259,224 (Nektar Therapeutics) and U.S. Pat. No.
7,267,941 (Nektar Therapeutics and National Institutes of Health).
For example, a PEG can be prepared having ester linkages in the
polymer backbone that are subject to hydrolysis. This hydrolysis
results in cleavage of the polymer into fragments of lower
molecular weight. Appropriate hydrolytically unstable or weak
linkages include but are not limited to carboxylate ester,
phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether,
imines, orthoesters, peptides and oligonucleotides, thioesters,
thiolesters, and carbonates. Hydrolytically degradable linkages
that may be contained within the polymer backbone include
carbamate, carbonate, sulfate, and acyloxyalkyl ether linkages;
imine linkages, resulting, for example, from reaction of an amine
and an aldehyde (see, e.g., Ouchi et al., Polymer Preprints,
38(1):582-3 (1997)); carbamate, phosphate ester, hydrazone, acetal,
ketal, or orthoester linkages, including
acetone-bis-(N-maleimidoethyl)ketal linkers (MK).
[0129] In a further embodiment, the polymer molecules contemplated
for use in the PEGylation approaches described herein are selected
from among water-soluble polymers or a mixture thereof. The polymer
may have a single reactive group, such as an active ester for
acylation or an aldehyde for alkylation, so that the degree of
polymerization may be controlled. The water-soluble polymer, or
mixture thereof if desired, may be selected from the group
consisting of, for example, PEG, monomethoxy-PEG, PEO, dextran,
poly-(N-vinyl pyrrolidone), propylene glycol homopolymers, fatty
acids, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), HPMA, FLEXIMAR.TM., and
polyvinyl alcohol, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, tresyl
monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonate
PEG, cellulose, other carbohydrate-based polymers, or mixtures
thereof. In certain embodiments, the polymer selected is
water-soluble so that the protein to which it is attached does not
precipitate in an aqueous environment, such as a physiological
environment. The polymer is, in various aspects, branched or
unbranched. In one embodiment, for therapeutic use of the
end-product preparation, the polymer is pharmaceutically
acceptable. Methods for generating peptides comprising a PEG moiety
are well-known in the art. See, for example, U.S. Pat. No.
5,824,784.
[0130] In one embodiment, the reactive aldehyde is
PEG-propionaldehyde, which is water-stable, or mono-C1-C10 alkoxy
or aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714). As
used herein, PEG is meant to encompass any of the forms of PEG that
have been used to derivatize other proteins, such as mono-(C1-C10)
alkoxy- or aryloxy-polyethylene glycol. In some embodiments, the
polymer is branched or unbranched. In one embodiment, for
therapeutic use of the end-product preparation, the polymer is
pharmaceutically acceptable.
[0131] A protein bound to at least one physiologically acceptable
polymer molecule includes a protein covalently bound or
non-covalently bound by interactions such as ionic, hydrophobic,
affinity, bioaffinity interactions, to one or more polymer
molecules. In one embodiment, the polymer molecule is coupled to
the protein by use of bifunctional reagents and via a spacer arm.
In a related embodiment, the polymer molecule is coupled to the
protein by affinity interaction. For example, the protein is
biotinylated and avidin or streptavidin conjugated polymer
molecules is bound to the protein. Further, polyclonal or
monoclonal antibodies as well as fragments thereof are bound to a
polymer molecule, and then this complex is bound to the protein.
Polymer molecules are bound to the protein also by enzymatic
methods such as, for example, the transfer of saccharides with
polyglycosyltransferase (U.S. Pat. No. 6,379,933) or
glycopegylation (US 2004 0132640). Another approach is the binding
of polymer molecules to the protein on the basis of their
biological function, like for example the binding of PEGylated
collagens or collagen fragments to the A1 and A3 domains of the VWF
protein. F or this purpose, in certain aspects, collagens from type
I and III, e.g. from human placenta, showing a strong interaction
with the VWF are used. The binding of the polymer molecule is
irreversible or reversible under physiological conditions after an
in vivo-application of the protein.
[0132] In one example of the present invention, in step (a) the
protein bound to at least one physiologically acceptable polymer
molecule is immobilized on a substrate or carrier matrix, for
example by an antibody being capable of specifically binding to
said protein.
[0133] A substrate or carrier matrix does not have any specific
limitations, and relates, for example, to an insoluble polymer
material, which can be an organic polymer, such as polyamide or a
vinyl polymer (e.g. poly(meth)acrylate, polystyrene and polyvinyl
alcohol, or derivatives thereof), a natural polymer such as
cellulose, dextrane, agarose, chitin and polyamino acids, or an
inorganic polymer, such as glass or metallohydroxide. In certain
embodiments, the substrate is in the form of a microcarrier,
particles, membranes, strips, paper, film, pearls, beads or plates,
such as microtiter plates. In one aspect, the protein bound to at
least one physiologically acceptable polymer molecule is
immobilized on the substrate directly by covalent coupling or via a
carrier such as a linker molecule or an antibody immobilized on the
substrate.
Detectable Labels
[0134] In some embodiments, the protein or polymer useful in the
method of the invention is labeled to facilitate its detection. A
"label" or a "detectable moiety" is a composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
chemical, or other physical means.
[0135] Depending on the screening assay employed, the protein or
fragment thereof, or the polymer,or a portion thereof is labelled.
The particular label or detectable group used is not a critical
aspect of the invention, as long as it does not significantly
interfere with the biological activity of the conjugate. The
detectable group is any material having a detectable physical or
chemical property. Thus, a label is any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means.
[0136] Examples of labels suitable for use in the present invention
include, but are not limited to, fluorescent dyes (e.g.,
fluorescein isothiocyanate, Texas red, rhodamine, and the like),
radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g.,
horse radish peroxidase, alkaline phosphatase and others commonly
used in an ELISA), and colorimetric labels such as colloidal gold
or colored glass or plastic beads (e.g., polystyrene,
polypropylene, latex, etc.).
