U.S. patent application number 12/342646 was filed with the patent office on 2009-07-30 for recombinant vwf formulations.
This patent application is currently assigned to BAXTER INTERNATIONAL INC.. Invention is credited to PETER MATTHIESSEN, KURT SCHNECKER, HANS-PETER SCHWARZ, PETER TURECEK.
Application Number | 20090192076 12/342646 |
Document ID | / |
Family ID | 40591819 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090192076 |
Kind Code |
A1 |
MATTHIESSEN; PETER ; et
al. |
July 30, 2009 |
RECOMBINANT VWF FORMULATIONS
Abstract
The present invention provides long-term stable pharmaceutical
formulations of recombinant von-Willebrand Factor (rVWF) and
methods for making and administering said formulations.
Inventors: |
MATTHIESSEN; PETER; (VIENNA,
AT) ; TURECEK; PETER; (KLOSTERNEUBURG, AT) ;
SCHWARZ; HANS-PETER; (VIENNA, AT) ; SCHNECKER;
KURT; (VIENNA, AT) |
Correspondence
Address: |
BAXTER HEALTHCARE CORPORATION
ONE BAXTER PARKWAY, DF2-2E
DEERFIELD
IL
60015
US
|
Assignee: |
BAXTER INTERNATIONAL INC.
DEERFIELD
IL
BAXTER HEALTHCARE SA
WALLISELLEN
|
Family ID: |
40591819 |
Appl. No.: |
12/342646 |
Filed: |
December 23, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61017418 |
Dec 28, 2007 |
|
|
|
61017881 |
Dec 31, 2007 |
|
|
|
Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 38/36 20130101;
A61K 47/20 20130101; A61P 7/04 20180101; A61K 47/10 20130101; A61K
47/12 20130101; A61K 47/26 20130101; A61K 9/0019 20130101; A61K
47/22 20130101; A61K 47/02 20130101; A61K 38/1709 20130101; A61K
9/08 20130101; A61K 47/18 20130101 |
Class at
Publication: |
514/7 |
International
Class: |
A61K 38/16 20060101
A61K038/16 |
Claims
1. A stable liquid pharmaceutical formulation of a recombinant von
Willebrand Factor (rVWF) comprising: (a) a rVWF; (b) a buffering
agent; (c) one or more salts; (d) optionally a stabilizing agent;
and (e) optionally a surfactant; wherein said rVWF comprises a
polypeptide selected from the group consisting of: a) the amino
acid sequence set out in SEQ ID NO: 3; b) a biologically active
analog, fragment or variant of a); c) a polypeptide encoded by the
polynucleotide set out in SEQ ID NO: 1; d) a biologically active
analog, fragment or variant of c); and e) a polypeptide encoded by
a polynucleotide that hybridizes to the polynucleotide set out in
SEQ ID NO: 1 under moderately stringent hybridization conditions;
wherein said buffer is comprised of a pH buffering agent in a range
of about 0.1 mM to about 500 mM and wherein the pH is in a range of
about 2.0 to about 12.0; wherein said salt is at a concentration of
about 1 to 500 mM; wherein said stabilizing agent is at a
concentration of about 0.1 to 1000 mM; and wherein said surfactant
is at a concentration of about 0.01 g/L to 0.5 g/L.
2. The formulation of claim 1 wherein the rVWF comprises the amino
acid sequence set out in SEQ ID NO: 3.
3. The formulation of claim 1 wherein the buffering agent is
selected from the group consisting of sodium citrate, glycine,
histidine, Tris and combinations of these agents.
4. The formulation of claim 3 wherein the buffering agent is sodium
citrate at a concentration of 15 mM.
5. The formulation of claim 1 wherein pH is in the range of
6.0-8.0.
6. The formulation of claim 5 wherein pH is in the range of
6.5-7.3.
7. The formulation of claim 4 wherein the pH is 7.0.
8. The formulation of claim 1 wherein the buffering agent is
citrate and the pH is 7.0.
9. The formulation of claim 1 wherein the salt is selected from the
group consisting of calcium chloride, sodium chloride and magnesium
chloride.
10. The formulation of claim 9 wherein the salt is at a
concentration range of 0.5 to 300 mM.
11. The formulation of claim 10 wherein the salt is calcium
chloride at a concentration of 10 mM.
12. The formulation of claim 1 wherein the rVWF comprises the amino
acid sequence set out in SEQ ID NO: 3; wherein the buffering agent
is citrate and the pH is 7.0; and wherein the salt is calcium
chloride at a concentration of 10 mM.
13. The formulation of claim 1 wherein the rVWF comprises the amino
acid sequence set out in SEQ ID NO: 3; wherein the buffering agent
is sodium citrate at a concentration of 15 mM and the pH is 7.0;
and wherein the salt is calcium chloride at a concentration of 10
mM and NaCl at a concentration of 100 mM.
14. The formulation of claim 3 wherein the one or more buffering
agents is histidine and Tris at a concentration of 3.3 mM each.
15. The formulation of claim 3 wherein the pH is 7.0.
16. The formulation of claim 9 wherein the one or more salts is
sodium chloride at a concentration of 30 mM and calcium chloride at
a concentration of 0.56 mM.
17. The formulation of claim 1 wherein the stabilizing agent is
selected from the group consisting of mannitol, lactose, sorbitol,
xylitol, sucrose, trehalose, mannose, maltose, lactose, glucose,
raffinose, cellobiose, gentiobiose, isomaltose, arabinose,
glucosamine, fructose and combinations of these stabilizing
agents.
18. The formulation of claim 17 wherein the stabilizing agents are
trehalose at a concentration of 7.8 mM and mannitol at a
concentration of 58.6 mM.
19. The formulation of claim 1 wherein the surfactant is selected
from the group consisting of digitonin, Triton X-100, Triton X-114,
TWEEN-20, TWEEN-80 and combinations of these surfactants.
20. The formulation of claim 1 wherein the surfactant is TWEEN-80
at 0.03 g/L.
21. The formulation of claim 1 wherein the rVWF comprises amino
acid sequence set out in SEQ ID NO: 3; wherein the buffering agents
are histidine at a concentration of 3.3 mM and Tris at a
concentration of 3.3 mM at pH 7.0; wherein the salts are sodium
chloride at a concentration of 30 mM and calcium chloride at a
concentration of 0.56 mM; wherein the stabilizing agents are
trehalose at a concentration of 7.8 mM and mannitol at a
concentration of 58.6 mM.; and wherein the surfactant is TWEEN-80
at 0.03 g/L.
Description
[0001] This application claims priority of U.S. Provisional
Application No. 61/017,418, filed Dec. 28, 2007, and U.S.
Provisional Application No. 61/017,881 filed Dec. 31, 2007, each of
which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] Generally, the invention relates to formulations of
recombinant VWF and methods for making a composition comprising
recombinant VWF.
BACKGROUND OF THE INVENTION
[0003] Von Willebrand factor (VWF) is a glycoprotein circulating in
plasma as a series of multimers ranging in size from about 500 to
20,000 kD. Multimeric forms of VWF are composed of 250 kD
polypeptide subunits linked together by disulfide bonds. VWF
mediates initial platelet adhesion to the sub-endothelium of the
damaged vessel wall. Only the larger multimers exhibit hemostatic
activity. It is assumed that endothelial cells secrete large
polymeric forms of VWF and those forms of VWF which have a low
molecular weight (low molecular weight VWF) arise from proteolytic
cleavage. The multimers having large molecular masses are stored in
the Weibel-Pallade bodies of endothelial cells and liberated upon
stimulation.
[0004] VWF is synthesized by endothelial cells and megakaryocytes
as prepro-VWF that consists to a large extent of repeated domains.
Upon cleavage of the signal peptide, pro-VWF dimerizes through
disulfide linkages at its C-terminal region. The dimers serve as
protomers for multimerization, which is governed by disulfide
linkages between the free end termini. The assembly to multimers is
followed by the proteolytic removal of the propeptide sequence
(Leyte et al., Biochem. J. 274 (1991), 257-261).
[0005] The primary translation product predicted from the cloned
cDNA of VWF is a 2813-residue precursor polypeptide (prepro-VWF).
The prepro-VWF consists of a 22 amino acid signal peptide and a 741
amino acid propeptide, with the mature VWF comprising 2050 amino
acids (Ruggeri Z. A., and Ware, J., FASEB J., 308-316 (1993)).
[0006] Defects in VWF are causal to Von Willebrand disease (VWD),
which is characterized by a more or less pronounced bleeding
phenotype. VWD type 3 is the most severe form in which VWF is
completely missing, and VWD type 1 relates to a quantitative loss
of VWF and its phenotype can be very mild. VWD type 2 relates to
qualitative defects of VWF and can be as severe as VWD type 3. VWD
type 2 has many sub forms, some being associated with the loss or
the decrease of high molecular weight multimers. Von Willebrand
syndrome type 2a (VWS-2A) is characterized by a loss of both
intermediate and large multimers. VWS-2B is characterized by a loss
of highest-molecular-weight multimers. Other diseases and disorders
related to VWF are known in the art.
[0007] US. Pat. Nos. 6,531,577, 7,166,709, and European Patent
Application No. 04380188.5, describe plasma-derived VWF
formulations. However, in addition to quantity and purity issues
with plasma-derived VWF, there is also a risk of blood-born
pathogens (e.g., viruses and Variant Creutzfeldt-Jakob disease
(vCJD).
[0008] Thus there exists a need in the art to develop a stable
pharmaceutical formulation comprising recombinant VWF.
SUMMARY OF THE INVENTION
[0009] The present invention provides formulations useful for
compositions comprising recombinant VWF, resulting in a highly
stable pharmaceutical composition. The stable pharmaceutical
composition is useful as a therapeutic agent in the treatment of
individuals suffering from disorders or conditions that can benefit
from the administration of recombinant VWF.
[0010] In one embodiment, the invention provides a stable liquid
pharmaceutical formulation of a recombinant von Willebrand Factor
(rVWF) comprising: (a) a rVWF; (b) a buffering agent; (c) one or
more salts; (d) optionally a stabilizing agent; and (e) optionally
a surfactant; wherein the rVWF comprises a polypeptide selected
from the group consisting of: a) the amino acid sequence set out in
SEQ ID NO: 3; b) a biologically active analog, fragment or variant
of a); c) a polypeptide encoded by the polynucleotide set out in
SEQ ID NO: 1; d) a biologically active analog, fragment or variant
of c); and e) a polypeptide encoded by a polynucleotide that
hybridizes to the polynucleotide set out in SEQ ID NO: 1 under
moderately stringent hybridization conditions; wherein the buffer
is comprised of a pH buffering agent in a range of about 0.1 mM to
about 500 mM and wherein the pH is in a range of about 2.0 to about
12.0; wherein the salt is at a concentration of about 1 to 500 mM;
wherein the stabilizing agent is at a concentration of about 0.1 to
1000 mM; and wherein the surfactant is at a concentration of about
0.01 g/L to 0.5 g/L.
[0011] In another embodiment, the aforementioned formulation is
provided wherein the rVWF comprises the amino acid sequence set out
in SEQ ID NO: 3. In another embodiment, an aforementioned
formulation is provided wherein the buffering agent is selected
from the group consisting of sodium citrate, glycine, histidine,
Tris and combinations of these agents. In yet another embodiment,
an aforementioned formulation is provided wherein the buffering
agent is citrate. In still another embodiment of the invention, the
aforementioned formulation is provided wherein pH is in the range
of 6.0-8.0, or 6.5-7.3. In a related embodiment, the aforementioned
formulation is provided wherein the pH is 7.0. In another
embodiment, an aforementioned formulation is provided wherein the
buffering agent is citrate and the pH is 7.0.
[0012] In still another embodiment, an aforementioned formulation
is provided wherein the salt is selected from the group consisting
of calcium chloride, sodium chloride and magnesium chloride. In
another embodiment, the aforementioned formulation is provided
wherein the salt is at a concentration range of 0.5 to 300 mM. In
another embodiment, the aforementioned formulation is provided
wherein the salt is calcium chloride at a concentration of 10
mM.
[0013] In another embodiment, an aforementioned formulation is
provided wherein the rVWF comprises the amino acid sequence set out
in SEQ ID NO: 3; wherein the buffering agent is citrate and the pH
is 7.0; and wherein the salt is calcium chloride at a concentration
of 10 mM. In still another embodiment, an aforementioned
formulation is provided wherein the rVWF comprises the amino acid
sequence set out in SEQ ID NO: 3; wherein the buffering agent is
sodium citrate and the pH is 7.0; and wherein the salt is calcium
chloride at a concentration of 10 mM and NaCl at a concentration of
100 mM.
[0014] Other formulations are also contemplated by the instant
invention. For example, in one embodiment, an aforementioned
formulation is provided wherein the one or more buffering agents is
histidine and Tris at a concentration of 3.3 mM each. In another
embodiment, the aforementioned formulation is provided wherein the
pH is 7.0. In yet another embodiment, an aforementioned formulation
is provided wherein the first salt is sodium chloride at a
concentration of 30 mM and the second salt is calcium chloride at a
concentration of 0.56 mM.
[0015] In still another embodiment of the invention, an
aforementioned formulation is provided wherein the stabilizing
agent is selected from the group consisting of mannitol, lactose,
sorbitol, xylitol, sucrose, trehalose, mannose, maltose, lactose,
glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose,
glucosamine, fructose and combinations of these stabilizing agents.
In another embodiment, the aforementioned formulation is provided
wherein the stabilizing agents are trehalose at a concentration of
7.8 mM and mannitol at a concentration of 58.6 mM.
[0016] In another embodiment, an aforementioned formulation is
provided wherein the surfactant is selected from the group
consisting of digitonin, Triton X-100, Triton X-114, TWEEN-20,
TWEEN-80 and combinations of these surfactants. In another
embodiment, the aforementioned formulation is provided wherein the
surfactant is TWEEN-80 at 0.03 g/L.
[0017] In one embodiment of the invention, an aforementioned
formulation is provided wherein the rVWF comprises amino acid
sequence set out in SEQ ID NO: 3; wherein the buffering agents are
histidine at a concentration of 3.3 mM and Tris at a concentration
of 3.3 mM at pH 7.0; wherein the first salt is sodium chloride at a
concentration of 30 mM and the second salt is calcium chloride at a
concentration of 0.56 mM; wherein the stabilizing agents are
trehalose at a concentration of 7.8 mM mannitol at a concentration
of 58.6 mM; and wherein the surfactant is TWEEN-80 at 0.03 g/L.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 shows that rVWF is not stable in Advate buffer after
26 weeks, due to the presence of glutathione.
[0019] FIG. 2 shows that rVWF is stable in Advate 1:3 buffer for up
to 12 weeks at 4.degree. C.
[0020] FIG. 3 shows that the stability of a citrate-based
formulation is better than Advate 1:3 buffer formulation containing
0.1M glutathione.
