U.S. patent application number 16/258488 was filed with the patent office on 2021-10-07 for lyophilized recombinant vwf formulations.
The applicant listed for this patent is Baxalta GmbH, Baxalta Incorporated. Invention is credited to Eva Haidweger, Kurt Schnecker, Peter Turecek.
Application Number | 20210308227 16/258488 |
Document ID | / |
Family ID | 1000005851539 |
Filed Date | 2021-10-07 |
United States Patent
Application |
20210308227 |
Kind Code |
A9 |
Schnecker; Kurt ; et
al. |
October 7, 2021 |
LYOPHILIZED RECOMBINANT VWF FORMULATIONS
Abstract
The present invention provides long-term stable pharmaceutical
formulations of lyophilized recombinant von-Willebrand Factor
(rVWF) and methods for making and administering said
formulations.
Inventors: |
Schnecker; Kurt; (Vienna,
AT) ; Haidweger; Eva; (Vienna, AT) ; Turecek;
Peter; (Klosterneuburg, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baxalta Incorporated
Baxalta GmbH |
Bannockburn
Zug |
IL |
US
CH |
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|
Prior
Publication: |
|
Document Identifier |
Publication Date |
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US 20190142907 A1 |
May 16, 2019 |
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Family ID: |
1000005851539 |
Appl. No.: |
16/258488 |
Filed: |
January 25, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14939364 |
Nov 12, 2015 |
10232022 |
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16258488 |
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12603064 |
Oct 21, 2009 |
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14939364 |
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14693078 |
Apr 22, 2015 |
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12603064 |
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12342646 |
Dec 23, 2008 |
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14693078 |
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61107273 |
Oct 21, 2008 |
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61017881 |
Dec 31, 2007 |
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61017418 |
Dec 28, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/22 20130101;
A61K 47/183 20130101; A61K 47/12 20130101; A61K 47/26 20130101;
A61K 9/19 20130101; A61K 9/08 20130101; A61K 38/16 20130101; A61K
38/36 20130101; A61K 38/17 20130101 |
International
Class: |
A61K 38/36 20060101
A61K038/36; A61K 47/22 20060101 A61K047/22; A61K 38/17 20060101
A61K038/17; A61K 47/26 20060101 A61K047/26; A61K 9/08 20060101
A61K009/08; A61K 47/12 20060101 A61K047/12; A61K 38/16 20060101
A61K038/16; A61K 9/19 20060101 A61K009/19; A61K 47/18 20060101
A61K047/18 |
Claims
1. A stable lyophilized pharmaceutical formulation of a recombinant
von Willebrand Factor (rVWF) comprising: (a) a rVWF; (b) one or
more buffering agents; (c) one or more amino acids; (d) one or more
stabilizing agents; and (e) one or more surfactants; wherein said
rVWF comprises a polypeptide encoded by the polynucleotide set out
in SEQ ID NO: 1, wherein the polypeptide causes agglutination of
stabilized platelets in the presence of ristocetin, or of binding
to Factor VIII; wherein said buffer comprises a pH buffering agent
selected from the group consisting of citrate and HEPES at 15 mM
and said pH is in a range of about 2.0 to about 12.0; said amino
acid is selected from the group consisting of glycine, lysine, and
histidine at a concentration of about 1 to about 500 mM; said
stabilizing agent is at a concentration of about 0.1 to about 1000
mM and 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;
and said surfactant is at a concentration of about 0.01 g/L to
about 0.5 g/L.
2. The formulation of claim 1 wherein the buffering agent is
citrate.
3. The formulation of claim 1 wherein pH is in the range of about
6.0 to about 8.0.
4. The formulation of claim 3 wherein pH is in the range of about
6.5 to about 7.5.
5. The formulation of claim 3 wherein the pH is about 7.3.
6. The formulation of claim 1 wherein the buffering agent is
citrate and the pH is about 7.3.
7. The formulation of claim 1 wherein the amino acid is at a
concentration range of about 1 mM to about 300 mM.
8. The formulation of claim 7 wherein the amino acid is glycine at
a concentration of about 15 mM.
9. The formulation of claim 1 wherein the buffering agent is
citrate and the pH is about 7.3; and wherein the amino acid is
glycine at a concentration of about 15 mM.