[0137] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. Preferably, the label in one embodiment is covalently
bound to the biopolymer using an isocyanate reagent for conjugating
an active agent according to the invention. In one aspect of the
invention, the bifunctional isocyanate reagents of the invention
are used to conjugate a label to a biopolymer to form a label
biopolymer conjugate without an active agent attached thereto. The
label biopolymer conjugate may be used as an intermediate for the
synthesis of a labeled conjugate according to the invention or may
be used to detect the biopolymer conjugate. As indicated above, a
wide variety of labels are used, with the choice of label depending
on sensitivity required, ease of conjugation with the desired
component of the assay, stability requirements, available
instrumentation, and disposal provisions. Non-radioactive labels
are often attached by indirect means. Generally, a ligand molecule
(e.g., biotin) is covalently bound to the molecule. The ligand
binds to another molecules (e.g., streptavidin) molecule, which is
either inherently detectable or covalently bound to a signal
system, such as a detectable enzyme, a fluorescent compound, or a
chemiluminescent compound.
[0138] In certain aspects, the conjugates are conjugated directly
to signal generating compounds, e.g., by conjugation with an enzyme
or fluorophore. Enzymes suitable for use as labels include, but are
not limited to, hydrolases, particularly phosphatases, esterases
and glycosidases, or oxidotases, particularly peroxidases.
Fluorescent compounds, i.e., fluorophores, suitable for use as
labels include, but are not limited to, fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
etc. Further examples of suitable fluorophores include, but are not
limited to, eosin, TRITC-amine, quinine, fluorescein W, acridine
yellow, lissamine rhodamine, B sulfonyl chloride erythroscein,
ruthenium (tris, bipyridinium), Texas Red, nicotinamide adenine
dinucleotide, flavin adenine dinucleotide, etc. Chemiluminescent
compounds suitable for use as labels include, but are not limited
to, luciferin and 2,3-dihydrophthalazinediones, e.g., luminol. For
a review of various labelling or signal producing systems that are
used in the methods of the present invention, see U.S. Pat. No.
4,391,904.
[0139] Means for detecting labels are well known to those of skill
in the art. Thus, for example, where the label is radioactive,
means for detection include a scintillation counter (e.g.,
radioimmunoassay, scintillation proximity assay) (Pitas et al.,
Drug Metab Dispos. 34:906-12, 2006) or photographic film, as in
autoradiography. Where the label is a fluorescent label, it may be
detected by exciting the fluorochrome with the appropriate
wavelength of light and detecting the resulting fluorescence (e.g.,
ELISA, immunoblot, flow cytometry, or other methods known in the
art). The fluorescence may be detected visually, by the use of
electronic detectors such as charge coupled devices (CCDs) or
photomultipliers and the like. Similarly, enzymatic labels may be
detected by providing the appropriate substrates for the enzyme and
detecting the resulting reaction product. Colorimetric or
chemiluminescent labels may be detected simply by observing the
color associated with the label. Other labeling and detection
systems suitable for use in the methods of the present invention
will be readily apparent to those of skill in the art.
[0140] In one embodiment the label, the protein:polymer conjugate
or the polymer:protein complex conjugate contemplated for use in
the method are linked to a solid support, such as a substrate or
carrier matrix, including but not limited to, a filter, a
microcarrier, a particle, a membrane, a strip, paper, a film, a
bead or a plate, or any other carrier matrix known in the art.
[0141] It is further contemplated that the labeled compounds may be
labeled and interact in solution. For example, the capture antibody
may be labeled with a fluorescent resonance energy transfer (FRET)
donor molecule and the target molecule is labeled with a FRET
acceptor molecule such that the molecules are in proximity when
binding occurs. Alternatively, the target molecule may be labeled
with the FRET donor and the antibody molecule the FRET acceptor.
Another possibility is to separate quenching and fluorescent
molecule both present on the antibody or target when target and
antibody hybridize. The target molecule is only close enough for
its label to emit if it is interacting with the reagent. This
produces a system where the molecule only emits when it interacts
with the reagent (direct monitoring). In one embodiment, a narrow
band pass filter is used to block all wavelengths except that of
the molecule's label. FRET molecule pairs are commercially
available in the art (e.g., from Invitrogen, Carlsbad, Calif.), and
may be used according to the manufacturer's protocol. FRET
emissions are detected using optical imaging techniques, such as a
CCD camera.
[0142] Another method of detecting antibody-antigen interactions is
to label it with an electron donor. This donor label would give
electrons to an electrical contact to which the reagent is bound.
See, for example, Ghindilis, A. (Biochem Soc Trans. 28:84-9, 2000)
and Dai et al. (Cancer Detect Prey. 29:233-40, 2005) which describe
enzymes useful in and methods for electro immunoassays. The
electron contact would then be read by an A to D (analog to
digital) converter and quantified. The higher the electron count
the more interactions took place.
[0143] One embodiment of a label capable of single molecule
detection is the use of plasmon-resonant particles (PRPs) as
optical reporters, as described in Schultz et al., Proc. Nat'l
Acad. Sci., 97:996-1001 (2000), incorporated herein by reference.
PRPs are metallic nanoparticles, typically 40-100 nm in diameter,
which scatter light elastically with remarkable efficiency because
of a collective resonance of the conduction electrons in the metal
(i.e., the surface plasmon resonance). The magnitude, peak
wavelength, and spectral bandwidth of the plasmon resonance
associated with a nanoparticle are dependent on the particle's
size, shape, and material composition, as well as the local
environment. By influencing these parameters during preparation,
PRPs are formed that have scattering peak anywhere in the visible
range of the spectrum. For spherical PRPs, both the peak scattering
wavelength and scattering efficiency increase with larger radius,
providing a means for producing differently colored labels.
Populations of silver spheres, for example, are reproducibly
prepared for which the peak scattering wavelength is within a few
nanometers of the targeted wavelength, by adjusting the final
radius of the spheres during preparation. Because PRPs are bright,
yet nanosized, they are used as indicators for single-molecule
detection; that is, the presence of a bound PRP in a field of view
can indicate a single binding event.
[0144] It is contemplated that the assay and the detection are
useful to determine the number of polymers bound to a protein or
protein complex, or to determine the extent of free polymer in a
solution, such as serum or plasma. The detectable signal observed
in the method correlates with the number of polymers bound to the
protein or protein complex, or free in solution when compared to a
standard having a known amount of polymer.