[0021] FIG. 4 shows that rVWF concentration is stable over 26 weeks
in Advate buffer.
[0022] FIG. 5 shows that rVWF concentration is stable over time in
Advate 1:3 buffer.
[0023] FIG. 6 shows that rVWF concentration is stable over time in
citrate-based buffer.
[0024] FIG. 7 shows that most excipients increase the unfolding
temperature of rVWF by about 1 or 2.degree. C.
[0025] FIG. 8 shows that 10 mM CaCl.sub.2 increases unfolding
temperature of rVWF by about 8.degree. C. to about 67.degree.
C.
[0026] FIG. 9 shows that the effect of CaCl.sub.2 is similar at pH
7.3 and pH 6.5.
DETAILED DESCRIPTION OF THE INVENTION
Definition of Terms
[0027] 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).
[0028] 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.
[0029] 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.
[0030] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0031] The term "comprising," with respect to a peptide compound,
means that a compound may include additional amino acids at either
or both amino and carboxy termini of the given sequence. Of course,
these additional amino acids should not significantly interfere
with the activity of the compound. With respect to a composition of
the instant invention, the term "comprising" means that a
composition may include additional components. These additional
components should not significantly interfere with the activity of
the composition.
[0032] The term "pharmacologically active" means that a substance
so described is determined to have activity that affects a medical
parameter (e.g., but not limited to blood pressure, blood
cell-count, cholesterol level) or disease state (e.g., but not
limited to cancer, autoimmune disorders).
[0033] As used herein the terms "express," "expressing" and
"expression" mean allowing or causing the information in a gene or
DNA sequence to become manifest, for example, producing a protein
by activating the cellular functions involved in transcription and
translation of a corresponding gene or DNA sequence. A DNA sequence
is expressed in or by a cell to form an "expression product" such
as a protein. The expression product itself, e.g. the resulting
protein, may also be said to be "expressed." An expression product
can be characterized as intracellular, extracellular or secreted.
The term "intracellular" means inside a cell. The term
"extracellular" means outside a cell, such as a transmembrane
protein. A substance is "secreted" by a cell if it appears in
significant measure outside the cell, from somewhere on or inside
the cell.
[0034] As used herein a "polypeptide" refers to a polymer composed
of amino acid residues, structural variants, related
naturally-occurring structural variants, and synthetic
non-naturally occurring analogs thereof linked via peptide bonds.
Synthetic polypeptides can be prepared, for example, using an
automated polypeptide synthesizer. The term "protein" typically
refers to large polypeptides. The term "peptide" typically refers
to short polypeptides.
[0035] As used herein a "fragment" of a polypeptide is meant to
refer to any portion of a polypeptide or protein smaller than the
full-length polypeptide or protein expression product.
[0036] As used herein an "analog" refers to any of two or more
polypeptides substantially similar in structure and having the same
biological activity, but can have varying degrees of activity, to
either the entire molecule, or to a fragment thereof. Analogs
differ in the composition of their amino acid sequences based on
one or more mutations involving substitution of one or more amino
acids for other amino acids. Substitutions can be 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.
[0037] As used herein a "variant" refers to a polypeptide, protein
or analog thereof that is modified to comprise additional chemical
moieties not normally a part of the molecule. Such moieties may
modulate the molecule's solubility, absorption, biological
half-life, etc. The moieties may 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. For example, the variant may be a blood clotting
factor having a chemical modification which confers a longer
half-life in vivo to the protein. In various aspects, polypeptides
are modified by glycosylation, pegylation, and/or
polysialylation.
[0038] Recombinant VWF
[0039] The polynucleotide and amino acid sequences of prepro-VWF
are set out in SEQ ID NO:1 and SEQ ID NO:2, respectively, and are
available at GenBank Accession Nos. NM.sub.--000552 and
NP.sub.--000543, respectively. The amino acid sequence
corresponding to the mature VWF protein is set out in SEQ ID NO: 3
(corresponding to amino acids 764-2813 of the full length
prepro-VWF amino acid sequence).
[0040] One form of useful rVWF has at least the property of in
vivo-stabilizing, e.g. binding, of at least one Factor VIII (FVIII)
molecule and 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 lb-binding domain and
binding sites for collagen and heparin (Pietu et al., Biochem.
Biophys. Res. Commun. 164: 1339-1347, 1989). The determination of
the ability of a VWF to stabilize at least one FVIII molecule can
be carried out in VWF-deficient mammals according to methods known
in the state in the art.
[0041] The rVWF of the present invention may be produced by any
method known in the art. One specific example is disclosed in
WO86/06096 published on Oct. 23, 1986 and U.S. patent application
Ser. No. 07/559,509, filed on Jul. 23, 1990, which is incorporated
herein by reference with respect to the methods of producing
recombinant VWF. Thus, methods are 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)
introducing recombinant DNA into procaryotic or eucaryotic cells by
transfection, e.g. via electroporation or microinjection, (iii)
cultivating said transformed cells, e.g. in a continuous or
batchwise manner, (iv) expressing VWF, e.g. constitutively or upon
induction, and (v) isolating said VWF, e.g. from the culture medium
or by harvesting the transformed cells, in order to (vi) obtain
purified rVWF, e.g. via anion exchange chromatography or affinity
chromatography. A recombinant VWF may be made in transformed host
cells using recombinant DNA techniques well known in the art. For
instance, sequences coding for the polypeptide could be excised
from DNA using suitable restriction enzymes.
[0042] Alternatively, the DNA molecule could be synthesized using
chemical synthesis techniques, such as the phosphoramidate method.
Also, a combination of these techniques could be used.
[0043] The invention also provides vectors encoding polypeptides of
the invention in an appropriate host. The vector comprises the
polynucleotide that encodes the polypeptide operatively linked to
appropriate expression control sequences. Methods of effecting this
operative linking, either before or after the polynucleotide is
inserted into the vector, are well known. Expression control
sequences include promoters, activators, enhancers, operators,
ribosomal binding sites, start signals, stop signals, cap signals,
polyadenylation signals, and other signals involved with the
control of transcription or translation. The resulting vector
having the polynucleotide therein is used to transform an
appropriate host. This transformation may be performed using
methods well known in the art.
[0044] Any of a large number of available and well-known host cells
may be used in the practice of this invention. The selection of a
particular host is dependent upon a number of factors recognized by
the art, including, for example, compatibility with the chosen
expression vector, toxicity of the peptides encoded by the DNA
molecule, rate of transformation, ease of recovery of the peptides,
expression characteristics, bio-safety and costs. A balance of
these factors must be struck with the understanding that not all
host cells are equally effective for the expression of a particular
DNA sequence. Within these general guidelines, useful microbial
host cells include bacteria, yeast and other fungi, insects,
plants, mammalian (including human) cells in culture, or other
hosts known in the art.
[0045] Next, the transformed host is cultured and purified. Host
cells may be cultured under conventional fermentation conditions so
that the desired compounds are expressed. Such fermentation
conditions are well known in the art. Finally, the polypeptides are
purified from culture by methods well known in the art.
[0046] Depending on the host cell utilized to express a compound of
the invention, carbohydrate (oligosaccharide) groups may
conveniently be attached to sites that are known to be
glycosylation sites in proteins. Generally, O-linked
oligosaccharides are attached to serine (Ser) or threonine (Thr)
residues while N-linked oligosaccharides are attached to asparagine
(Asn) residues when they are part of the sequence Asn-X-Ser/Thr,
where X can be any amino acid except proline. X is preferably one
of the 19 naturally occurring amino acids not counting proline. The
structures of N-linked and O-linked oligosaccharides and the sugar
residues found in each type are different. One type of sugar that
is commonly found on both is N-acetylneuraminic acid (referred to
as sialic acid). Sialic acid is usually the terminal residue of
both N-linked and O-linked oligosaccharides and, by virtue of its
negative charge, may confer acidic properties to the glycosylated
compound. Such site(s) may be incorporated in the linker of the
compounds of this invention and are preferably glycosylated by a
cell during recombinant production of the polypeptide compounds
(e.g., in mammalian cells such as CHO, BHK, COS). However, such
sites may further be glycosylated by synthetic or semi-synthetic
procedures known in the art.
[0047] Alternatively, the compounds may be made by synthetic
methods. For example, solid phase synthesis techniques may be used.
Suitable techniques are well known in the art, and include those
described in Merrifield (1973), Chem. Polypeptides, pp. 335-61
(Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem.
Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414;
Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat.
No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2:
105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2:
257-527. Solid phase synthesis is the preferred technique of making
individual peptides since it is the most cost-effective method of
making small peptides.
[0048] Fragments, Variants and Analogs of VWF
[0049] Methods for preparing polypeptide fragments, variants or
analogs are well-known in the art.
[0050] Fragments of a polypeptide are prepared using, without
limitation, enzymatic cleavage (e.g., trypsin, chymotrypsin) and
also using recombinant means to generate a polypeptide fragments
having a specific amino acid sequence. Polypeptide fragments may be
generated comprising a region of the protein having a particular
activity, such as a multimerization domain or any other
identifiable VWF domain known in the art.
[0051] Methods of making polypeptide analogs are also well-known.
Amino acid sequence analogs of a polypeptide can be substitutional,
insertional, addition or deletion analogs. Deletion analogs,
including fragments of a polypeptide, lack one or more residues of
the native protein which are not essential for function or
immunogenic activity. Insertional analogs involve the addition of,
e.g., amino acid(s) at a non-terminal point in the polypeptide.
This analog may include insertion of an immunoreactive epitope or
simply a single residue. Addition analogs, including fragments of a
polypeptide, include the addition of one or more amino acids at
either of both termini of a protein and include, for example,
fusion proteins.
[0052] Substitutional analogs typically exchange one amino acid of
the wild-type for another at one or more sites within the protein,
and may be designed to modulate one or more properties of the
polypeptide without the loss of other functions or properties. In
one aspect, substitutions are conservative substitutions. 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).
[0053] Analogs may be substantially homologous or substantially
identical to the recombinant VWF from which they are derived.
Preferred analogs are those which retain at least some of the
biological activity of the wild-type polypeptide, e.g. blood
clotting activity.
[0054] Polypeptide variants contemplated include polypeptides
chemically modified by such techniques as ubiquitination,
glycosylation, including polysialation, conjugation to therapeutic
or diagnostic agents, labeling, 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 same or
essentially the same binding properties of non-modified molecules
of the invention. Such chemical modification may include direct or
indirect (e.g., via a linker) attachment of an agent to the VWF
polypeptide. In the case of indirect attachment, it is contemplated
that the linker may be hydrolyzable or non-hydrolyzable.
[0055] Preparing pegylated polypeptide analogs will generally
comprise the steps of (a) reacting the polypeptide with
polyethylene glycol (such as a reactive ester or aldehyde
derivative of PEG) under conditions whereby the binding construct
polypeptide becomes attached to one or more PEG groups, and (b)
obtaining the reaction product(s). In general, the optimal reaction
conditions for the acylation reactions will be determined based on
known parameters and the desired result. For example, the larger
the ratio of PEG: protein, the greater the percentage of
poly-pegylated product. In some embodiments, the binding construct
will have a single PEG moiety at the N-terminus. Polyethylene
glycol (PEG) may be attached to the blood clotting factor to
provide a longer half-life in vivo. The PEG group may be of any
convenient molecular weight and may be linear or branched. The
average molecular weight of the PEG ranges from about 2 kiloDalton
("kD") to about 100 kDa, from about 5 kDa to about 50 kDa, or from
about 5 kDa to about 10 kDa. The PEG groups are attached to the
blood clotting factor via acylation or reductive alkylation through
a natural or engineered reactive group on the PEG moiety (e.g., an
aldehyde, amino, thiol, or ester group) to a reactive group on the
blood clotting factor (e.g., an aldehyde, amino, or ester group) or
by any other technique known in the art.
[0056] Methods for preparing polysialylated polypeptide are
described in United States Patent Publication 20060160948,
Fernandes et Gregoriadis; Biochim. Biophys. Acta 1341: 26-34, 1997,
and Saenko et al., Haemophilia 12:42-51, 2006. Briefly, a solution
of colominic acid containing 0.1 M NaIO.sub.4 is stirred in the
dark at room temperature to oxidize the CA. The activated CA
solution is dialyzed against, e.g., 0.05 M sodium phosphate buffer,
pH 7.2 in the dark and this solution was added to a rVWF solution
and incubated for 18 h at room temperature in the dark under gentle
shaking. Free reagents can then be separated from the
rVWF-polysialic acid conjugate by ultrafiltration/diafiltration.
Conjugation of rVWF with polysialic acid may also be achieved using
glutaraldehyde as cross-linking reagent (Migneault et al.,
Biotechniques 37: 790-796, 2004).
[0057] It is further contemplated that a polypeptide of the
invention may be a fusion protein with a second agent which is a
polypeptide. In one embodiment, the second agent which is a
polypeptide, without limitation, is an enzyme, a growth factor, an
antibody, 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. In a related embodiment, the second agent is a blood
clotting factor such as Factor VIII, Factor VII, Factor IX. The
fusion protein contemplated is made by chemical or recombinant
techniques well-known in the art.
[0058] It is also contemplated that prepro-VWF and pro-VWF
polypeptides may provide a therapeutic benefit in the formulations
of the present invention. For example, U.S. Pat. No. 7,005,502
describes a pharmaceutical preparation comprising substantial
amounts of pro-VWF that induces thrombin gerneation in vitro. In
addition to recombinant, biologically active fragments, variants,
or analogs of the naturally-occurring mature VWF, the present
invention contemplates the use of recombinant biologically active
fragments, variants, or analogs of the prepro-VWF (set out in SEQ
ID NO:2) or pro-VWF polypeptides (amino acid residues 23 to 764 of
SEQ ID NO: 2) in the formulations described herein.
[0059] Polynucleotides encoding fragments, variants and analogs may
be readily generated by a worker of skill to encode biologically
active fragments, variants, or analogs of the naturally-occurring
molecule that possess the same or similar biological activity to
the naturally-occurring molecule. These polynucleotides can be
prepared using PCR techniques, digestion/ligation of DNA encoding
molecule, and the like. 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, using any method known in
the art, including, but not limited to site-specific mutagenesis.
As used herein, the phrase "moderately stringent hybridization
conditions" means, for example, hybridization at 42.degree. C. in
50% formamide and washing at 60.degree. C. in 0.1.times.SSC, 0.1%
SDS. It is understood by those of skill in the art that variation
in these conditions occurs based on the length and GC nucleotide
base content of the sequences to be hybridized. Formulas standard
in the art are appropriate for determining exact hybridization
conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
[0060] Formulations and Excipients in General
[0061] Excipients are additives that are included in a formulation
because they either impart or enhance the stability and delivery of
a drug product. Regardless of the reason for their inclusion,
excipients are an integral component of a drug product and
therefore need to be safe and well tolerated by patients. For
protein drugs, the choice of excipients is particularly important
because they can affect both efficacy and immunogenicity of the
drug. Hence, protein formulations need to be developed with
appropriate selection of excipients that afford suitable stability,
safety, and marketability.