10. The formulation of claim 1 wherein the stabilizing agents are
trehalose at a concentration of about 10 g/L and mannitol at a
concentration of about 20 g/L.
11. 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.
12. The formulation of claim 11 wherein the surfactant is TWEEN-80
at about 0.01 g/L.
13. The formulation of claim 1 wherein the buffering agent is
citrate at a concentration of about 15 mM at about pH 7.3; wherein
the amino acid is glycine at a concentration of about 15 mM;
wherein the stabilizing agents are trehalose at a concentration of
about 10 g/L and mannitol at a concentration of about 20 g/L; and
wherein the surfactant is TWEEN-80 at about 0.1 g/L.
14. A stable lyophilized pharmaceutical formulation of a
recombinant von Willebrand Factor (rVWF) comprising: (a) a rVWF;
(b) one or more buffering agents; (c) one or more amino acids; (d)
one or more stabilizing agents; and (e) one or more surfactants;
wherein the formulation is prepared by lyophilizing a solution
comprising: (a) said rVWF comprising a polypeptide encoded by the
polynucleotide set out in SEQ ID NO: 1; (b) said buffer comprising
a pH buffering agent in a range of about 0.1 mM to about 500 mM and
having a pH in a range of about 2.0 to about 12.0; wherein the
buffering agent is citrate; (c) said amino acid at a concentration
of about 1 to about 500 mM; wherein the amino acid is glycine; (d)
said stabilizing agent at a concentration of about 0.1 to about
1000 mM; wherein the one or more stabilizing agents is mannitol and
trehalose; and (e) said surfactant at a concentration of about 0.01
g/L to about 0.5 g/L; wherein the surfactant is TWEEN-80.
15. A stable lyophilized pharmaceutical formulation of a
recombinant von Willebrand Factor (rVWF) comprising: (a) a rVWF;
(b) one or more buffering agents; (c) one or more amino acids; (d)
one or more stabilizing agents; and (e) one or more surfactants;
wherein the formulation is prepared by lyophilizing a solution
comprising: (a) said rVWF comprising a polypeptide encoded by the
polynucleotide set out in SEQ ID NO: 1; (b) said buffer comprising
a pH buffering agent in a range of about 0.1 mM to about 500 mM and
having a pH in a range of about 6.5 to about 7.5; wherein the
buffering agent is citrate; (c) said amino acid at a concentration
of about 1 to about 500 mM; wherein the amino acid is glycine; (d)
said stabilizing agent at a concentration of about 0.1 to about
1000 mM; wherein the one or more stabilizing agents is mannitol and
trehalose; and (e) said surfactant at a concentration of about 0.01
g/L to about 0.5 g/L, wherein the surfactant is TWEEN-80.
Description
PRIORITY
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/939,364, filed Nov. 12, 2015 which is a continuation of
U.S. patent application Ser. No. 12/603,064, filed Oct. 21, 2009,
now abandoned, which claims priority to U.S. Provisional
Application No. 61/107,273, filed Oct. 21, 2008.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 25, 2019, is named 008073_5131US03_SL.txt and is 54,313
bytes in size.
FIELD OF THE INVENTION
[0003] Generally, the invention relates to formulations of
lyophilized recombinant VWF and methods for making a lyophilized
composition comprising recombinant VWF.
BACKGROUND OF THE INVENTION
[0004] 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.
[0005] 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).
[0006] 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)).
[0007] 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.
[0008] U.S. 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). Further, VWF is known to form aggregates during stress
conditions.
[0009] Thus there exists a need in the art to develop a stable
pharmaceutical formulation comprising recombinant VWF.
SUMMARY OF THE INVENTION
[0010] The present invention provides formulations useful for
lyophilization of 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.
[0011] In one embodiment, a stable lyophilized pharmaceutical
formulation of a recombinant von Willebrand Factor (rVWF) is
provided comprising: (a) a rVWF; (b) one or more buffering agents;
(c) one or more amino acids; (d) one or more stabilizing agents;
and (e) one or more surfactants; the rVWF comprising 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; the buffer is
comprising of a pH buffering agent in a range of about 0.1 mM to
about 500 mM and the pH is in a range of about 2.0 to about 12.0;
the amino acid is at a concentration of about 1 to about 500 mM;
the stabilizing agent is at a concentration of about 0.1 to about
1000 mM; and the surfactant is at a concentration of about 0.01 g/L
to about 0.5 g/L.