[0145] Therefore, in one embodiment, the invention provides a
method for determining the number of physiologically acceptable
polymer molecules bound to a protein or a protein complex or free
in solution comprising, contacting said polymer with an antibody
that specifically binds said polymer, wherein the number of
polymers bound by the antibody correlates with levels of antibody
detected bound when compared to a known control.
[0146] In an alternate embodiment, the invention contemplates a
method for determining the number of physiologically acceptable
polymer molecules bound to a protein or a protein complex,
contacting said protein or protein complex with an antibody that
specifically binds said protein or protein complex, wherein the
number of polymers bound by the antibody correlates with levels of
antibody detected bound when compared to a known control.
[0147] In related embodiments, the method of the invention is
carried out using an other detection regimens, for example, wherein
the protein and polymer specific antibodies are used in any order
as follows, wherein the first antibody listed is the antibody bound
to the carrier matrix and the second antibody bound in the antibody
that is detectable. Exemplary assays useful to detect the number of
polymers bound to a protein or protein complex include an
anti-polymer-anti-protein detection method, an
anti-protein-anti-polymer detection method, or an
anti-polymer-anti-polymer detection method, wherein the
anti-polymer antibody is the same antibody for each binding step,
or is a different polymer-specific antibody for each step. In a
related embodiment, the assay is carried out using only an
anti-polymer specific antibody or an anti-protein-specific
antibody.
Kits
[0148] As an additional aspect, the invention includes kits which
comprise one or more compounds or compositions packaged in a manner
which facilitates their use to practice methods of the invention.
In one embodiment, such a kit includes a composition comprising a
protein or protein complex conjugated to a physiologically
acceptable polymer, such as PEGylated Factor VIII, and an antibody
or other molecule that specifically detects the water soluble
polymer on the 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 related embodiments, the binding agent is
a soluble receptor, a ligand, a cofactor or another agent that
specifically binds the protein, protein complex or polymer. The kit
may optionally include reagents and buffers for preparation of the
samples for detection of the polymer-protein complex. Preferably,
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
modified blood factor composition.
[0149] In one embodiment of the present invention, the method
includes an Enzyme Linked Immunosorbent Assay (ELISA) comprising
the following steps:
[0150] (i) immobilizing an antibody being capable of specifically
binding to a protein bound to at least one physiologically
acceptable polymer molecule to an ELISA plate;
[0151] (ii) binding the protein of interest to the immobilized
antibody; and
[0152] (iii)detecting the amount of physiologically acceptable
polymer molecule bound to the protein by an antibody being capable
of specifically binding to a physiologically acceptable polymer
molecule bound to said protein of interest.
[0153] The present invention will be further illustrated in the
following examples, without any limitation thereto.
EXAMPLES
Example 1
Direct Enzyme Linked Immunosorbent Assay (ELISA) on the Antigen
HSAP-2-SS (PEGylated Human Serum Albumin (hSA))
[0154] To determine if polyclonal antibodies to PEG generated using
a PEGylated antigen injected into animals, human serum albumin
(hSA) was linked to PEG and the protein conjugate injected into
rabbits. The amount of anti-PEG antibody was then measured.
[0155] In brief, a polyclonal antibody is generated by immunization
of rabbits (Richter AW et al. 1983; Int Arch Allergy Appl Immunol
70:124-31) with PEG covalently bound to human serum albumin (HSA).
Rabbits are inoculated with preparations of the antigen HSAP-2-h-SS
with about 380 .mu.g/ml protein and a PEG concentration of 250
.mu.g/ml. Serum samples of all animals are taken before the start
and after 3 and 4 weeks and are subsequently tested for detectable
antibody formation against the antigen HSAP-2-h-SS. The antigen
HSAP-2-h-SS (PEGylated hSA) is coated in 0.1 M carbonate at pH 9.6
at 1 .mu.g/ml. The samples are diluted in PBS-gelatin buffer and
incubated with the wells and subsequently with a goat anti-rabbit
IgG-HRP antibody using Single Incubation Multilayer Immune
Technique (SIMIT) (Naser, W., J Immunol Methods. 129:151-7, 1990).
In SIMIT, the ligand (e.g., antibody) and ligand binding agent
(e.g., anti-antibody) are co-incubated in order that during a
single incubation step, multiple layers of immunoreactants are
formed thereby resulting in enhanced assay sensitivity. An antibody
formation against the antigen HSAP-2-h-SS is detectable. The
antigen can be coated directly on plate and there is an increase of
titer with time of immunization FIG. 1A).
[0156] More specifically, PEGylated hSA was prepared according to
Abuchowski et al (J Biol Chem 252: 3578-81, 1977). The PEGylated
hSA had higher molecular weight as shown by high-performance
size-exclusion chromatography and SDS-PAGE. Serum samples of all
animals were taken before the start and after 3 and 4 weeks and
pooled. These pooled samples were subsequently tested for antibody
formation against the immunization antigen by a direct ELISA.
Briefly, the PEGylated hSA was coated in 0.1 M sodium carbonate
buffer, pH 9.6 at a concentration of 1 .mu.g/mL to 96-well
polystyrene microplates (Nunc Maxisorp F96). The pooled rabbit
serum samples were diluted in phosphate-buffered saline (PBS)
containing 1 mg/mL gelatin and incubated with the wells and
subsequently with a goat anti-rabbit IgG-HRP antibody. An antibody
formation against the immunization antigen was detectable. In
addition, there was an increase of titer with time of immunization
(FIG. 1B). The same method was used to measure the antibody titers
in samples obtained in another immunization study. Table 1 shows
the blank-corrected optical densities (OD) of samples taken at the
start and after 36 and 50 days. Also in this case, the results for
the sample dilutions 1/50 and 1/100 demonstrate the formation of
IgG against the immunization antigen that increased with time.
TABLE-US-00003 TABLE 1 Anti-PEG IgG titers after immunization with
PEGylated hSA Dilution 1/50 Dilution 1/100 Rabbit d 0 d 36 d 50 d 0
d 36 d 50 1 0.000 0.699 0.651 0.000 0.480 0.260 2 0.000 0.420 0.329
0.000 0.233 0.116 3 0.000 0.162 0.084 0.000 0.098 0.022 4 0.000
0.440 0.343 0.000 0.212 0.116 5 0.000 0.423 0.408 0.000 0.196 0.115
6 0.003 0.152 0.115 0.002 0.114 0.079 Mean 0.001 0.383 0.322 0.000
0.222 0.118
[0157] These results show that a PEG conjugated hSA protein induces
the production of polyclonal antibodies from subject animals.