[0062] The principal challenge in developing formulations for
therapeutic proteins is stabilizing the product against the
stresses of manufacturing, shipping and storage. The role of
formulation excipients is to provide stabilization against these
stresses. Excipients may also be employed to reduce viscosity of
high concentration protein formulations in order to enable their
delivery and enhance patient convenience. In general, excipients
can be classified on the basis of the mechanisms by which they
stabilize proteins against various chemical and physical stresses.
Some excipients are used to alleviate the effects of a specific
stress or to regulate a particular susceptibility of a specific
protein. Other excipients have more general effects on the physical
and covalent stabilities of proteins. The excipients described
herein are organized either by their chemical type or their
functional role in formulations. Brief descriptions of the modes of
stabilization are provided when discussing each excipient type.
[0063] Given the teachings and guidance provided herein, those
skilled in the art will know what amount or range of excipient can
be included in any particular formulation to achieve a
biopharmaceutical formulation of the invention that promotes
retention in stability of the biopharmaceutical (e.g., a
polypeptide). For example, the amount and type of a salt to be
included in a biopharmaceutical formulation of the invention can be
selected based on the desired osmolality (i.e., isotonic, hypotonic
or hypertonic) of the final solution as well as the amounts and
osmolality of other components to be included in the formulation.
Similarly, by exemplification with reference to the type of polyol
or sugar included in a formulation, the amount of such an excipient
will depend on its osmolality.
[0064] By way of example, inclusion of about 5% sorbitol can
achieve isotonicity while about 9% of a sucrose excipient is needed
to achieve isotonicity. Selection of the amount or range of
concentrations of one or more excipients that can be included
within a biopharmaceutical formulation of the invention has been
exemplified above by reference to salts, polyols and sugars.
However, those skilled in the art will understand that the
considerations described herein and further exemplified by
reference to specific excipients are equally applicable to all
types and combinations of excipients including, for example, salts,
amino acids, other tonicity agents, surfactants, stabilizers,
bulking agents, cryoprotectants, lyoprotectants, anti-oxidants,
metal ions, chelating agents and/or preservatives.
[0065] Further, where a particular excipient is reported in molar
concentration, those skilled in the art will recognize that the
equivalent percent (%) w/v (e.g., (grams of substance in a solution
sample/mL of solution).times.100%) of solution is also
contemplated.
[0066] Of course, a person having ordinary skill in the art would
recognize that the concentrations of the excipients described
herein share an interdependency within a particular formulation. By
way of example, the concentration of a bulking agent may be lowered
where, e.g., there is a high polypeptide concentration or where,
e.g., there is a high stabilizing agent concentration. In addition,
a person having ordinary skill in the art would recognize that, in
order to maintain the isotonicity of a particular formulation in
which there is no bulking agent, the concentration of a stabilizing
agent would be adjusted accordingly (i.e., a "tonicifying" amount
of stabilizer would be used). Common excipients are known in the
art and can be found in Powell et al., Compendium of Excipients fir
Parenteral Formulations (1998), PDA J. Pharm. Sci. Technology,
52:238-311.
[0067] Buffers and Buffering Agents
[0068] The stability of a pharmacologically active polypeptide
formulation is usually observed to be maximal in a narrow pH range.
This pH range of optimal stability needs to be identified early
during pre-formulation studies. Several approaches, such as
accelerated stability studies and calorimetric screening studies,
have been demonstrated to be useful in this endeavor (Remmele R. L.
Jr., et al., Biochemistry, 38(16): 5241-7 (1999)). Once a
formulation is finalized, the drug product must be manufactured and
maintained throughout its shelf-life. Hence, buffering agents are
almost always employed to control pH in the formulation.
[0069] Organic acids, phosphates and Tris have been employed
routinely as buffers in protein formulations. The buffer capacity
of the buffering species is maximal at a pH equal to the pKa and
decreases as pH increases or decreases away from this value. Ninety
percent of the buffering capacity exists within one pH unit of its
pKa. Buffer capacity also increases proportionally with increasing
buffer concentration.
[0070] Several factors need to be considered when choosing a
buffer. First and foremost, the buffer species and its
concentration need to be defined based on its pKa and the desired
formulation pH. Equally important is to ensure that the buffer is
compatible with the polypeptide and other formulation excipients,
and does not catalyze any degradation reactions. A third important
aspect to be considered is the sensation of stinging and irritation
the buffer may induce upon administration. For example, citrate is
known to cause stinging upon injection (Laursen T, et al., Basic
Clin Pharmacol Toxicol., 98(2): 218-21 (2006)). The potential for
stinging and irritation is greater for drugs that are administered
via the subcutaneous (SC) or intramuscular (IM) routes, where the
drug solution remains at the site for a relatively longer period of
time than when administered by the IV route where the formulation
gets diluted rapidly into the blood upon administration. For
formulations that are administered by direct IV infusion, the total
amount of buffer (and any other formulation component) needs to be
monitored. One has to be particularly careful about potassium ions
administered in the form of the potassium phosphate buffer, which
can induce cardiovascular effects in a patient (Hollander-Rodriguez
J C, et al., Am. Fam. Physician., 73(2): 283-90 (2006)).
[0071] The buffer system present in the compositions is selected to
be physiologically compatible and to maintain a desired pH of the
pharmaceutical formulation. In one embodiment, the pH of the
solution is between pH 2.0 and pH 12.0. For example, the pH of the
solution may be 2.0, 2.3, 2.5, 2.7, 3.0, 3.3, 3.5, 3.7, 4.0, 4.3,
4.5, 4.7, 5.0, 5.3, 5.5, 5.7, 6.0, 6.3, 6.5, 6.7, 7.0, 7.3, 7.5,
7.7, 8.0, 8.3, 8.5, 8.7, 9.0, 9.3, 9.5, 9.7, 10.0, 10.3, 10.5,
10.7, 11.0, 11.3, 11.5, 11.7, or 12.0.
[0072] The pH buffering compound may be present in any amount
suitable to maintain the pH of the formulation at a predetermined
level. In one embodiment, the pH buffering concentration is between
0.1 mM and 500 mM (1 M). For example, it is contemplated that the
pH buffering agent is at least 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2,
1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500 mM.
[0073] Exemplary pH buffering agents used to buffer the formulation
as set out herein include, but are not limited to glycine,
histidine, glutamate, succinate, phosphate, acetate, citrate, Tris
and amino acids or mixtures of amino acids, including, but not
limited to aspartate, histidine, and glycine.
[0074] Salts
[0075] Salts are often added to increase the ionic strength of the
formulation, which can be important for protein solubility,
physical stability, and isotonicity. Salts can affect the physical
stability of proteins in a variety of ways. Ions can stabilize the
native state of proteins by binding to charged residues on the
protein's surface. Alternatively, salts can stabilize the denatured
state by binding to peptide groups along the protein backbone
(--CONH--). Salts can also stabilize the protein native
conformation by shielding repulsive electrostatic interactions
between residues within a protein molecule. Salts in protein
formulations can also shield attractive electrostatic interactions
between protein molecules that can lead to protein aggregation and
insolubility. In formulations provided, the salt concentration is
between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300,
and 500 mM.
[0076] Stabilizers and Bulking Agents
[0077] In the present pharmaceutical formulations, a stabilizer (or
a combination of stabilizers) may be added to prevent or reduce
storage-induced aggregation and chemical degradation. A hazy or
turbid solution upon reconstitution indicates that the protein has
precipitated or at least aggregated. The term "stabilizer" means an
excipient capable of preventing aggregation or other physical
degradation, as well as chemical degradation (for example,
autolysis, deamidation, oxidation, etc.) in an aqueous state.
Stabilizers that are conventionally employed in pharmaceutical
compositions include, but are not limited to, sucrose, trehalose,
mannose, maltose, lactose, glucose, raffinose, cellobiose,
gentiobiose, isomaltose, arabinose, glucosamine, fructose,
mannitol, sorbitol, glycine, arginine HCL, poly-hydroxy compounds,
including polysaccharides such as dextran, starch, hydroxyethyl
starch, cyclodextrins, N-methylpyrollidene, cellulose and
hyaluronic acid, sodium chloride, [Carpenter et al., Develop. Biol.
Standard 74:225, (1991)]. In the present formulations, the
stabilizer is incorporated in a concentration of about 0.1, 0.5,
0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90,
100, 200, 500, 700, 900, or 1000 mM.
[0078] If desired, the formulations also include appropriate
amounts of bulking and osmolarity regulating agents. Bulking agents
include, for example, mannitol, glycine, sucrose, polymers such as
dextran, polyvinylpyrolidone, carboxymethylcellulose, lactose,
sorbitol, trehalose, or xylitol. In one embodiment, the bulking
agent is mannitol. The bulking agent is incorporated in a
concentration of about 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000
mM.
[0079] Surfactants
[0080] Protein molecules have a high propensity to interact with
surfaces making them susceptible to adsorption and denaturation at
air-liquid, vial-liquid, and liquid-liquid (silicone oil)
interfaces. This degradation pathway has been observed to be
inversely dependent on protein concentration and results in either
the formation of soluble and insoluble protein aggregates or the
loss of protein from solution via adsorption to surfaces. In
addition to container surface adsorption, surface-induced
degradation is exacerbated with physical agitation, as would be
experienced during shipping and handling of the product.
[0081] Surfactants are commonly used in protein formulations to
prevent surface-induced degradation. Surfactants are amphipathic
molecules with the capability of out-competing proteins for
interfacial positions. Hydrophobic portions of the surfactant
molecules occupy interfacial positions (e.g., air/liquid), while
hydrophilic portions of the molecules remain oriented towards the
bulk solvent. At sufficient concentrations (typically around the
detergent's critical micellar concentration), a surface layer of
surfactant molecules serve to prevent protein molecules from
adsorbing at the interface. Thereby, surface-induced degradation is
minimized. The most commonly used surfactants are fatty acid esters
of sorbitan polyethoxylates, i.e. polysorbate 20 and polysorbate
80. The two differ only in the length of the aliphatic chain that
imparts hydrophobic character to the molecules, C-12 and C-18,
respectively. Accordingly, polysorbate-80 is more surface-active
and has a lower critical micellar concentration than
polysorbate-20.
[0082] Detergents can also affect the thermodynamic conformational
stability of proteins. Here again, the effects of a given detergent
excipient will be protein specific. For example, polysorbates have
been shown to reduce the stability of some proteins and increase
the stability of others. Detergent destabilization of proteins can
be rationalized in terms of the hydrophobic tails of the detergent
molecules that can engage in specific binding with partially or
wholly unfolded protein states. These types of interactions could
cause a shift in the conformational equilibrium towards the more
expanded protein states (i.e. increasing the exposure of
hydrophobic portions of the protein molecule in complement to
binding polysorbate). Alternatively, if the protein native state
exhibits some hydrophobic surfaces, detergent binding to the native
state may stabilize that conformation.
[0083] Another aspect of polysorbates is that they are inherently
susceptible to oxidative degradation. Often, as raw materials, they
contain sufficient quantities of peroxides to cause oxidation of
protein residue side-chains, especially methionine. The potential
for oxidative damage arising from the addition of stabilizer
emphasizes the point that the lowest effective concentrations of
excipients should be used in formulations. For surfactants, the
effective concentration for a given protein will depend on the
mechanism of stabilization. It has been postulated that if the
mechanism of surfactant stabilization is related to preventing
surface-denaturation the effective concentration will be around the
detergent's critical micellar concentration. Conversely, if the
mechanism of stabilization is associated with specific
protein-detergent interactions, the effective surfactant
concentration will be related to the protein concentration and the
stoichiometry of the interaction (Randolph T. W., et al., Pharm
Biotechnol., 13:159-75 (2002)).
[0084] Surfactants may also be added in appropriate amounts to
prevent surface related aggregation phenomenon during freezing and
drying [Chang, B, J. Pharm. Sci. 85:1325, (1996)]. Exemplary
surfactants include anionic, cationic, nonionic, zwitterionic, and
amphoteric surfactants including surfactants derived from
naturally-occurring amino acids. Anionic surfactants include, but
are not limited to, sodium lauryl sulfate, dioctyl sodium
sulfosuccinate and dioctyl sodium sulfonate, chenodeoxycholic acid,
N-lauroylsarcosine sodium salt, lithium dodecyl sulfate,
1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium
deoxycholate, and glycodeoxycholic acid sodium salt. Cationic
surfactants include, but are not limited to, benzalkonium chloride
or benzethonium chloride, cetylpyridinium chloride monohydrate, and
hexadecyltrimethylammonium bromide. Zwitterionic surfactants
include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12.
Non-ionic surfactants include, but are not limited to, digitonin,
Triton X-100, Triton X-114, TWEEN-20, and TWEEN-80. Surfactants
also include, but are not limited to lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and
60, glycerol monostearate, polysorbate 40, 60, 65 and 80, soy
lecithin and other phospholipids such as dioleyl phosphatidyl
choline (DOPC), dimyristoylphosphatidyl glycerol (DMPG),
dimyristoylphosphatidyl choline (DMPC), and (dioleyl phosphatidyl
glycerol) DOPG; sucrose fatty acid ester, methyl cellulose and
carboxymethyl cellulose. Compositions comprising these surfactants,
either individually or as a mixture in different ratios, are
therefore further provided. In the present formulations, the
surfactant is incorporated in a concentration of about 0.01 to
about 0.5 g/L.
[0085] Other Common Excipient Components
[0086] Amino Acids
[0087] Amino acids have found versatile use in protein formulations
as buffers, bulking agents, stabilizers and antioxidants. Histidine
and glutamic acid are employed to buffer protein formulations in
the pH range of 5.5-6.5 and 4.0-5.5 respectively. The imidazole
group of histidine has a pKa=6.0 and the carboxyl group of glutamic
acid side chain has a pKa of 4.3 which makes these amino acids
suitable for buffering in their respective pH ranges. Glutamic acid
is particularly useful in such cases (e.g., Stemgen.RTM.).
Histidine is commonly found in marketed protein formulations (e.g.,
Xolair.RTM., Herceptin.RTM., Recombinate.RTM.), and this amino acid
provides an alternative to citrate, a buffer known to sting upon
injection. Interestingly, histidine has also been reported to have
a stabilizing effect, as observed in formulations with ABX-IL8 (an
IgG2 antibody), with respect to aggregation when used at high
concentrations in both liquid and lyophilized presentations (Chen
B, et al., Pharm Res., 20(12): 1952-60 (2003)). Histidine (up to 60
mM) was also observed to reduce the viscosity of a high
concentration formulation of this antibody. However, in the same
study, the authors observed increased aggregation and discoloration
in histidine containing formulations during freeze-thaw studies of
the antibody in stainless steel containers. The authors attributed
this to an effect of iron ions leached from corrosion of steel
containers. Another note of caution with histidine is that it
undergoes photo-oxidation in the presence of metal ions (Tomita M,
et al., Biochemistry, 8(12): 5149-60 (1969)). The use of methionine
as an antioxidant in formulations appears promising; it has been
observed to be effective against a number of oxidative stresses
(Lam X M, et al., J Pharm Sci., 86(11): 1250-5 (1997)).
[0088] The amino acids glycine, proline, serine and alanine have
been shown to stabilize proteins by the mechanism of preferential
exclusion. Glycine is also a commonly used bulking agent in
lyophilized formulations (e.g., Neumega.RTM., Genotropin.RTM.,
Humatrope.RTM.). Arginine has been shown to be an effective agent
in inhibiting aggregation and has been used in both liquid and
lyophilized formulations (e.g., Activase.RTM., Avonex.RTM.,
Enbrel.RTM. liquid). Furthermore, the enhanced efficiency of
refolding of certain proteins in the presence of arginine has been
attributed to its suppression of the competing aggregation reaction
during refolding.
[0089] Antioxidants
[0090] Oxidation of protein residues arises from a number of
different sources. Beyond the addition of specific antioxidants,
the prevention of oxidative protein damage involves the careful
control of a number of factors throughout the manufacturing process
and storage of the product such as atmospheric oxygen, temperature,
light exposure, and chemical contamination. The most commonly used
pharmaceutical antioxidants are reducing agents,
oxygen/free-radical scavengers, or chelating agents. Antioxidants
in therapeutic protein formulations are water-soluble and remain
active throughout the product shelf-life. Reducing agents and
oxygen/free-radical scavengers work by ablating active oxygen
species in solution. Chelating agents such as EDTA are effective by
binding trace metal contaminants that promote free-radical
formation. For example, EDTA was utilized in the liquid formulation
of acidic fibroblast growth factor to inhibit the metal ion
catalyzed oxidation of cysteine residues. EDTA has been used in
marketed products like Kineret.RTM. and Ontak.RTM..
[0091] In addition to the effectiveness of various excipients to
prevent protein oxidation, the potential for the antioxidants
themselves to induce other covalent or physical changes to the
protein is of concern. For example, reducing agents can cause
disruption of intramolecular disulfide linkages, which can lead to
disulfide shuffling. In the presence of transition metal ions,
ascorbic acid and EDTA have been shown to promote methionine
oxidation in a number of proteins and peptides (Akers M J, and
Defelippis M R. Peptides and Proteins as Parenteral Solutions. In:
Pharmaceutical Formulation Development of Peptides and Proteins.
Sven Frokjaer, Lars Hovgaard, editors. Pharmaceutical Science.
Taylor and Francis, UK (1999)); Fransson J. R., J. Pharm. Sci.
86(9): 4046-1050 (1997); Yin J, et al., Pharm Res., 21(12): 2377-83
(2004)). Sodium thiosulfate has been reported to reduce the levels
of light and temperature induced methionine-oxidation in rhuMab
HER2; however, the formation of a thiosulfate-protein adduct was
also reported in this study (Lam X M, Yang J Y, et al., J Pharm
Sci. 86(11): 1250-5 (1997)). Selection of an appropriate
antioxidant is made according to the specific stresses and
sensitivities of the protein.
[0092] Metal Ions
[0093] In general, transition metal ions are undesired in protein
formulations because they can catalyze physical and chemical
degradation reactions in proteins. However, specific metal ions are
included in formulations when they are co-factors to proteins and
in suspension formulations of proteins where they form coordination
complexes (e.g., zinc suspension of insulin). Recently, the use of
magnesium ions (10-120 mM) has been proposed to inhibit the
isomerization of aspartic acid to isoaspartic acid (WO
2004039337).
[0094] Two examples where metal ions confer stability or increased
activity in proteins are human deoxyribonuclease (rhDNase,
Pulmozyme.RTM.), and Factor VIII. In the case of rhDNase, Ca.sup.+2
ions (up to 100 mM) increased the stability of the enzyme through a
specific binding site (Chen B, et al., J Pharm Sci., 88(4): 477-82
(1999)). In fact, removal of calcium ions from the solution with
EGTA caused an increase in deamidation and aggregation. However,
this effect was observed only with Ca.sup.+2 ions; other divalent
cations Mg.sup.+2, Mn.sup.+2 and Zn.sup.+2 were observed to
destabilize rhDNase. Similar effects were observed in Factor VIII.
Ca.sup.+2 and Sr.sup.+2 ions stabilized the protein while others
like Mg.sup.+2, Mn.sup.+2 and Zn.sup.+2, Cu.sup.+2 and Fe.sup.+2
destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm., 155,
121-131 (1997). In a separate study with Factor VIII, a significant
increase in aggregation rate was observed in the presence of
Al.sup.+3 ions (Derrick T S, et al., J. Pharm. Sci., 93(10):
2549-57 (2004)). The authors note that other excipients like buffer
salts are often contaminated with Al.sup.+3 ions and illustrate the
need to use excipients of appropriate quality in formulated
products.
[0095] Preservatives
[0096] Preservatives are necessary when developing multi-use
parenteral formulations that involve more than one extraction from
the same container. Their primary function is to inhibit microbial
growth and ensure product sterility throughout the shelf-life or
term of use of the drug product. Commonly used preservatives
include benzyl alcohol, phenol and m-cresol. Although preservatives
have a long history of use, the development of protein formulations
that includes preservatives can be challenging. Preservatives
almost always have a destabilizing effect (aggregation) on
proteins, and this has become a major factor in limiting their use
in multi-dose protein formulations (Roy S, et al., J Pharm Sci.,
94(2): 382-96 (2005)).
[0097] To date, most protein drugs have been formulated for
single-use only. However, when multi-dose formulations are
possible, they have the added advantage of enabling patient
convenience, and increased marketability. A good example is that of
human growth hormone (hGH) where the development of preserved
formulations has led to commercialization of more convenient,
multi-use injection pen presentations. At least four such pen
devices containing preserved formulations of hGH are currently
available on the market. Norditropin.RTM. (liquid, Novo Nordisk),
Nutropin AQ.RTM. (liquid, Genentech) & Genotropin
(lyophilized--dual chamber cartridge, Pharmacia & Upjohn)
contain phenol while Somatrope.RTM. (Eli Lilly) is formulated with
m-cresol.
[0098] Several aspects need to be considered during the formulation
development of preserved dosage forms. The effective preservative
concentration in the drug product must be optimized. This requires
testing a given preservative in the dosage form with concentration
ranges that confer anti-microbial effectiveness without
compromising protein stability. For example, three preservatives
were successfully screened in the development of a liquid
formulation for interleukin-1 receptor (Type I), using differential
scanning calorimetry (DSC). The preservatives were rank ordered
based on their impact on stability at concentrations commonly used
in marketed products (Remmele R L Jr., et al., Pharm Res., 15(2):
200-8 (1998)).
[0099] Some preservatives can cause injection site reactions, which
is another factor that needs consideration when choosing a
preservative. In clinical trials that focused on the evaluation of
preservatives and buffers in Norditropin, pain perception was
observed to be lower in formulations containing phenol and benzyl
alcohol as compared to a formulation containing m-cresol
(Kappelgaard A. M., Horm Res. 62 Suppl 3:98-103 (2004)).
Interestingly, among the commonly used preservative, benzyl alcohol
possesses anesthetic properties (Minogue S C, and Sun D A., Anesth
Analg., 100(3): 683-6 (2005)).
[0100] Lyophilization
[0101] It is also contemplated that the formulations comprising a
VWF polypeptide of the invention may be lyophilized prior to
administration. Lyophilization is carried out using techniques
common in the art and should be optimized for the composition being
developed [Tang et al., Pharm Res. 21:191-200, (2004) and Chang et
al., Pharm Res. 13:243-9 (1996)].
[0102] A lyophilization cycle is, in one aspect, composed of three
steps: freezing, primary drying, and secondary drying [A. P.
Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977)]. In
the freezing step, the solution is cooled to initiate ice
formation. Furthermore, this step induces the crystallization of
the bulking agent. The ice sublimes in the primary drying stage,
which is conducted by reducing chamber pressure below the vapor
pressure of the ice, using a vacuum and introducing heat to promote
sublimation. Finally, adsorbed or bound water is removed at the
secondary drying stage under reduced chamber pressure and at an
elevated shelf temperature. The process produces a material known
as a lyophilized cake. Thereafter the cake can be reconstituted
with either sterile water or suitable diluent for injection.
[0103] The lyophilization cycle not only determines the final
physical state of the excipients but also affects other parameters
such as reconstitution time, appearance, stability and final
moisture content. The composition structure in the frozen state
proceeds through several transitions (e.g., glass transitions,
wettings, and crystallizations) that occur at specific temperatures
and can be used to understand and optimize the lyophilization
process. The glass transition temperature (Tg and/or Tg') can
provide information about the physical state of a solute and can be
determined by differential scanning calorimetry (DSC). Tg and Tg'
are an important parameter that must be taken into account when
designing the lyophilization cycle. For example, Tg' is important
for primary drying. Furthermore, in the dried state, the glass
transition temperature provides information on the storage
temperature of the final product.
[0104] Methods of Preparation
[0105] The present invention further contemplates methods for the
preparation of pharmaceutical formulations. A variety of aqueous
carriers, e.g., sterile water for injection, water with
preservatives for multi dose use, or water with appropriate amounts
of surfactants (for example, polysorbate-20), 0.4% saline, 0.3%
glycine, or aqueous suspensions may contain the active compound in
admixture with excipients suitable for the manufacture of aqueous
suspensions. In various aspects, such excipients are suspending
agents, for example sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethyl-eneoxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl,
p-hydroxybenzoate.
[0106] Administration
[0107] To administer compositions to human or test animals, in one
aspect, the compositions comprises one or more pharmaceutically
acceptable carriers. The phrases "pharmaceutically" or
"pharmacologically" acceptable refer to molecular entities and
compositions that are stable, inhibit protein degradation such as
aggregation and cleavage products, and in addition do not produce
allergic, or other adverse reactions when administered using routes
well-known in the art, as described below. "Pharmaceutically
acceptable carriers" include any and all clinically useful
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like,
including those agents disclosed above.
[0108] The pharmaceutical formulations may be administered orally,
topically, transdermally, parenterally, by inhalation spray,
vaginally, rectally, or by intracranial injection. The term
parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intracisternal injection, or infusion
techniques. Administration by intravenous, intradermal,
intramuscular, intramammary, intraperitoneal, intrathecal,
retrobulbar, intrapulmonary injection and or surgical implantation
at a particular site is contemplated as well. Generally,
compositions are essentially free of pyrogens, as well as other
impurities that could be harmful to the recipient.
[0109] Single or multiple administrations of the compositions can
be carried out with the dose levels and pattern being selected by
the treating physician. For the prevention or treatment of disease,
the appropriate dosage will depend on the type of disease to be
treated, as defined above, the severity and course of the disease,
whether drug is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the drug, and the discretion of the attending
physician.
[0110] Kits
[0111] As an additional aspect, the invention includes kits which
comprise one or more pharmaceutical formulations packaged in a
manner which facilitates their use for administration to subjects.
In one embodiment, such a kit includes pharmaceutical formulation
described herein (e.g., a composition comprising a therapeutic
protein or peptide), packaged in a container such as a sealed
bottle or vessel, with a label affixed to the container or included
in the package that describes use of the compound or composition in
practicing the method. In one embodiment, the pharmaceutical
formulation is packaged in the container such that the amount of
headspace in the container (e.g., the amount of air between the
liquid formulation and the top of the container) is very small.
Preferably, the amount of headspace is negligible (i.e., almost
none). In one embodiment, the kit contains a first container having
a therapeutic protein or peptide composition and a second container
having a physiologically acceptable reconstitution solution for the
composition. In one aspect, the pharmaceutical formulation is
packaged in a unit dosage form. The kit may further include a
device suitable for administering the pharmaceutical formulation
according to a specific route of administration. Preferably, the
kit contains a label that describes use of the pharmaceutical
formulations.
[0112] Dosages
[0113] The dosage regimen involved in a method for treating a
condition described herein will be determined by the attending
physician, considering various factors which modify the action of
drugs, e.g. the age, condition, body weight, sex and diet of the
patient, the severity of any infection, time of administration and
other clinical factors. By way of example, a typical dose of a
recombinant VWF of the present invention is approximately 50 U/kg,
equal to 500 .mu.g/kg.
[0114] Formulations of the invention may be administered by an
initial bolus followed by a continuous infusion to maintain
therapeutic circulating levels of drug product. As another example,
the inventive compound may be administered as a one-time dose.
Those of ordinary skill in the art will readily optimize effective
dosages and administration regimens as determined by good medical
practice and the clinical condition of the individual patient. The
frequency of dosing will depend on the pharmacokinetic parameters
of the agents and the route of administration. The optimal
pharmaceutical formulation will be determined by one skilled in the
art depending upon the route of administration and desired dosage.
See for example, Remington's Pharmaceutical Sciences, 18th Ed.
(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, the
disclosure of which is hereby incorporated by reference. Such
formulations may influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of the administered
agents. Depending on the route of administration, a suitable dose
may be calculated according to body weight, body surface area or
organ size. Appropriate dosages may be ascertained through use of
established assays for determining blood level dosages in
conjunction with appropriate dose-response data. The final dosage
regimen will be determined by the attending physician, considering
various factors which modify the action of drugs, e.g. the drug's
specific activity, the severity of the damage and the
responsiveness of the patient, the age, condition, body weight, sex
and diet of the patient, the severity of any infection, time of
administration and other clinical factors. As studies are
conducted, further information will emerge regarding the
appropriate dosage levels and duration of treatment for various
diseases and conditions.
[0115] The following examples are not intended to be limiting but
only exemplary of specific embodiments of the invention.
Example 1
Shaking Experiments
[0116] In order to determine the amount of precipitation of rVWF in
various formulations, the percent recovery of rVWF following
turbulent shaking was tested under a variety of conditions.
[0117] rVWF in Advate buffer (90 mM NaCl, 1.68 mM CaCl.sub.2, 10 mM
L-histidine, 1 mM tris, 0.26 mM glutathione, 23.4 mM trehalose,
175.7 mM mannitol, and 0.1 g/L TWEEN-80, pH 7.0) or Advate 1:3
buffer (Advate buffer diluted 3-fold in water) was subjected to
turbulent shaking on a shaker at room temperature (RT) for 0 min, 1
min, 2.5 hrs, or 4 days, and percent recovery of the rVWF was
measured relative to the starting material prior to shaking. As
shown in Table 1, losses of about 40-80% were observed in the
Advate buffer while losses of about 20-30% were observed in the
Advate 1:3 buffer. VWF antigen VWF:Ag corresponds to the amount of
VWF which can be detected in an VWF-specific ELISA using polyclonal
anti-VWF antibody, while VWF:RCo corresponds to the amount of VWF
which causes agglutination of stabilized platelets in the presence
of ristocetin. In both cases human reference plasma calibrated
against the actual WHO standard was used as standard (1 ml of
reference plasma usually contains 1U VWF).