Example 2
Inhibition of the Direct ELISA on the Antigen HSAP-2-SS by PEG
[0158] To determine if the binding of the anti-PEG antibody was
specific for PEG, the ability of free PEG to interfere with
antibody binding was assessed.
[0159] In brief, rabbits are immunized with the antigen HSAP-2-SS
and serum samples are prepared as described above (Example 1). The
antigen HSAP-2-h-SS is coated on a surface in 0.1 M carbonate at pH
9.6 at 1 .mu.g/ml. The samples are diluted in PBS-gelatin buffer or
PBS-gelatin-1% PEG 5000 buffer (+1% PEG) and incubated with the
wells and subsequently with a goat anti-rabbit IgG-HRP antibody
(SIMIT). The binding of the antibody to the antigen (=PEGylated
hSA) obtained by the immunization of rabbits can be inhibited by
the addition of PEG 5000 to the sample dilution buffer (FIG.
2A).
[0160] More specifically, the anti-PEG specificity of the antisera
obtained by immunization with the PEGylated hSA was checked with an
inhibition study. Plates (Example 1) were coated with the
immunization antigen PEGylated hSA at a concentration of 10
.mu.g/mL. Pooled rabbit serum samples taken 3 and 4 weeks after the
start of the immunization were diluted in PBS-gelatin to obtain
dilution series ranging from 1/100 to 1/100,000. PEG 5000 was added
at a concentration of 10 mg/mL to inhibit the binding to PEGylated
hSA. Bound rabbit IgG was detected by using a goat anti-rabbit
IgG-peroxidase conjugate and the peroxidase substrate Sureblue.
Polyethylene glycol (PEG) 5000 decreased the binding of rabbit IgG
to the plate-immobilized PEGylated hSA (FIG. 2B)
[0161] These results demonstrate that the IgG contained in the
rabbit serum specifically recognized and bound to PEG. Residual
binding of rabbit IgG in the presence of PEG was caused by
antibodies directed towards hSA. These non-PEG-specific IgGs were
adsorbed by affinity chromatography on immobilized hSA.
Example 3
Direct ELISA on a PEG-Modified Plate
[0162] To determine if the anti-PEG antibody would bind PEG bound
directly to the plastic, a direct PEG ELISA was developed.
[0163] In brief, rabbits are immunized with the antigen HSAP-2-SS
and serum samples are prepared as described above (Example 1). A
substrate (NUNC Maxisorp F96) is coated with mPEG-NPC 5000 at 1
mg/ml in 15 mM HEPES 2 hours at room temperature and then blocked
with PBS-gelatin (5 mg/ml). The samples are diluted in PBS-gelatin
buffer and incubated with the wells and subsequently with a goat
anti-rabbit IgG-HRP antibody (SIMIT). A binding of the antibodies
present in the serum samples to a PEG-modified plate (NUNC Maxisorp
F96) is detected (FIG. 3).
[0164] More specifically, rabbits were immunized with PEGylated hSA
and serum samples were prepared as described above (Example 1).
Plates (Example 1) were coated with mPEG-p-nitrophenyl carbonate
(NPC; SunBio, Korea) 5000 at 1 mg/ml in 15 mM HEPES at room
temperature for 2 hours and then blocked with PBS-gelatin (5
mg/ml). The serum samples were diluted with PBS-gelatin buffer,
incubated with the wells and subsequently with a goat anti-rabbit
IgG-peroxidase. A clear binding of IgG present in the rabbit serum
samples to the PEG-modified plate was detected (FIG. 3). When the
same procedure was carried out with polylysine- and
NH.sub.2-activated plates (Costar), no reaction could be
observed.
[0165] These results demonstrate that the anti-PEG IgG contained in
the rabbit serum samples recognized and bound to PEG.
Example 4
[0166] Direct ELISA on VWF and PEG-VWF To determine if the anti-PEG
antibody will bind PEGylated proteins other than the immunization
antigen, the anti-PEG antibodies were used in an ELISA with
PEGylated von Willebrand Factor.
[0167] In brief, rabbits are immunized with the antigen HSAP-2-SS
and serum samples were prepared as described above (Example 1). A
substrate is coated with PEGylated VWF (PEG-VWF) in 0.1 M carbonate
at pH 9.6, another substrate is coated with recombinant VWF
(rVWF-12) in 0.1 M carbonate at pH 9.6. The samples are diluted in
PBS-gelatin buffer incubated with the wells and subsequently with a
goat anti-rabbit IgG-HRP antibody (SIMIT). The PEGylation of VWF is
determined as an increase in molecular weight confirmed by
SDS-PAGE. The binding of the antibodies present in the serum
samples to PEGylated recombinant VWF (rVWF) is detected. No binding
of the antibodies present in the serum samples to rVWF is observed
(FIG. 4A).
[0168] More specifically, rabbit serum samples (see Example 1) were
allowed to react with plate-immobilized rVWF and PEGylated rVWF.
PEGylated rVWF was prepared by using the PEGylation reagent as
described by Kozlowski et al (BioDrug 5: 419-29, 2001). Both
proteins were coated to polystyrene plates (Example 1). The rabbit
serum samples, taken before the immunization and after 3 weeks,
were diluted in PBS-gelatin buffer, incubated with the wells and
subsequently with a goat anti-rabbit IgG-HRP antibody. The binding
of the IgG present in the rabbit serum samples to plate-immobilized
PEGylated rVWF was detected, although the rabbits were immunized
with PEGylated hSA. No binding of the IgG present in the rabbit
serum samples to rVWF was observed (FIG. 4B).
[0169] These experiments demonstrate that the anti-PEG antibodies
do not non-specifically bind non-PEGylated protein.
Example 5
ELISA for the Detection of VWF-PEGylation
[0170] To determine the ability of the anti-PEG antibody to detect
PEGylated protein, such as PEGylated VWF, a VWF-PEG ELISA was
developed.
[0171] In brief, a substrate (NUNC Maxisorp F96) is coated with
anti-VWF antibody and incubated with decreasing amounts of
PEGylated VWF followed by an incubation with an anti-PEG peroxidase
conjugate. The bound peroxidase is detected by a color reaction
with SureBlue and the signal intensity is correlated with the
concentration of PEGylated VWF in the dilution (FIG. 5).
[0172] More specifically, the following example describes a
protein-PEG ELISA that uses a protein-specific antibody, preferably
derived from rabbit, in combination with an enzyme-conjugated
anti-PEG IgG, preferably derived from rabbits, for the detection
and the measurement of a PEGylated protein. Basically, the
PEGylated protein is captured by the plate-immobilized anti-protein
antibody and then allowed to react with an anti-PEG IgG-peroxidase
conjugate. Rabbit anti-human VWF (DakoCytomation A-0082) was
diluted 1/500 in sodium carbonate buffer, pH 9.6 and coated to a
polystyrene plate (Example 1). Alternatively, any monoclonal
antibody can be used in an appropriate dilution. Washing was done
with PBS, the dilution buffer contained gelatin at 5 mg/mL. rVWF
(sample A) and various PEGylated rVWF preparations (samples E, F,
G) were diluted with dilution buffer to a VWF:Ag concentration of
0.85 mU/mL. Sample A represents the native rVWF before modification
whereas the preparations E, F and G were prepared using the
PEGylation reagent PEG-SS-SK in the molar concentrations of 1 mM,
2.5 mM and 7.5 mM. Five further 1+1 dilutions were prepared and
incubated with the plate-immobilized anti-VWF IgG. Bound PEGylated
rVWF was detected by reaction with the anti-PEG IgG peroxidase
conjugate and the peroxidase substrate SureBlue. Table 2 shows the
slopes and the regression coefficients for the dose-response curves
of the different preparations measured. Obviously, non-PEGylated
rVWF (sample A) showed no response, whereas the linear
dose-response curves of the three PEGylated rVWF samples E, F and G
had clearly differing slopes.
TABLE-US-00004 TABLE 2 Slope and correlation coefficients of dose-
response curves of the rVWF-PEG ELISA Sample A Sample E Sample F
Sample G slope 0.000 0.4771 2.0523 4.6259 correlation coefficient
n.a. 1.000 0.992 0.995
[0173] The three PEGylated rVWF preparations showed increased
molecular weight on SDS PAGE (FIG. 5) as compared to the
non-PEGylated rVWF. In addition, higher PEG to rVWF ratios applied
for the PEGylation resulted in increased molecular weights of the
PEGylated rVWF preparations and in steeper dose-response curves.
Thus, the design described not only specifically detected
protein-bound PEG, but also allowed the differentiation of
preparations with different degrees of PEGylation.
Example 6
Specificity of the rVWF-PEG ELISA as Shown by the Inhibition With
PEG
[0174] In order to assess the specificity of the PEG assay, an
inhibition study study was carried out.
[0175] The assay was done as described above (see Example 5) using
the PEGylated rVWF preparation G with the highest degree of
PEGylation. The diluted PEGylated rVWF sample (0.85 mU/mL) was
incubated with the plate-immobilized anti-VWF antibody and then
with the anti-PEG IgG-peroxidase conjugate in the presence of PEG
5000 (50 mg/mL to 0.024 mg/mL). PEG 5000 causes a clear
dose-dependent inhibition (FIG. 6) with an IC.sub.50 of 0.18
.mu.g/mL.
Example 7
Description of a PEG-PEG ELISA
[0176] This example describes a PEG-PEG ELISA that uses the
polyclonal rabbit anti-PEG IgG for capturing and detecting
PEGylated proteins or free PEG.
[0177] Anti-albumin-depleted rabbit anti-PEG IgG was coated in 0.1
M sodium carbonate, pH 9.6 overnight to polystyrene plates (Example
1). The blocking of the plates was done with PBS, pH 6.1 containing
2% non-fat dry milk and 2 mM benzamidine, at 37.degree. C. for 3
hours. Tween 20 or other polyethoxy-containing detergents were not
used for the whole assay. Blocking buffer was used to prepare
dilution series for the following samples: mPEG2-20K-NHS (stable
20K PEGylation reagent as described by Kozlowski et al [Biodrug
2001; 5: 419-29]) and stable PEGylated rVWF (9.8 .mu.g bound PEG
per IU VWF:Ag), prepared by using this reagent; 20K-PEG2-FMOC-NHS
(branched "releasable" 20K PEG reagent, as described in
US2008/0234193) and releasable 20K-PEGylated rVWF (8.2 .mu.g bound
PEG per IU VWF:Ag) prepared by using this reagent. The PEG reagents
were dissolved in distilled water at a concentration of 10 mg/mL
and kept at room temperature overnight to hydrolyze the active
N-hydroxysuccine imide (NHS) group. The samples' dilutions were
allowed to bind to the plate-immobilized anti PEG antibody at room
temperature for 1 hour. The plates were then washed and anti-PEG
IgG peroxidase was applied. Finally, bound peroxidase activity was
measured. All samples showed linear dose-response curves (FIG. 7),
although with different sensitivities. The PEGylated rVWF
preparations could be measured in the low ng range of bound PEG.
The non-conjugated free PEG reagents after hydrolysis could also be
measured with this assay design but higher PEG concentrations were
required for the linear dose-response relation.
[0178] These findings demonstrated that the anti-PEG IgG obtained
by immunization of rabbits with 5K PEGylated hSA (i) binds not only
to 5 k PEG used for the immunization and (ii) binds to a repeating
epitope presented on the PEG chain and not to the protein-PEG
linkage region. By employing a pretreatment for the removal of
protein-bound PEG, this assay design is useful for the measurement
of free, non-conjugated PEG as it remains, for example, in the
reaction mixture after PEGylation. In addition, this assay is also
useful to measure the amounts of non-bound PEG in the purified
PEG-protein conjugate.
Example 8
Specificity of the PEG-PEG ELISA
[0179] The specificity of the PEG-PEG ELISA described above was
shown using the assay conditions described above (Example 7). In
addition, a non-PEGylated rVWF sample was analyzed using the
PEG-PEG assay and showed no response, even at more than100-times
higher VWF:Ag concentration (FIG. 8). These results demonstrate the
specificity of the anti-PEG antibody and the PEG-PEG assay.