TABLE-US-00001 TABLE 1 Influence of turbulent shaking time on rVWF
recovery Turbulent shaking at VWF:Ag Recovery VWF:RCo Recovery
RCo/VWF:Ag rVWF RT [U/ml] [%] [U/ml] [%] [U/U] Advate 0 min 213
100% 104 100% 0.49 1 min 120 56% 2.5 hr 139 65% 4 d 37 17% 7 7%
0.19 Advate 0 min 206 100% 134 100% 0.65 1:3 1 min 152 74% 2.5 hr
170 82% 4 d 138 67% 131 98% 0.95
[0118] The effect of freeze/thawing and lyophilization was also
tested in the shaking experiments. Freezing was performed at
-20.degree. C. in an -20.degree. C. cold room or on dry ice,
thawing in both cases at RT and both started from the liquid
formulations. As for lyophilization, the formulated VWF samples
described herein were frozen within a pilot scale lyophilizer at
<=-40.degree. C. and were lyophilized using a standard Iyo
program. Shaking was performed directly with the liquid
formulations (2 ml in 5 ml vials). As shown in Table 2, percent
recovery of rVWF was higher in Advate 1:3 buffer compared to Advate
buffer.
TABLE-US-00002 TABLE 2 VWF:Ag VWF:RCo VWF:Ag recovery VWF:RCo
recovery RCo:Ag RVWF [U/ml] [%] [U/ml] [%] [U/U] Advate Frozen 213
100% 104 100% 0.49 Frozen - 3x 229 107% 84 81% 0.37 at -20.degree.
C. Frozen - 3x 231 108% 72 69% 0.31 with dry ice Lyo 242 113% 61
59% 0.25 Starting 213 100% 104 100% 0.49 material Heavily 37.0 17%
7.2 6.9% 0.19 shaken for 4 days at RT Advate Frozen 206 100% 134
100% 0.65 1:3 Frozen - 3x 184 89% 132 99% 0.72 at -20.degree. C.
Frozen - 3x 195 94% 128 96% 0.66 with dry ice Lyo 195 94% 107 80%
0.55 Starting 206 100% 134 100% 0.65 material Heavily 138 67% 131
98% 0.95 shaken for 4 days at RT
[0119] Percent recovery was also measured in the shaking
experiments with rVWF being stored in syringes with headspace and
without headspace. Interestingly, when rVWF is stored in syringes
without headspace and shaken as described above, no rVWF
precipitation was observed. In contrast, when rVWF is stored in
syringes with headspace, some precipitation was observed.
[0120] In summary, turbulent shaking resulted in at least 30% loss
of rVWF in Advate buffer or Advate 1:3 buffer, with Advate buffer
showing higher loss of recovery compared to Advate 1:3 buffer.
Interestingly, the same precipitates observed in the turbulent
shaking experiments were not observed when rVWF was stored and
transported .about.5000 km in an automobile (representing the
expected shaking during transport). Precipitation of rVWF could be
eliminated by storage in syringes without headspace.
Example 2
Stability of Recombinant VWF
[0121] The stability of rVWF was tested by assessing the activity
level of rVWF present in a various formulations.
[0122] As shown in FIG. 1, rVWF is not stable in Advate buffer
after 26 weeks due to the presence of 0.3 mM glutathione. As shown
in FIG. 2, however, rVWF is more stable in Advate 1:3 buffer (e.g.,
for up 12 weeks at 4.degree. C.)
[0123] As shown in FIG. 3, the stability of a citrate-based
formulation (15 mM sodium citrate, 10 mM CaCl.sub.2, 100 mM NaCl,
pH 7.0) is better than Advate 1:3 buffer formulation containing
0.1M glutathione.
[0124] Likewise, the concentration of rVWF was measured over time
in various buffers. As shown in FIG. 4, FIG. 5 and FIG. 6, rVWF
concentration is stable over time in Advate buffer, Advate 1:3
buffer, and citrate-based buffer, respectively.
Example 4
Characterization of the Liquid Formulations
[0125] Differential scanning calorimetry (DSC) was used to assess
the extent of protein (rVWF) unfolding in various buffers. As shown
in Table 3, Advate buffer pH 7.0 is the optimum for
stabilization.
[0126] DSC is a thermoanalytical technique in which the difference
in the amount of heat required to increase the temperature of a
sample and references are measured as a function of temperature.
The result of a DSC experiment is a curve of heat flux versus
temperature or versus time.
[0127] The Differential Scanning Calorimeter can scan through a
range of temperatures while heating and cooling and it determines a
phase transition, i.e. melting, crystallization, or glass
transition, by measuring the amount of heat needed to reach a set
temperature. The calorimeter was calibrated with a set of pure
metals (zinc, indium, and tin) that have a known heat capacity, Cp
and melting point, Tm. The respective reference buffer was placed
into the reference capillary and the rVWF sample was placed into
the sample capillary of the instrument.
TABLE-US-00003 TABLE 3 Unfolding temperature in various buffers Lot
Buffer pH T unfold [.degree. C] rVWF161A Advate 7.0 66.0 rVWF161B
Immunate 6.8 64.5 rVWF161C Citrate 6.8 61.2 rVWF161D NovoSeven 6.8
64.9 rVWF158 Hepes 7.4 61.3
Buffer components and concentrations:
TABLE-US-00004 A) Advate: 5.26 g/l NaCl pH = 7.0 0.248 g/l CaCl2 32
g/l D-Mannitol 8 g/l Trehalose 1.56 g/l L-Histidine 1.2 g/l Tris
0.08 g/l Glutadione red. B) Immunate: 5.25 g/l Glycin pH = 6.8 2.2
g/l NaCl 5.25 g/l NaCit3 5.25 g/l Lysin-HCl 0.62 g/l CaCl2 C)
Citrat: 3 g/l Glycin pH = 6.8 2.92 g/l NaCl 2.5 g/l NaCit3 30 g/l
D-Mannitol 10 g/l Trehalose D) NovoSeven: 0.75 g/l Glycin pH = 6.8
2.92 g/l NaCl 1.47 g/l CaCl2 30 g/l D-Mannitol
[0128] rVWF158: 20 mM Hepes, 150 mM NaCl, 5 g/L sucrose, pH 7.4
[0129] Further, as shown in FIG. 7, most formulation excipients
increase the unfolding temperature by about 1-2.degree. C. FIG. 8
shows that 10 mM CaCl.sub.2 increases the unfolding temperature by
.about.8.degree. C. to .about.67.degree. C., an unfolding
temperature which can also be reached by Advate buffer. This effect
of CaCl.sub.2 is similar at pH 7.3 and 6.5, as shown in FIG. 9.
Finally, the effect of trehalose and sucrose were analyzed on the
unfolding temperature. Compared to citrate alone, neither trehalose
nor sucrose increased the unfolding temperature of rVWF. A summary
of the unfolding temperature (Tmax) data for rVWF in the presence
of various excipients is set out in Table 4.
TABLE-US-00005 TABLE 4 15 mM Sodium 15 mM 50 mM Citrate buffer --
15 mM Tris Glycine NaCl .DELTA.H [kJ/mol] 128494.3 656259.7
157352.2 124985.8 Unfolding T 58.6 59.1 61 [.degree. C.] - Peak 1
Peak 2 65.2 68.5 65.5 Peak 3 80.4 80.1 81 Peak 4 15 mM Sodium 15 mM
20.52 g/L 10.26 g/L Citrate buffer Histidine Mannitol Trehalose
.DELTA.H [kJ/mol] 134044.5 1588590.1 612235.9 Unfolding T 59.2 58.5
58.5 [.degree. C.] - Peak 1 Peak 2 65.2 65.5 71.3 Peak 3 79.3 78.2
81.5 Peak 4 88.5 92.7 0.25 mM 15 mM Sodium 32 g/L Sac- Citrate
buffer 1 mM CaCl.sub.2 10 mM CaCl.sub.2 Saccharose charose .DELTA.H
[kJ/mol] 266008.2 308171.3 115082.4 246904.6 Unfolding T 64.5 67.2
59.2 60 [.degree. C.] - Peak 1 Peak 2 66 67 Peak 3 81 83.1 81.1
81.7 Peak 4 91.8 93 32 g/L 15 mM Sodium 0.1 g/L Raf-
Na.sub.2HPO.sub.4/ 7.8 mM Citrate buffer TWEEN-80 finose
NaHPO.sub.4 Trehalose .DELTA.H [kJ/mol] 338792.7 127329.2 197967.5
135573.3 Unfolding T 58.7 60.1 61.4 58.4 [.degree. C.] - Peak 1
Peak 2 64.4 65.8 65.4 Peak 3 81.6 80.3 80.4 80.4 Peak 4 89.2
[0130] In addition to the various buffers, DSC was used to assess
unfolding temperature of rVWF at various pH values in Advate
buffer. The results are shown in Table 5, below. Advate buffer pH
7.0 is the optimum for stabilization (i.e., highest unfolding
temperature; Peak 1) of rVWF.
TABLE-US-00006 TABLE 5 pH Peak 1 Peak 2 5.0 59.5 62.0 6.0 65.2 75.4
7.0 67.2 82.8 8.0 66.6 85.6 9.0 65.0 84.9
[0131] The fluorescence spectrum of rVWF in Advate buffer and
Advate 1:3 buffer was assessed after storage at various
temperatures for various lengths of time. No (or only slight)
change in fluorescence spectrum was observed after storage at
40.degree. C. from 0 to 28 days in either Advate or Advate 1:3
buffers. No difference was observed at other temperatures.
[0132] Likewise, degradation of rVWF was assessed using
gelfiltration (Superose 6). While some degradation was observed
after 26 weeks at 4.degree. C. in Advate buffer, almost no
degradation of rVWF in Advate 1:3 buffer was observed after 26
weeks at 4.degree. C. At 40.degree. C., glutathione increased the
amount of degradation over time (albeit to a slower extent in
Advate 1:3 buffer).
[0133] Based on the above Examples, Advate 1:3 buffer offers an
advantage with respect to freeze/thawing and recovery after
lyophilization as compared to the undiluted Advate buffer.
Moreover, Advate 1:3 buffer can stabilize (e.g., maintain
biological activity) rVWF activity during incubation at 40.degree.
C. better that Advate buffer. rVWF in Advate 1:3 buffer is stable
for 4 weeks of incubation at 4.degree. C. Finally, DSC has
demonstrated that pH 7.0 is optimum for preventing degradation of
rVWF (i.e., showed the highest unfolding temperature).
[0134] Thus, in view of the data presented herein, a formulation
was proposed for rVWF including 15 mM citrate (or glycine or
hitidine), 10 mM CaCl.sub.2, pH 6.5-7.3, adjusted to the desired
osmolarity by NaCl. For example, in one embodiment, the
citrate-based formula is 15 mM sodium citrate, 10 mM CaCl.sub.2,
100 mM NaCl, pH 7.0.
[0135] Alternatively, an Advate or Advate 1:3 buffer, without
glutathione, is also contemplated: Advate: 90 mM NaCl, 1.68 mM
CaCl.sub.2, 10 mM L-histidine, 10 mM Tris, 0.26 mM glutathione,
23.4 mM trehalose, 175.7 mM mannitol, and 0.1 g/L TWEEN-80, pH 7.0;
Advate 1:3: 30 mM NaCl, 0.56 mM CaCl.sub.2, 3.3 mM L-histidine, 3.3
mM tris, 7.8 mM trehalose, 58.6 mM mannitol, and 0.03 g/L TWEEN-80,
ph 7.0.