Example 9
Description of a PEG-Protein ELISA for the Measurement of Stable
PEGylated rVWF
[0180] To determine if a PEG-specific ELISA would be a sensitive
detection method when the anti-PEG antibody was used as the capture
antibody, a PEG-protein ELISA was developed which uses an anti-PEG
antibody for capturing the PEGylated protein and a protein-specific
antibody for detecting the bound PEGylated protein.
[0181] Albumin-depleted anti-PEG IgG was diluted to about 50
.mu.g/mL with 0.1 M carbonate buffer, pH 9.6 and coated to the
wells of 96-well polystyrene microplate (Nunc Maxisorp F96). The
wells were then blocked with dilution buffer (3% non-fat dry milk
in PBS, 2 mM benzamidine; pH 6.1) at 37.degree. C. for two hours.
Serial dilutions of the samples were then loaded and incubated with
the wells at room temperature for 60 min. After washing, rabbit
anti-human VWF-peroxidase (DakoCytomation) was added and bound
peroxidase activity was measured with SureBlue. Alternatively, the
peroxidase conjugate was added to the samples and incubated without
a preceding washing step using the single incubation multilayer
immune technique (SIMIT). A stable 20K-PEGylated rVWF preparation
(see Example 7) was used. The robustness of the PEG-VWF ELISA assay
was shown by diluting this preparation in Von Willebrand deficient
(VWD) mouse plasma (final concentration of VWF in plasma was 90%)
and by the addition of PEG reagent (final concentration of PEG
reagent: 1 mg/mL at 0.5 IU PEGylated VWF) as described in Example 7
and rVWF (final concentration of rVWF: 7 IU at 5 IU PEGylated
rVWF). Linear dose-response curves were obtained for all samples in
the range of 27 to 1.7 ng/mL bound PEG (FIG. 9) when using the
sequential assay format, but also for the SIMIT format.
[0182] Neither the presence of non-conjugated PEG reagent nor a
surplus of non-PEGylated rVWF impaired the assay. Also, the matrix
of VWD mouse plasma did not interfere. Thus, the assay demonstrates
robust and sensitive detection of PEG-protein conjugates
Example 10
Description of a PEG-Protein ELISA for the Measurement of
Releasable PEGylated rVWF
[0183] The robustness study described above (see Example 9) was
also done with a releasable 20K PEGylated rVWF preparation (see
Example 7). Similar results were obtained for the releasable
20K-PEGylated rVWF preparation with a linear range of 21 to 1.3
ng/mL (FIG. 10) and no interference of any of the compounds was
detected. These data demonstrated that the linker used to attach
the PEG moiety to the protein had no impact on the
detection/measurement of the PEG-protein conjugate.
Example 11
Specificity of the PEG-Protein ELISA for Protein-Bound PEG
[0184] The specificity of the PEG-protein ELISA was shown by the
direct measurement of the non-conjugated PEG reagents and PEGylated
rVWF preparations as described above.
[0185] In both cases, stable and releasable reagents and conjugates
were used. Both PEGylated rVWF preparations showed similar,
dose-dependent responses, whereas both reagents, measured at
10-times higher concentrations, did not show dose-dependent signals
(FIG. 11). These data demonstrate that the PEG-protein ELISA
specifically detects and measures PEG-protein conjugates.
Example 12
Specificity of a PEG-rFVIII ELISA
[0186] To determine if the PEG ELISA described herein could be used
for additional blood clotting factors, the general applicable
principle of the PEG-protein ELISA was shown by analyzing a
PEGylated rFVIII preparation using the assay conditions as
described above (Example 9).
[0187] An anti-human FVIII peroxidase (Cedarlane) was used instead
of an anti-human VWF peroxidase for detecting plate-bound PEGylated
rFVIII. Results showed that the PEG-rFVIII ELISA was specific
because non-PEGylated rFVIII did not show any signal even when
analyzed at 1000-times higher FVIII:Ag concentrations (FIG.
12).
Example 13
PEG-rFVIII ELISA With Stable and Releasable PEGylated rFVIII
[0188] The specificity of the PEG ELISA was also measured for
stable and releaseable preparations of PEG-FVIII.
[0189] Albumin-depleted anti-PEG IgG was diluted to about 50
.mu.g/mL with 0.1 M carbonate buffer, pH 9.6 and coated to the
wells of a 96-well polystyrene microplate. The wells were then
blocked with dilution buffer (3% non-fat dry milk in PBS, 2 mM
benzamidine; pH 6.1) at room temperature for two hours. Serial
dilutions of the samples were then loaded and incubated with the
wells at room temperature for 60 min. After washing, sheep
anti-human FVIII-peroxidase (Cedarlane) was added and bound
peroxidase activity was measured with SureBlue. A stable and a
releasable 20K-PEGylated rFVIII preparation were used. These
preparations had concentrations of bound PEG of 115 .mu.g/mL and
301 .mu.g/mL, respectively. Table 3 shows the measuring data
obtained on analysis of these samples and gives the characteristics
of the regression curves.
TABLE-US-00005 TABLE 3 PEG-rFVIII ELISA with stable and releasable
PEGylated rFVIII Releasable PEGylated rFVIII Stable PEGylated
rFVIII Day 1 Day 2 ng PEG/mL OD ng PEG/mL plate 1 plate 2 plate 1
plate 2 57.6 1.181 75.2 0.698 0.674 0.761 0.883 28.8 0.732 37.6
0.363 0.351 0.382 0.527 14.4 0.432 18.8 0.182 0.175 0.200 0.250 7.2
0.237 9.4 0.097 0.087 0.104 0.125 3.6 0.149 4.7 0.046 0.045 0.049
0.062 slope 0.7600 slope 0.9751 0.9822 0.9791 0.9740 r 0.9992 r
0.9997 0.9999 0.9996 0.9985
[0190] The analysis of both the stable PEGylated and the releasable
PEGylated rFVIII preparation resulted in linear dose-response
curves in the nanogram range of bound PEG. In addition, the assay
had good reproducibility as shown for the releasable PEGylated
rFVIII preparation, which allows for accurate measurement of
PEGylated FVIII.
Example 14
Influence of Different Anti-FVIII Peroxidase Conjugates on the
Assay Performance
[0191] The influence of different anti-FVIII peroxidase conjugates
on the assay performance was investigated.
[0192] The PEG-rFVIII ELISA was carried out as described above (see
Example 13). Detection of anti-human FVIII peroxidase conjugates
from Asserachrom and Cedarlane were compared in the same assay
(FIG. 13). In both cases, linear dose-response relations were
obtained between signal and FVIII:Ag levels of the samples,
confirming that both conjugates could be used interchangeably.
[0193] These results suggest that the PEG ELISA is useful with any
preparation of anti-protein antibody conjugate available at an
appropriate selectivity.
Example 15
Performance of the PEG-FVIII ELISA in FVIII-Deficient Mouse Plasma
and Rat Plasma
[0194] The efficacy and sensitivity of the PEG-rFVIII ELISA was
investigated in FVIII-deficient mouse plasma and in rat plasma.
[0195] A releasable PEGylated rFVIII preparation was spiked at a
concentration equivalent to 0.5 .mu.g bound PEG/mL in the plasma of
the animals or in dilution buffer. The resulting dose-response
curves of these samples (FIG. 14) were very similar in buffer and
in the animal plasma. In addition, stable PEGylated rFVIII was
spiked to FVIII-deficient mouse plasma, diluted 1/10 and 1/20, at
levels of bound PEG of 50 ng/mL. Recoveries of 99.8% and 97.9% of
the spiked concentrations were measured. This demonstrated that the
PEG-rFVIII ELISA is useful for monitoring the pharmacokinetic of
releasable PEGylated rFVIII at high sensitivity and specificity
without requiring any specific sample pretreatment other than
appropriate sample dilution. Similar data were obtained when
samples with PEGylated rVWF were analyzed.
Example 16
Measurement of Releasable PEGylated rFVIII Preparations With
Different Degree of PEGylation
[0196] Releasable PEGylated rFVIII preparations with different
degree of PEGylation were analyzed with the PEG-FVIII ELISA.
[0197] The ELISA was done as described above (see Example 13). In
addition, the FVIII:Ag levels of these preparations were measured
using a commercially available FVIII ELISA kit. The degree of
PEGylation of these preparations was measured with a HPLC-based
method and was expressed as mol bound PEG per mol FVIII. The
PEGylated FVIII preparation was added to dilution buffer or to
FVIII-deficient mouse plasma and these samples were measured with
the PEG-FVIII ELISA The concentrations of bound PEG measured with
the PEG rFVIII ELISA were then normalized to the FVIII:Ag
concentrations of these samples and expressed as .mu.g bound PEG
per U FVIII:Ag. These FVIII:Ag-normalized PEG concentrations
correlated well in buffer and in the plasma of FVIII-deficient mice
with the degree of PEGylation as measured for the different
preparations with the HPLC-based method (FIG. 15).
[0198] These results show that the PEG-rFVIII ELISA could
discriminate between PEGylated rFVIII preparations according to
their degree of PEGylation, and comparison of the absorbance of the
samples to a known standard indicates the degree of PEGylation of
the protein sample. Additionally, these results are achieved in
buffer and also in the matrix of FVIII deficient mouse plasma as
the assay does not require any specific sample pretreatement except
appropriate dilution of the test samples. This provides a method to
measure PEGylated protein or other PEG levels in the serum of a
patient receiving PEGylated therapeutic protein.
Example 17
Influence of Free PEG on the PEG-FVIII ELISA
[0199] The possible interference of free PEG on the PEG ELISA assay
was investigated in a PEG concentration range up to 1000
.mu.g/mL.
[0200] A releasable PEGylated rFVIII preparation was mixed with
20K-PEG2-FMOC-NHS to yield final concentrations of 20, 100, 200,
500 and 1000 .mu.g/mL. The PEG reagent was dissolved in distilled
water and kept overnight to destroy the NHS reactivity before it
was added to the PEGylated rFVIII preparation. The dose-response
curves obtained for these samples were highly similar (FIG. 16) and
their slopes differed less than 10%.
[0201] This assays shows that even high levels of free PEG had no
influence on detection levels of the PEG-rFVIII ELISA.
Example 18
Measurement of PEG Release From a Releasable PEGylated rFVIII
[0202] As shown above, the PEG ELISA measures release of the PEG
polymer from the protein-PEG conjugate. To determine if the assay
can measure the rate of release, a releasable PEGylated rFVIII
preparation kept at conditions triggering the release of
protein-bound PEG was used to measure PEG release over time.
[0203] The levels of free PEG were measured with size-exclusion
chromatography. The levels of protein-bound PEG were measured with
the PEG-FVIII ELISA and related to the FVIII:Ag concentrations of
these samples. The FVIII:Ag normalized FVIII-bound PEG levels
correlated well with the levels of free PEG (see FIG. 17).
[0204] These experiments demonstrated that the PEG-FVIII ELISA was
capable of monitoring the release of PEG from a releasable
PEGylated rFVIII preparation. This assay is useful to measure the
release kinetics of PEGylated protein in vivo to patients receiving
PEGylated FVIII or other PEGylated therapeutic protein.
Example 19
Detection of PEGylated rFVIIa in Normal Pooled Rat Plasma
[0205] Alternative methods to determine the levels of PEGylation of
a protein or protein complex include detection of the
protein-polymer complex based on molecular weight of the complex
itself. This type of assay is carried out using sodium
dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)
isolation of the protein and detection of PEG molecules on the
protein using an anti-PEG Western blot detection method.
[0206] To determine the detection PEGylated protein in plasma using
this technique, samples of PEGylated FVIII were diluted in rat
plasma and PEGylated protein levels were measured.