Sequence CWU 1
1
318833DNAArtificial SequenceSynthetic Polynucleotide 1agctcacagc
tattgtggtg ggaaagggag ggtggttggt ggatgtcaca gcttgggctt 60tatctccccc
agcagtgggg actccacagc ccctgggcta cataacagca agacagtccg
120gagctgtagc agacctgatt gagcctttgc agcagctgag agcatggcct
agggtgggcg 180gcaccattgt ccagcagctg agtttcccag ggaccttgga
gatagccgca gccctcattt 240gcaggggaag atgattcctg ccagatttgc
cggggtgctg cttgctctgg ccctcatttt 300gccagggacc ctttgtgcag
aaggaactcg cggcaggtca tccacggccc gatgcagcct 360tttcggaagt
gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg
420cagttacctc ctggcagggg gctgccagaa acgctccttc tcgattattg
gggacttcca 480gaatggcaag agagtgagcc tctccgtgta tcttggggaa
ttttttgaca tccatttgtt 540tgtcaatggt accgtgacac agggggacca
aagagtctcc atgccctatg cctccaaagg 600gctgtatcta gaaactgagg
ctgggtacta caagctgtcc ggtgaggcct atggctttgt 660ggccaggatc
gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa
720gacctgcggg ctgtgtggca actttaacat ctttgctgaa gatgacttta
tgacccaaga 780agggaccttg acctcggacc cttatgactt tgccaactca
tgggctctga gcagtggaga 840acagtggtgt gaacgggcat ctcctcccag
cagctcatgc aacatctcct ctggggaaat 900gcagaagggc ctgtgggagc
agtgccagct tctgaagagc acctcggtgt ttgcccgctg 960ccaccctctg
gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg
1020tgctgggggg ctggagtgcg cctgccctgc cctcctggag tacgcccgga
cctgtgccca 1080ggagggaatg gtgctgtacg gctggaccga ccacagcgcg
tgcagcccag tgtgccctgc 1140tggtatggag tataggcagt gtgtgtcccc
ttgcgccagg acctgccaga gcctgcacat 1200caatgaaatg tgtcaggagc
gatgcgtgga tggctgcagc tgccctgagg gacagctcct 1260ggatgaaggc
ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta
1320ccctcccggc acctccctct ctcgagactg caacacctgc atttgccgaa
acagccagtg 1380gatctgcagc aatgaagaat gtccagggga gtgccttgtc
acaggtcaat cacacttcaa 1440gagctttgac aacagatact tcaccttcag
tgggatctgc cagtacctgc tggcccggga 1500ttgccaggac cactccttct
ccattgtcat tgagactgtc cagtgtgctg atgaccgcga 1560cgctgtgtgc
acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa
1620actgaagcat ggggcaggag ttgccatgga tggccaggac gtccagctcc
ccctcctgaa 1680aggtgacctc cgcatccagc atacagtgac ggcctccgtg
cgcctcagct acggggagga 1740cctgcagatg gactgggatg gccgcgggag
gctgctggtg aagctgtccc ccgtctatgc 1800cgggaagacc tgcggcctgt
gtgggaatta caatggcaac cagggcgacg acttccttac 1860cccctctggg
ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg
1920ggactgccag gacctgcaga agcagcacag cgatccctgc gccctcaacc
cgcgcatgac 1980caggttctcc gaggaggcgt gcgcggtcct gacgtccccc
acattcgagg cctgccatcg 2040tgccgtcagc ccgctgccct acctgcggaa
ctgccgctac gacgtgtgct cctgctcgga 2100cggccgcgag tgcctgtgcg
gcgccctggc cagctatgcc gcggcctgcg cggggagagg 2160cgtgcgcgtc
gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt
2220gtacctgcag tgcgggaccc cctgcaacct gacctgccgc tctctctctt
acccggatga 2280ggaatgcaat gaggcctgcc tggagggctg cttctgcccc
ccagggctct acatggatga 2340gaggggggac tgcgtgccca aggcccagtg
cccctgttac tatgacggtg agatcttcca 2400gccagaagac atcttctcag
accatcacac catgtgctac tgtgaggatg gcttcatgca 2460ctgtaccatg
agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct
2520gtctcatcgc agcaaaagga gcctatcctg tcggcccccc atggtcaagc
tggtgtgtcc 2580cgctgacaac ctgcgggctg aagggctcga gtgtaccaaa
acgtgccaga actatgacct 2640ggagtgcatg agcatgggct gtgtctctgg
ctgcctctgc cccccgggca tggtccggca 2700tgagaacaga tgtgtggccc
tggaaaggtg tccctgcttc catcagggca aggagtatgc 2760ccctggagaa
acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa
2820ctgcacagac catgtgtgtg atgccacgtg ctccacgatc ggcatggccc
actacctcac 2880cttcgacggg ctcaaatacc tgttccccgg ggagtgccag
tacgttctgg tgcaggatta 2940ctgcggcagt aaccctggga cctttcggat
cctagtgggg aataagggat gcagccaccc 3000ctcagtgaaa tgcaagaaac
gggtcaccat cctggtggag ggaggagaga ttgagctgtt 3060tgacggggag
gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga
3120gtctggccgg tacatcattc tgctgctggg caaagccctc tccgtggtct
gggaccgcca 3180cctgagcatc tccgtggtcc tgaagcagac ataccaggag
aaagtgtgtg gcctgtgtgg 3240gaattttgat ggcatccaga acaatgacct
caccagcagc aacctccaag tggaggaaga 3300ccctgtggac tttgggaact
cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt 3360gcctctggac
tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga
3420ttcctcctgt agaatcctta ccagtgacgt cttccaggac tgcaacaagc
tggtggaccc 3480cgagccatat ctggatgtct gcatttacga cacctgctcc
tgtgagtcca ttggggactg 3540cgcctgcttc tgcgacacca ttgctgccta
tgcccacgtg tgtgcccagc atggcaaggt 3600ggtgacctgg aggacggcca
cattgtgccc ccagagctgc gaggagagga atctccggga 3660gaacgggtat
gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg
3720tcagcaccct gagccactgg cctgccctgt gcagtgtgtg gagggctgcc
atgcccactg 3780ccctccaggg aaaatcctgg atgagctttt gcagacctgc
gttgaccctg aagactgtcc 3840agtgtgtgag gtggctggcc ggcgttttgc
ctcaggaaag aaagtcacct tgaatcccag 3900tgaccctgag cactgccaga
tttgccactg tgatgttgtc aacctcacct gtgaagcctg 3960ccaggagccg
ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct
4020gtatgtggag gacatctcgg aaccgccgtt gcacgatttc tactgcagca
ggctactgga 4080cctggtcttc ctgctggatg gctcctccag gctgtccgag
gctgagtttg aagtgctgaa 4140ggcctttgtg gtggacatga tggagcggct
gcgcatctcc cagaagtggg tccgcgtggc 4200cgtggtggag taccacgacg
gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc 4260gtcagagctg
cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac
4320cagcgaggtc ttgaaataca cactgttcca aatcttcagc aagatcgacc
gccctgaagc 4380ctcccgcatc accctgctcc tgatggccag ccaggagccc
caacggatgt cccggaactt 4440tgtccgctac gtccagggcc tgaagaagaa
gaaggtcatt gtgatcccgg tgggcattgg 4500gccccatgcc aacctcaagc
agatccgcct catcgagaag caggcccctg agaacaaggc 4560cttcgtgctg
agcagtgtgg atgagctgga gcagcaaagg gacgagatcg ttagctacct
4620ctgtgacctt gcccctgaag cccctcctcc tactctgccc cccgacatgg
cacaagtcac 4680tgtgggcccg gggctcttgg gggtttcgac cctggggccc
aagaggaact ccatggttct 4740ggatgtggcg ttcgtcctgg aaggatcgga
caaaattggt gaagccgact tcaacaggag 4800caaggagttc atggaggagg
tgattcagcg gatggatgtg ggccaggaca gcatccacgt 4860cacggtgctg
cagtactcct acatggtgac tgtggagtac cccttcagcg aggcacagtc
4920caaaggggac atcctgcagc gggtgcgaga gatccgctac cagggcggca
acaggaccaa 4980cactgggctg gccctgcggt acctctctga ccacagcttc
ttggtcagcc agggtgaccg 5040ggagcaggcg cccaacctgg tctacatggt
caccggaaat cctgcctctg atgagatcaa 5100gaggctgcct ggagacatcc
aggtggtgcc cattggagtg ggccctaatg ccaacgtgca 5160ggagctggag
aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct
5220cccccgagag gctcctgacc tggtgctgca gaggtgctgc tccggagagg
ggctgcagat 5280ccccaccctc tcccctgcac ctgactgcag ccagcccctg
gacgtgatcc ttctcctgga 5340tggctcctcc agtttcccag cttcttattt
tgatgaaatg aagagtttcg ccaaggcttt 5400catttcaaaa gccaatatag
ggcctcgtct cactcaggtg tcagtgctgc agtatggaag 5460catcaccacc
attgacgtgc catggaacgt ggtcccggag aaagcccatt tgctgagcct
5520tgtggacgtc atgcagcggg agggaggccc cagccaaatc ggggatgcct
tgggctttgc 5580tgtgcgatac ttgacttcag aaatgcatgg tgccaggccg
ggagcctcaa aggcggtggt 5640catcctggtc acggacgtct ctgtggattc
agtggatgca gcagctgatg ccgccaggtc 5700caacagagtg acagtgttcc
ctattggaat tggagatcgc tacgatgcag cccagctacg 5760gatcttggca
ggcccagcag gcgactccaa cgtggtgaag ctccagcgaa tcgaagacct
5820ccctaccatg gtcaccttgg gcaattcctt cctccacaaa ctgtgctctg
gatttgttag 5880gatttgcatg gatgaggatg ggaatgagaa gaggcccggg
gacgtctgga ccttgccaga 5940ccagtgccac accgtgactt gccagccaga
tggccagacc ttgctgaaga gtcatcgggt 6000caactgtgac cgggggctga
ggccttcgtg ccctaacagc cagtcccctg ttaaagtgga 6060agagacctgt
ggctgccgct ggacctgccc ctgcgtgtgc acaggcagct ccactcggca
6120catcgtgacc tttgatgggc agaatttcaa gctgactggc agctgttctt
atgtcctatt 6180tcaaaacaag gagcaggacc tggaggtgat tctccataat
ggtgcctgca gccctggagc 6240aaggcagggc tgcatgaaat ccatcgaggt
gaagcacagt gccctctccg tcgagctgca 6300cagtgacatg gaggtgacgg
tgaatgggag actggtctct gttccttacg tgggtgggaa 6360catggaagtc
aacgtttatg gtgccatcat gcatgaggtc agattcaatc accttggtca
6420catcttcaca ttcactccac aaaacaatga gttccaactg cagctcagcc
ccaagacttt 6480tgcttcaaag acgtatggtc tgtgtgggat ctgtgatgag
aacggagcca atgacttcat 6540gctgagggat ggcacagtca ccacagactg
gaaaacactt gttcaggaat ggactgtgca 6600gcggccaggg cagacgtgcc
agcccatcct ggaggagcag tgtcttgtcc ccgacagctc 6660ccactgccag
gtcctcctct taccactgtt tgctgaatgc cacaaggtcc tggctccagc
6720cacattctat gccatctgcc agcaggacag ttgccaccag gagcaagtgt
gtgaggtgat 6780cgcctcttat gcccacctct gtcggaccaa cggggtctgc
gttgactgga ggacacctga 6840tttctgtgct atgtcatgcc caccatctct
ggtctacaac cactgtgagc atggctgtcc 6900ccggcactgt gatggcaacg
tgagctcctg tggggaccat ccctccgaag gctgtttctg 6960ccctccagat
aaagtcatgt tggaaggcag ctgtgtccct gaagaggcct gcactcagtg
7020cattggtgag gatggagtcc agcaccagtt cctggaagcc tgggtcccgg
accaccagcc 7080ctgtcagatc tgcacatgcc tcagcgggcg gaaggtcaac
tgcacaacgc agccctgccc 7140cacggccaaa gctcccacgt gtggcctgtg
tgaagtagcc cgcctccgcc agaatgcaga 7200ccagtgctgc cccgagtatg
agtgtgtgtg tgacccagtg agctgtgacc tgcccccagt 7260gcctcactgt
gaacgtggcc tccagcccac actgaccaac cctggcgagt gcagacccaa
7320cttcacctgc gcctgcagga aggaggagtg caaaagagtg tccccaccct
cctgcccccc 7380gcaccgtttg cccacccttc ggaagaccca gtgctgtgat
gagtatgagt gtgcctgcaa 7440ctgtgtcaac tccacagtga gctgtcccct
tgggtacttg gcctcaactg ccaccaatga 7500ctgtggctgt accacaacca
cctgccttcc cgacaaggtg tgtgtccacc gaagcaccat 7560ctaccctgtg
ggccagttct gggaggaggg ctgcgatgtg tgcacctgca ccgacatgga
7620ggatgccgtg atgggcctcc gcgtggccca gtgctcccag aagccctgtg
aggacagctg 7680tcggtcgggc ttcacttacg ttctgcatga aggcgagtgc
tgtggaaggt gcctgccatc 7740tgcctgtgag gtggtgactg gctcaccgcg
gggggactcc cagtcttcct ggaagagtgt 7800cggctcccag tgggcctccc
cggagaaccc ctgcctcatc aatgagtgtg tccgagtgaa 7860ggaggaggtc
tttatacaac aaaggaacgt ctcctgcccc cagctggagg tccctgtctg
7920cccctcgggc tttcagctga gctgtaagac ctcagcgtgc tgcccaagct
gtcgctgtga 7980gcgcatggag gcctgcatgc tcaatggcac tgtcattggg
cccgggaaga ctgtgatgat 8040cgatgtgtgc acgacctgcc gctgcatggt
gcaggtgggg gtcatctctg gattcaagct 8100ggagtgcagg aagaccacct
gcaacccctg ccccctgggt tacaaggaag aaaataacac 8160aggtgaatgt
tgtgggagat gtttgcctac ggcttgcacc attcagctaa gaggaggaca
8220gatcatgaca ctgaagcgtg atgagacgct ccaggatggc tgtgatactc
acttctgcaa 8280ggtcaatgag agaggagagt acttctggga gaagagggtc
acaggctgcc caccctttga 8340tgaacacaag tgtctggctg agggaggtaa
aattatgaaa attccaggca cctgctgtga 8400cacatgtgag gagcctgagt
gcaacgacat cactgccagg ctgcagtatg tcaaggtggg 8460aagctgtaag
tctgaagtag aggtggatat ccactactgc cagggcaaat gtgccagcaa
8520agccatgtac tccattgaca tcaacgatgt gcaggaccag tgctcctgct
gctctccgac 8580acggacggag cccatgcagg tggccctgca ctgcaccaat
ggctctgttg tgtaccatga 8640ggttctcaat gccatggagt gcaaatgctc
ccccaggaag tgcagcaagt gaggctgctg 8700cagctgcatg ggtgcctgct
gctgcctgcc ttggcctgat ggccaggcca gagtgctgcc 8760agtcctctgc
atgttctgct cttgtgccct tctgagccca caataaaggc tgagctctta
8820tcttgcaaaa ggc 883322783PRTArtificial SequenceSynthetic
Polypeptide 2Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Leu Ile
Leu Pro Gly1 5 10 15Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser
Thr Ala Arg Cys20 25 30Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe
Asp Gly Ser Met Tyr35 40 45Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu
Ala Gly Gly Cys Gln Lys50 55 60Arg Ser Phe Ser Ile Ile Gly Asp Phe
Gln Asn Gly Lys Arg Val Ser65 70 75 80Leu Ser Val Tyr Leu Gly Glu
Phe Phe Asp Ile His Leu Phe Val Asn85 90 95Gly Thr Val Thr Gln Gly
Asp Gln Arg Val Ser Met Pro Tyr Ala Ser100 105 110Lys Leu Glu Thr
Glu Ala Gly Tyr Tyr Lys Leu Ser Gly Glu Ala Tyr115 120 125Gly Phe
Val Ala Arg Ile Asp Gly Ser Gly Asn Phe Gln Val Leu Leu130 135
140Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly Leu Cys Gly Asn Phe
Asn145 150 155 160Ile Phe Ala Glu Asp Asp Phe Met Thr Gln Glu Gly
Thr Leu Thr Ser165 170 175Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala
Leu Ser Ser Gly Glu Gln180 185 190Trp Cys Glu Arg Pro Ser Ser Ser
Cys Asn Ile Ser Ser Gly Glu Met195 200 205Gln Lys Gly Leu Trp Glu
Gln Cys Gln Leu Leu Lys Ser Thr Ser Val210 215 220Phe Ala Arg Cys
His Pro Leu Val Asp Pro Glu Pro Phe Cys Glu Lys225 230 235 240Thr
Leu Cys Glu Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu245 250
255Leu Glu Tyr Ala Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr
Gly260 265 270Trp Thr Asp His Ser Ala Cys Ser Pro Val Cys Pro Ala
Gly Met Glu275 280 285Tyr Arg Gln Cys Val Ser Pro Cys Ala Arg Thr
Cys Gln Ser Leu His290 295 300Ile Asn Glu Met Cys Gln Glu Arg Cys
Val Asp Gly Cys Ser Cys Pro305 310 315 320Glu Gly Gln Leu Leu Asp
Glu Gly Leu Cys Val Glu Ser Thr Glu Cys325 330 335Pro Cys Val His
Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser340 345 350Arg Asp
Cys Asn Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser355 360
365Asn Glu Glu Cys Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His
Phe370 375 380Lys Ser Phe Asp Asn Arg Tyr Phe Thr Phe Ser Gly Ile
Cys Gln Tyr385 390 395 400Leu Leu Ala Arg Asp Cys Gln Asp His Ser
Phe Ser Ile Val Ile Glu405 410 415Thr Val Gln Cys Ala Asp Asp Arg
Asp Ala Val Cys Thr Arg Ser Val420 425 430Thr Val Arg Leu Pro Gly
Leu His Asn Ser Leu Val Lys Leu Lys His435 440 445Gly Ala Gly Val
Ala Met Asp Gly Gln Asp Val Gln Leu Pro Leu Leu450 455 460Lys Gly
Asp Leu Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu465 470 475
480Ser Tyr Gly Glu Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg
Leu485 490 495Leu Val Lys Leu Ser Pro Val Tyr Ala Gly Lys Thr Cys
Gly Leu Cys500 505 510Gly Asn Tyr Asn Gly Asn Gln Gly Asp Asp Phe
Leu Thr Pro Ser Gly515 520 525Leu Ala Glu Pro Arg Val Glu Asp Phe
Gly Asn Ala Trp Lys Leu His530 535 540Gly Asp Cys Gln Asp Leu Gln
Lys Gln His Ser Asp Pro Cys Ala Leu545 550 555 560Asn Pro Arg Met
Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr565 570 575Ser Pro
Thr Phe Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr580 585
590Leu Arg Asn Cys Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg
Glu595 600 605Cys Leu Cys Gly Ser Tyr Ala Ala Ala Cys Ala Gly Arg
Gly Val Arg610 615 620Val Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu
Asn Cys Pro Lys Gly625 630 635 640Gln Val Tyr Leu Gln Cys Gly Thr
Pro Cys Asn Leu Thr Cys Arg Ser645 650 655Leu Ser Tyr Pro Asp Glu
Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys660 665 670Phe Cys Pro Pro
Met Asp Glu Arg Gly Asp Cys Val Pro Lys Ala Gln675 680 685Cys Pro
Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp Ile Phe690 695
700Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met His
Cys705 710 715 720Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp
Ala Val Leu Ser725 730 735Ser Pro Leu Ser His Arg Ser Lys Arg Ser
Leu Ser Cys Arg Pro Pro740 745 750Met Val Lys Leu Val Cys Pro Ala
Asp Asn Leu Arg Ala Glu Gly Leu755 760 765Glu Cys Thr Lys Thr Cys
Gln Asn Tyr Asp Leu Glu Cys Met Ser Met770 775 780Gly Cys Val Ser
Gly Cys Leu Cys Pro Pro Gly Met Val Arg His Glu785 790 795 800Asn
Arg Cys Glu Arg Cys Pro Cys Phe His Gln Gly Lys Glu Tyr Ala805 810
815Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr Cys Val Cys Arg
Asp820 825 830Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp Ala Thr
Cys Ser Thr835 840 845Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly
Leu Lys Tyr Leu Phe850 855 860Pro Gly Glu Cys Gln Tyr Val Leu Val
Gln Asp Tyr Cys Gly Ser Asn865 870 875 880Pro Gly Thr Phe Arg Ile
Leu Val Gly Asn Lys Gly Cys Ser His Pro885 890 895Ser Val Lys Cys
Lys Lys Arg Val Thr Ile Leu Val Glu Gly Gly Glu900 905 910Ile Glu
Leu Phe Asp Gly Glu Val Asn Val Lys Arg Pro Met Lys Asp915 920
925Glu Thr His Phe Glu Val Val Glu Ser Gly Arg Tyr Ile Ile Leu
Leu930 935 940Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg His Leu
Ser Ile Ser945 950 955 960Val Val Leu Lys Gln Thr Tyr Gln Glu Lys
Val Cys Gly Leu Cys Gly965 970 975Asn Phe Asp Gly Ile Gln Asn Asn
Asp Leu Thr Ser Ser Asn Leu Gln980 985 990Val Glu Glu Asp Pro Val
Asp Phe Gly Asn Ser Trp Lys Val Ser Ser995 1000 1005Gln Cys Ala Asp
Thr Arg Lys Val Pro Leu Asp Ser Ser Pro Ala1010 1015 1020Thr Cys
His Asn Asn Ile Met Lys Gln Thr Met Val Asp Ser Ser1025 1030
1035Cys Arg Ile Leu Thr Ser Asp Val Phe Gln Asp Cys Asn Lys Leu1040
1045 1050Val Asp Pro Glu Pro Tyr Leu Asp Val Cys Ile Tyr Asp Thr
Cys1055 1060 1065Ser Cys
Glu Ser Ile Gly Asp Cys Ala Cys Phe Cys Asp Thr Ile1070 1075
1080Ala Ala Tyr Ala His Val Cys Ala Gln His Gly Lys Val Val Thr1085
1090 1095Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg
Asn1100 1105 1110Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr
Asn Ser Cys1115 1120 1125Ala Pro Ala Cys Gln Val Thr Cys Gln His
Pro Glu Pro Leu Ala1130 1135 1140Cys Pro Val Gln Cys Val Glu Gly
Cys His Ala His Cys Pro Pro1145 1150 1155Gly Lys Ile Leu Asp Glu
Leu Leu Gln Thr Cys Val Asp Pro Glu1160 1165 1170Asp Cys Pro Val
Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly1175 1180 1185Lys Lys
Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile1190 1195
1200Cys His Cys Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu1205
1210 1215Pro Gly Gly Leu Val Val Pro Pro Thr Asp Ala Pro Val Ser
Pro1220 1225 1230Thr Thr Leu Tyr Val Glu Asp Ile Ser Glu Pro Pro
Leu His Asp1235 1240 1245Phe Tyr Cys Ser Arg Leu Leu Asp Leu Val
Phe Leu Leu Asp Gly1250 1255 1260Ser Ser Arg Leu Ser Glu Ala Glu
Phe Glu Val Leu Lys Ala Phe1265 1270 1275Val Val Asp Met Met Glu
Arg Leu Arg Ile Ser Gln Lys Trp Val1280 1285 1290Arg Val Ala Val
Val Glu Tyr His Asp Gly Ser His Ala Tyr Ile1295 1300 1305Gly Leu
Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala1310 1315
1320Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala Ser Thr Ser Glu1325
1330 1335Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp
Arg1340 1345 1350Pro Glu Ala Ser Arg Ile Thr Leu Leu Leu Met Ala
Ser Gln Glu1355 1360 1365Pro Gln Arg Met Ser Arg Asn Phe Val Arg
Tyr Val Gln Gly Leu1370 1375 1380Lys Lys Lys Lys Val Ile Val Ile
Pro Val Gly Ile Gly Pro His1385 1390 1395Ala Asn Leu Lys Gln Ile
Arg Leu Ile Glu Lys Gln Ala Pro Glu1400 1405 1410Asn Lys Ala Phe
Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln1415 1420 1425Arg Asp
Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala1430 1435
1440Pro Pro Pro Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly1445
1450 1455Pro Gly Leu Leu Gly Val Ser Thr Leu Gly Pro Lys Arg Asn
Ser1460 1465 1470Met Val Leu Asp Val Ala Phe Val Leu Glu Gly Ser
Asp Lys Ile1475 1480 1485Gly Glu Ala Asp Phe Asn Arg Ser Lys Glu
Phe Met Glu Glu Val1490 1495 1500Ile Gln Arg Met Asp Val Gly Gln
Asp Ser Ile His Val Thr Val1505 1510 1515Leu Gln Tyr Ser Tyr Met
Val Thr Val Glu Tyr Pro Phe Ser Glu1520 1525 1530Ala Gln Ser Lys
Gly Asp Ile Leu Gln Arg Val Arg Glu Ile Arg1535 1540 1545Tyr Gln
Gly Gly Asn Arg Thr Asn Thr Gly Leu Ala Leu Arg Tyr1550 1555
1560Leu Ser Asp His Ser Phe Leu Val Ser Gln Gly Asp Arg Glu Gln1565
1570 1575Ala Pro Asn Leu Val Tyr Met Val Thr Gly Asn Pro Ala Ser
Asp1580 1585 1590Glu Ile Lys Arg Leu Pro Gly Asp Ile Gln Val Val
Pro Ile Gly1595 1600 1605Val Gly Pro Asn Ala Asn Val Gln Glu Leu
Glu Arg Ile Gly Trp1610 1615 1620Pro Asn Ala Pro Ile Leu Ile Gln
Asp Phe Glu Thr Leu Pro Arg1625 1630 1635Glu Ala Pro Asp Leu Val
Leu Gln Arg Cys Cys Ser Gly Glu Gly1640 1645 1650Leu Gln Ile Pro
Thr Leu Ser Pro Ala Pro Asp Cys Ser Gln Pro1655 1660 1665Leu Asp
Val Ile Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro Ala1670 1675
1680Ser Tyr Phe Asp Glu Met Lys Ser Phe Ala Lys Ala Phe Ile Ser1685
1690 1695Lys Ala Asn Ile Gly Pro Arg Leu Thr Gln Val Ser Val Leu
Gln1700 1705 1710Tyr Gly Ser Ile Thr Thr Ile Asp Val Pro Trp Asn
Val Val Pro1715 1720 1725Glu Lys Ala His Leu Leu Ser Leu Val Asp
Val Met Gln Arg Glu1730 1735 1740Gly Gly Pro Ser Gln Ile Gly Asp
Ala Leu Gly Phe Ala Val Arg1745 1750 1755Tyr Leu Thr Ser Glu Met
His Gly Ala Arg Pro Gly Ala Ser Lys1760 1765 1770Ala Val Val Ile
Leu Val Thr Asp Val Ser Val Asp Ser Val Asp1775 1780 1785Ala Ala
Ala Asp Ala Ala Arg Ser Asn Arg Val Thr Val Phe Pro1790 1795
1800Ile Gly Ile Gly Asp Arg Tyr Asp Ala Ala Gln Leu Arg Ile Leu1805
1810 1815Ala Gly Pro Ala Gly Asp Ser Asn Val Val Lys Leu Gln Arg
Ile1820 1825 1830Glu Asp Leu Pro Thr Met Val Thr Leu Gly Asn Ser
Phe Leu His1835 1840 1845Lys Leu Cys Ser Gly Phe Val Arg Ile Cys
Met Asp Glu Asp Gly1850 1855 1860Asn Glu Lys Arg Pro Gly Asp Val
Trp Thr Leu Pro Asp Gln Cys1865 1870 1875His Thr Val Thr Cys Gln
Pro Asp Gly Gln Thr Leu Leu Lys Ser1880 1885 1890His Arg Val Asn
Cys Asp Arg Gly Leu Arg Pro Ser Cys Pro Asn1895 1900 1905Ser Gln
Ser Pro Val Lys Val Glu Glu Thr Cys Gly Cys Arg Trp1910 1915
1920Thr Cys Pro Cys Val Cys Thr Gly Ser Ser Thr Arg His Ile Val1925
1930 1935Thr Phe Asp Gly Gln Asn Phe Lys Leu Thr Gly Ser Cys Ser
Tyr1940 1945 1950Val Leu Phe Gln Asn Lys Glu Gln Asp Leu Glu Val
Ile Leu His1955 1960 1965Asn Gly Ala Cys Ser Pro Gly Ala Arg Gln
Gly Cys Met Lys Ser1970 1975 1980Ile Glu Val Lys His Ser Ala Leu
Ser Val Glu Leu His Ser Asp1985 1990 1995Met Glu Val Thr Val Asn
Gly Arg Leu Val Ser Val Pro Tyr Val2000 2005 2010Gly Gly Asn Met
Glu Val Asn Val Tyr Gly Ala Ile Met His Glu2015 2020 2025Val Arg
Phe Asn His Leu Gly His Ile Phe Thr Phe Thr Pro Gln2030 2035
2040Asn Asn Glu Phe Gln Leu Gln Leu Ser Pro Lys Thr Phe Ala Ser2045
2050 2055Lys Thr Tyr Gly Leu Cys Gly Ile Cys Asp Glu Asn Gly Ala
Asn2060 2065 2070Asp Phe Met Leu Arg Asp Gly Thr Val Thr Thr Asp
Trp Lys Thr2075 2080 2085Leu Val Gln Glu Trp Thr Val Gln Arg Pro
Gly Gln Thr Cys Gln2090 2095 2100Pro Glu Gln Cys Leu Val Pro Asp
Ser Ser His Cys Gln Val Leu2105 2110 2115Leu Leu Pro Leu Phe Ala
Glu Cys His Lys Val Leu Ala Pro Ala2120 2125 2130Thr Phe Tyr Ala
Ile Cys Gln Gln Asp Ser Cys His Gln Glu Gln2135 2140 2145Val Cys
Glu Val Ile Ala Ser Tyr Ala His Leu Cys Arg Thr Asn2150 2155
2160Gly Val Cys Val Asp Trp Arg Thr Pro Asp Phe Cys Ala Met Ser2165
2170 2175Cys Pro Pro Ser Leu Val Tyr Asn His Cys Glu His Gly Cys
Pro2180 2185 2190Arg His Cys Asp Gly Asn Val Ser Ser Cys Gly Asp
His Pro Ser2195 2200 2205Glu Gly Cys Phe Cys Pro Pro Asp Lys Val
Met Leu Glu Gly Ser2210 2215 2220Cys Val Pro Glu Glu Ala Cys Thr
Gln Cys Ile Gly Glu Asp Gly2225 2230 2235Val Gln His Gln Phe Leu
Glu Ala Trp Val Pro Asp His Gln Pro2240 2245 2250Cys Gln Ile Cys
Thr Cys Leu Ser Gly Arg Lys Val Asn Cys Thr2255 2260 2265Thr Gln
Pro Cys Pro Thr Ala Lys Ala Pro Thr Cys Gly Leu Cys2270 2275
2280Glu Val Ala Arg Leu Arg Gln Asn Ala Asp Gln Cys Cys Pro Glu2285
2290 2295Tyr Glu Cys Val Cys Asp Pro Val Ser Cys Asp Leu Pro Pro
Val2300 2305 2310Pro His Cys Glu Arg Gly Leu Gln Pro Thr Leu Thr
Asn Pro Gly2315 2320 2325Glu Cys Arg Pro Asn Phe Thr Cys Ala Cys
Arg Lys Glu Glu Cys2330 2335 2340Lys Arg Val Ser Pro Pro Ser Cys
Pro Pro His Arg Leu Pro Thr2345 2350 2355Leu Arg Lys Thr Gln Cys
Cys Asp Glu Tyr Glu Cys Ala Cys Asn2360 2365 2370Cys Val Asn Ser
Thr Val Ser Cys Pro Leu Gly Tyr Leu Ala Ser2375 2380 2385Thr Ala
Thr Asn Asp Cys Gly Cys Thr Thr Thr Thr Cys Leu Pro2390 2395
2400Asp Lys Val Cys Val His Arg Ser Thr Ile Tyr Pro Val Gly Gln2405
2410 2415Phe Trp Glu Glu Gly Cys Asp Val Cys Thr Cys Thr Asp Met
Glu2420 2425 2430Asp Ala Val Met Gly Leu Arg Val Ala Gln Cys Ser
Gln Lys Pro2435 2440 2445Cys Glu Asp Ser Cys Arg Ser Gly Phe Thr
Tyr Val Leu His Glu2450 2455 2460Gly Glu Cys Cys Gly Arg Cys Leu
Pro Ser Ala Cys Glu Val Val2465 2470 2475Thr Gly Ser Pro Arg Gly
Asp Ser Gln Ser Ser Trp Lys Ser Val2480 2485 2490Gly Ser Gln Trp
Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg2495 2500 2505Val Lys
Glu Glu Val Phe Ile Gln Gln Arg Asn Val Ser Cys Pro2510 2515
2520Gln Leu Glu Val Pro Val Cys Pro Ser Gly Phe Gln Leu Ser Cys2525