[0207] Samples of 20-kDa-PEG-FVIIa and 40-kDa-PEG-FVIIa were
diluted to 100 .mu.g/ml, 50 .mu.g/ml, 25 .mu.g/ml, 12.5 .mu.g/ml
and 6.3 .mu.g/ml in rat plasma (Sprague Dawley), and subjected to
sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blot. Sampling buffer (NuPAGE LDS sample buffer,
Invitrogen) was added to 1 .mu.l of the product diluted in plasma
and loaded onto gradient (3-8%) tris-acetate SDS polyacrylamide
gels (NuPage Novex, 1.0 mm; Invitrogen). Electrophoresis was
performed in tris-acetate SDS running buffer under non-reducing
conditions. Proteins were blotted for 16 hours with 1.25 W at
+4.degree. C. onto polyvinylidene difluoride (PVDF, 0.2 .mu.m)
membranes (Sequi-Blot PVDF membrane, BIO-RAD, Richmond, Calif.,
USA). Afterwards, membranes were blocked in casein-TBS solution
(Pierce, Rockford, Ill., USA) for 1 hour at +37.degree. C.
[0208] Afterwards, the immunoblots were incubated with the
monoclonal rabbit anti-PEG antibody (Epitomics, Calif., USA),
diluted 1/1000 for 2 hours at room temperature. The antibody was
diluted in TBS +0.05% Tween20 (TBST) +10% casein-TBS. After 5
washing steps with TBST, each for 10 minutes, the secondary
antibody goat anti-rabbit IgG (H+L)- horseradish peroxidase (HRP)
conjugate was applied (DAKO Cytomation, Glostrup, Denmark), diluted
1/1000 in TBST/10% casein-TBS, for 1 hour at room temperature (RT).
After 5 washing steps with TBST, the blots were developed using the
enhanced chemiluminescence (ECL) Plus Detection Kit according to
the manual of the manufacturer (GE Healthcare, Buckinghamshire,
UK).
[0209] For the detection, a less sensitive ECL Western Blotting
Reagent was used to visualize the PEGylated proteins. Even with
this technique, the PEGylated protein was detectable in all applied
concentrations. The secondary antibody showed a cross-reaction with
the rat immunoglobulins (band marked with * in FIG. 18). This cross
reaction could be avoided by immunodepletion of the rat plasma for
the immunoglobulin prior application to the gel.
Example 20
Detection of PEGylated rFVIIa in Normal Human Plasma
[0210] To determine the detection of PEGylated protein in human
plasma, samples of PEGylated FVIII were diluted and PEGylated
protein levels were measured.
[0211] Samples of 20-kDa-PEG-FVIIa was diluted to 5 .mu.g/ml and
2.5 .mu.g/ml in pooled normal human (George King Bio-Medical)
plasma or in 5% HSA/HNa buffer (25 mM HEPES, 175 mM NaCl, pH 7.35).
The ECL plus detection system was used and the film was exposed for
a very short (30 seconds) time (FIG. 2B). For these samples,
SDS-PAGE using a 3-8% tris-acetate gradient gel was followed by
Western blot analysis. The ECL Plus Detection System was used to
visualize the bands.
[0212] For comparison, SDS-PAGE using 4-12% bis-tris gradient gels
followed by Western blot analysis of 100 and 50 ng of
20-kDa-PEG-FVIIa detected with anti-PEG antibody (diluted 1/300 in
TBS/0.05% non fat dry milk (BIO-RAD)) and a polyclonal sheep
anti-human FVII antibody (Affinity Biologicals, ON, Canada),
diluted 1/2000 in TBST/0.1% non fat dry milk. An alkaline
phosphatase (ALP) system was applied to visualize the proteins
(FIG. 19A).
[0213] There was no difference detectable whether the PEGylated
rFVIIa was diluted in buffer or in plasma, and only a weak cross
reaction with the human plasma was observed (FIG. 19B). These
results demonstrate that the method is appropriately sensitive to
detect low levels of conjugated protein in a sample comprising many
different proteins, such as human plasma, and is therefore useful
to detect polymer-conjugated protein a sample taken from a patient
receiving blood clotting factor to treat a clotting disorder.
Example 21
Detection of In Vitro PEG-Release of 20-kDa-PEG-rFVIIa in Normal
Human Plasma
[0214] PEGylation usually decreases the protein's biological
function. However, modifying the proteins with a reversibly-linked
PEG, which has the potential to dissociate from the protein over
time should allow liberation of the native protein, accompanied
with full restoration of its activity. This process is monitored by
measuring the increase of activity in the plasma over time.
However, the measured activity is depending on the rate of release
reaction and inactivation/elimination of the protein. This
invention is also suitable to measure the structural changes
including de-PEGylation of such a protein in a plasma matrix.
[0215] The releasable 20-kDa-PEG-rFVIIa conjugate was diluted to
0.023 .mu.g/ml in normal human plasma and incubated for 24 hours at
37.degree. C. The release of the PEG molecule was determined by
SDS-PAGE and Western Blot analysis using the specific anti-PEG
antibody as described in Example 1. As shown in FIG. 20 the amount
of di-PEGylated rFVIIa slightly decreases over time and completely
disappears after 24 hours incubation. In contrast, the mono-PEG
species shows a slight increase first and is still present after 24
hours. Thus, the methods detects sequential de-PEGylation of the
protein molecule.
[0216] These results illustrate that the present method allows for
the determination of the degree of water soluble polymer of the
surface of a protein or protein complex, and also allows for a
determination of the mechanism of release of a releasable
water-soluble polymer from the protein.
Example 22
Detection of PEGylated FVIII in Normal Human Plasma
[0217] To determine the ability of the present assay to detect a
change in the degree of PEGylation, two FVIII samples conjugated
with different PEG reagents exhibiting a differing PEGylation
degree were diluted in human plasma and the detection of the
molecules measured.
[0218] Samples were diluted in the range of 5 to 1 .mu.g/ml and
loaded onto 3-8% gradient tris-acetate SDS-polyacrylamide gels
followed by Western blot analysis. The PEGylation degree (PD) of
the stable 20-kDa PEG-FVIII conjugate is 3.7 (FIG. 21A), that of
the releasable one with the same PEG type is 6 (FIG. 21B).
[0219] As shown in FIG. 21, a higher PEGylation degree resulted in
a stronger signal using the same development conditions.
[0220] These results show that the new method to trace PEGylated
proteins in pharmacokinetic studies described herein can detect
changes in their domain structure and PEGylation degree.
[0221] Numerous modifications and variations in the invention as
set forth in the above illustrative examples are expected to occur
to those skilled in the art. Consequently only such limitations as
appear in the appended claims should be placed on the
invention.
* * * * *