2530 2535Lys Thr Ser Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met
Glu2540 2545 2550Ala Cys Met Leu Asn Gly Thr Val Ile Gly Pro Gly
Lys Thr Val2555 2560 2565Met Ile Asp Val Cys Thr Thr Cys Arg Cys
Met Val Gln Val Gly2570 2575 2580Val Ile Ser Gly Phe Lys Leu Glu
Cys Arg Lys Thr Thr Cys Asn2585 2590 2595Pro Cys Pro Leu Gly Tyr
Lys Glu Glu Asn Asn Thr Gly Glu Cys2600 2605 2610Cys Gly Arg Cys
Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly2615 2620 2625Gly Gln
Ile Met Thr Leu Lys Arg Asp Glu Thr Leu Gln Asp Gly2630 2635
2640Cys Asp Thr His Phe Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe2645
2650 2655Trp Glu Lys Arg Val Thr Gly Cys Pro Pro Phe Asp Glu His
Lys2660 2665 2670Cys Leu Ala Glu Gly Gly Lys Ile Met Lys Ile Pro
Gly Thr Cys2675 2680 2685Cys Asp Thr Cys Glu Glu Pro Glu Cys Asn
Asp Ile Thr Ala Arg2690 2695 2700Leu Gln Tyr Val Lys Val Gly Ser
Cys Lys Ser Glu Val Glu Val2705 2710 2715Asp Ile His Tyr Cys Gln
Gly Lys Cys Ala Ser Lys Ala Met Tyr2720 2725 2730Ser Ile Asp Ile
Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser2735 2740 2745Pro Thr
Arg Thr Glu Pro Met Gln His Cys Thr Asn Gly Ser Val2750 2755
2760Val Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys Ser Pro2765
2770 2775Arg Lys Cys Ser Lys278032050PRTArtificial
SequenceSynthetic Polypeptide 3Ser Leu Ser Cys Arg Pro Pro Met Val
Lys Leu Val Cys Pro Ala Asp1 5 10 15Asn Leu Arg Ala Glu Gly Leu Glu
Cys Thr Lys Thr Cys Gln Asn Tyr20 25 30Asp Leu Glu Cys Met Ser Met
Gly Cys Val Ser Gly Cys Leu Cys Pro35 40 45Pro Gly Met Val Arg His
Glu Asn Arg Cys Val Ala Leu Glu Arg Cys50 55 60Pro Cys Phe His Gln
Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys65 70 75 80Ile Gly Cys
Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr85 90 95Asp His
Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr100 105
110Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln
Tyr115 120 125Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr
Phe Arg Ile130 135 140Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser
Val Lys Cys Lys Lys145 150 155 160Arg Val Thr Ile Leu Val Glu Gly
Gly Glu Ile Glu Leu Phe Asp Gly165 170 175Glu Val Asn Val Lys Arg
Pro Met Lys Asp Glu Thr His Phe Glu Val180 185 190Val Glu Ser Gly
Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser195 200 205Val Val
Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr210 215
220Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile
Gln225 230 235 240Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu
Glu Asp Pro Val245 250 255Asp Phe Gly Asn Ser Trp Lys Val Ser Ser
Gln Cys Ala Asp Thr Arg260 265 270Lys Val Pro Leu Asp Ser Ser Pro
Ala Thr Cys His Asn Asn Ile Met275 280 285Lys Gln Thr Met Val Asp
Ser Ser Cys Arg Ile Leu Thr Ser Asp Val290 295 300Phe Gln Asp Cys
Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val305 310 315 320Cys
Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys325 330
335Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His
Gly340 345 350Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln
Ser Cys Glu355 360 365Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys
Glu Trp Arg Tyr Asn370 375 380Ser Cys Ala Pro Ala Cys Gln Val Thr
Cys Gln His Pro Glu Pro Leu385 390 395 400Ala Cys Pro Val Gln Cys
Val Glu Gly Cys His Ala His Cys Pro Pro405 410 415Gly Lys Ile Leu
Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp420 425 430Cys Pro
Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys435 440
445Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His
Cys450 455 460Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro
Gly Gly Leu465 470 475 480Val Val Pro Pro Thr Asp Ala Pro Val Ser
Pro Thr Thr Leu Tyr Val485 490 495Glu Asp Ile Ser Glu Pro Pro Leu
His Asp Phe Tyr Cys Ser Arg Leu500 505 510Leu Asp Leu Val Phe Leu
Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala515 520 525Glu Phe Glu Val
Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu530 535 540Arg Ile
Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp545 550 555
560Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser
Glu565 570 575Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser
Gln Val Ala580 585 590Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe
Gln Ile Phe Ser Lys595 600 605Ile Asp Arg Pro Glu Ala Ser Arg Ile
Thr Leu Leu Leu Met Ala Ser610 615 620Gln Glu Pro Gln Arg Met Ser
Arg Asn Phe Val Arg Tyr Val Gln Gly625 630 635 640Leu Lys Lys Lys
Lys Val Ile Val Ile Pro Val Gly Ile Gly Pro His645 650 655Ala Asn
Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn660 665
670Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg
Asp675 680 685Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala
Pro Pro Pro690 695 700Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val
Gly Pro Gly Leu Leu705 710 715 720Gly Val Ser Thr Leu Gly Pro Lys
Arg Asn Ser Met Val Leu Asp Val725 730 735Ala Phe Val Leu Glu Gly
Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn740 745 750Arg Ser Lys Glu
Phe Met Glu Glu Val Ile Gln Arg Met Asp Val Gly755 760 765Gln Asp
Ser Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr770 775
780Val Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu
Gln785 790 795 800Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg
Thr Asn Thr Gly805 810 815Leu Ala Leu Arg Tyr Leu Ser Asp His Ser
Phe Leu Val Ser Gln Gly820 825 830Asp Arg Glu Gln Ala Pro Asn Leu
Val Tyr Met
Val Thr Gly Asn Pro835 840 845Ala Ser Asp Glu Ile Lys Arg Leu Pro
Gly Asp Ile Gln Val Val Pro850 855 860Ile Gly Val Gly Pro Asn Ala
Asn Val Gln Glu Leu Glu Arg Ile Gly865 870 875 880Trp Pro Asn Ala
Pro Ile Leu Ile Gln Asp Phe Glu Thr Leu Pro Arg885 890 895Glu Ala
Pro Asp Leu Val Leu Gln Arg Cys Cys Ser Gly Glu Gly Leu900 905
910Gln Ile Pro Thr Leu Ser Pro Ala Pro Asp Cys Ser Gln Pro Leu
Asp915 920 925Val Ile Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro Ala
Ser Tyr Phe930 935 940Asp Glu Met Lys Ser Phe Ala Lys Ala Phe Ile
Ser Lys Ala Asn Ile945 950 955 960Gly Pro Arg Leu Thr Gln Val Ser
Val Leu Gln Tyr Gly Ser Ile Thr965 970 975Thr Ile Asp Val Pro Trp
Asn Val Val Pro Glu Lys Ala His Leu Leu980 985 990Ser Leu Val Asp
Val Met Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly995 1000 1005Asp Ala
Leu Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His1010 1015
1020Gly Ala Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr1025
1030 1035Asp Val Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala
Arg1040 1045 1050Ser Asn Arg Val Thr Val Phe Pro Ile Gly Ile Gly
Asp Arg Tyr1055 1060 1065Asp Ala Ala Gln Leu Arg Ile Leu Ala Gly
Pro Ala Gly Asp Ser1070 1075 1080Asn Val Val Lys Leu Gln Arg Ile
Glu Asp Leu Pro Thr Met Val1085 1090 1095Thr Leu Gly Asn Ser Phe
Leu His Lys Leu Cys Ser Gly Phe Val1100 1105 1110Arg Ile Cys Met
Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp1115 1120 1125Val Trp
Thr Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro1130 1135
1140Asp Gly Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg1145
1150 1155Gly Leu Arg Pro Ser Cys Pro Asn Ser Gln Ser Pro Val Lys
Val1160 1165 1170Glu Glu Thr Cys Gly Cys Arg Trp Thr Cys Pro Cys
Val Cys Thr1175 1180 1185Gly Ser Ser Thr Arg His Ile Val Thr Phe
Asp Gly Gln Asn Phe1190 1195 1200Lys Leu Thr Gly Ser Cys Ser Tyr
Val Leu Phe Gln Asn Lys Glu1205 1210 1215Gln Asp Leu Glu Val Ile
Leu His Asn Gly Ala Cys Ser Pro Gly1220 1225 1230Ala Arg Gln Gly
Cys Met Lys Ser Ile Glu Val Lys His Ser Ala1235 1240 1245Leu Ser
Val Glu Leu His Ser Asp Met Glu Val Thr Val Asn Gly1250 1255
1260Arg Leu Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu Val Asn1265
1270 1275Val Tyr Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu
Gly1280 1285 1290His Ile Phe Thr Phe Thr Pro Gln Asn Asn Glu Phe
Gln Leu Gln1295 1300 1305Leu Ser Pro Lys Thr Phe Ala Ser Lys Thr
Tyr Gly Leu Cys Gly1310 1315 1320Ile Cys Asp Glu Asn Gly Ala Asn
Asp Phe Met Leu Arg Asp Gly1325 1330 1335Thr Val Thr Thr Asp Trp
Lys Thr Leu Val Gln Glu Trp Thr Val1340 1345 1350Gln Arg Pro Gly
Gln Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys1355 1360 1365Leu Val
Pro Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu1370 1375
1380Phe Ala Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe Tyr Ala1385
1390 1395Ile Cys Gln Gln Asp Ser Cys His Gln Glu Gln Val Cys Glu
Val1400 1405 1410Ile Ala Ser Tyr Ala His Leu Cys Arg Thr Asn Gly
Val Cys Val1415 1420 1425Asp Trp Arg Thr Pro Asp Phe Cys Ala Met
Ser Cys Pro Pro Ser1430 1435 1440Leu Val Tyr Asn His Cys Glu His
Gly Cys Pro Arg His Cys Asp1445 1450 1455Gly Asn Val Ser Ser Cys
Gly Asp His Pro Ser Glu Gly Cys Phe1460 1465 1470Cys Pro Pro Asp
Lys Val Met Leu Glu Gly Ser Cys Val Pro Glu1475 1480 1485Glu Ala
Cys Thr Gln Cys Ile Gly Glu Asp Gly Val Gln His Gln1490 1495
1500Phe Leu Glu Ala Trp Val Pro Asp His Gln Pro Cys Gln Ile Cys1505
1510 1515Thr Cys Leu Ser Gly Arg Lys Val Asn Cys Thr Thr Gln Pro
Cys1520 1525 1530Pro Thr Ala Lys Ala Pro Thr Cys Gly Leu Cys Glu
Val Ala Arg1535 1540 1545Leu Arg Gln Asn Ala Asp Gln Cys Cys Pro
Glu Tyr Glu Cys Val1550 1555 1560Cys Asp Pro Val Ser Cys Asp Leu
Pro Pro Val Pro His Cys Glu1565 1570 1575Arg Gly Leu Gln Pro Thr
Leu Thr Asn Pro Gly Glu Cys Arg Pro1580 1585 1590Asn Phe Thr Cys
Ala Cys Arg Lys Glu Glu Cys Lys Arg Val Ser1595 1600 1605Pro Pro
Ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr1610 1615
1620Gln Cys Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys Val Asn Ser1625
1630 1635Thr Val Ser Cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr
Asn1640 1645 1650Asp Cys Gly Cys Thr Thr Thr Thr Cys Leu Pro Asp
Lys Val Cys1655 1660 1665Val His Arg Ser Thr Ile Tyr Pro Val Gly
Gln Phe Trp Glu Glu1670 1675 1680Gly Cys Asp Val Cys Thr Cys Thr
Asp Met Glu Asp Ala Val Met1685 1690 1695Gly Leu Arg Val Ala Gln
Cys Ser Gln Lys Pro Cys Glu Asp Ser1700 1705 1710Cys Arg Ser Gly
Phe Thr Tyr Val Leu His Glu Gly Glu Cys Cys1715 1720 1725Gly Arg
Cys Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro1730 1735
1740Arg Gly Asp Ser Gln Ser Ser Trp Lys Ser Val Gly Ser Gln Trp1745
1750 1755Ala Ser Pro Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg
Val1760 1765 1770Lys Glu Glu Val Phe Ile Gln Gln Arg Asn Val Ser
Cys Pro Gln1775 1780 1785Leu Glu Val Pro Val Cys Pro Ser Gly Phe
Gln Leu Ser Cys Lys1790 1795 1800Thr Ser Ala Cys Cys Pro Ser Cys
Arg Cys Glu Arg Met Glu Ala1805 1810 1815Cys Met Leu Asn Gly Thr
Val Ile Gly Pro Gly Lys Thr Val Met1820 1825 1830Ile Asp Val Cys
Thr Thr Cys Arg Cys Met Val Gln Val Gly Val1835 1840 1845Ile Ser
Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro1850 1855
1860Cys Pro Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys Cys1865
1870 1875Gly Arg Cys Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly
Gly1880 1885 1890Gln Ile Met Thr Leu Lys Arg Asp Glu Thr Leu Gln
Asp Gly Cys1895 1900 1905Asp Thr His Phe Cys Lys Val Asn Glu Arg
Gly Glu Tyr Phe Trp1910 1915 1920Glu Lys Arg Val Thr Gly Cys Pro
Pro Phe Asp Glu His Lys Cys1925 1930 1935Leu Ala Glu Gly Gly Lys
Ile Met Lys Ile Pro Gly Thr Cys Cys1940 1945 1950Asp Thr Cys Glu
Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg Leu1955 1960 1965Gln Tyr
Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp1970 1975
1980Ile His Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr Ser1985
1990 1995Ile Asp Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser
Pro2000 2005 2010Thr Arg Thr Glu Pro Met Gln Val Ala Leu His Cys
Thr Asn Gly2015 2020 2025Ser Val Val Tyr His Glu Val Leu Asn Ala
Met Glu Cys Lys Cys2030 2035 2040Ser Pro Arg Lys Cys Ser Lys2045
2050
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