[0012] In another embodiment, the rVWF comprises the amino acid
sequence set out in SEQ ID NO: 3. In still another embodiment, the
buffering agent is selected from the group consisting of citrate,
glycine, histidine, HEPES, Tris and combinations of these agents.
In yet another embodiment, the buffering agent is citrate. In
various embodiments, the pH is in the range of about 6.0 to about
8.0, about 6.5 to about 7.5, or about 7.3. In another embodiment,
the pH is about 7.3.
[0013] In another embodiment, the aforementioned amino acid is
selected from the group consisting of glycine, histidine, proline,
serine, alanine and arginine. In another embodiment, the amino acid
is at a concentration range of about 0.5 mM to about 300 mM. In
still another embodiment, the amino acid is glycine at a
concentration of about 15 mM.
[0014] In one embodiment of the invention, the rVWF comprises the
amino acid sequence set out in SEQ ID NO: 3; wherein the buffering
agent is citrate and the pH is about 7.3; and wherein the amino
acid is glycine at a concentration of about 15 mM.
[0015] In still another embodiment of the invention, the
aforementioned one or more stabilizing agents 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 one
embodiment, the stabilizing agents are trehalose at a concentration
of about 10 g/L mM and mannitol at a concentration of about 20
g/L.
[0016] In yet another embodiment of the invention, the
aforementioned 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 still another embodiment, the
surfactant is TWEEN-80 at about 0.01 g/L.
[0017] In another embodiment of the invention, the rVWF comprises
amino acid sequence set out in SEQ ID NO: 3; wherein the buffering
agent is citrate at a concentration of about 15 mM at about pH 7.3;
wherein the amino acid is glycine at a concentration of about 15
mM; wherein the stabilizing agents are trehalose at a concentration
of about 10 g/L and mannitol at a concentration of about 20 g/L.;
and wherein the surfactant is TWEEN-80 at about 0.1 g/L.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 shows ANCOVA analysis of pooled VWF:RCo activity in
lots evaluated for stability (stored at 5.degree. C..+-.3.degree.
C.).
[0019] FIG. 2 shows the increase in residual moisture in rVWF FDP
stored at 5.degree. C..+-.3.degree. C.
[0020] FIG. 3 shows the increase in residual moisture in rVWF FDP
stored at 40.degree. C..+-.2.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
Definition of Terms
[0021] 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).
[0022] 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.
[0023] 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.
[0024] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0025] 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.
[0026] 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).
[0027] 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.
[0028] 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 are prepared, for example, using an
automated polypeptide synthesizer. The term "protein" typically
refers to large polypeptides. The term "peptide" typically refers
to short polypeptides.
[0029] 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.
[0030] 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, deletion, insertion
and/or addition 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.
[0031] 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 and without limitation, in one aspect
the variant is 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.
Recombinant VWF
[0032] 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_000552 and NP_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).
[0033] 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 the A2 domain thus resistant to proteolysis (Lankhof et
al., Thromb. Haemost. 77: 1008-1013, 1997), and a 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 is,
in one aspect, carried out in VWF-deficient mammals according to
methods known in the state in the art.
[0034] The rVWF of the present invention is 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 the transformed cells, e.g. in a continuous or
batchwise manner, (iv) expressing VWF, e.g. constitutively or upon
induction, and (v) isolating the 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 is, in one aspect, 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.
Alternatively, the DNA molecule is, in another aspect, synthesized
using chemical synthesis techniques, such as the phosphoramidate
method. Also, in still another aspect, a combination of these
techniques is used.
[0035] 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.
[0036] Any of a large number of available and well-known host cells
are 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, without limitation, bacteria, yeast and other
fungi, insects, plants, mammalian (including human) cells in
culture, or other hosts known in the art.
[0037] Transformed host cells are 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 media or the
host cells themselves by methods well known in the art.
[0038] Depending on the host cell utilized to express a compound of
the invention, carbohydrate (oligosaccharide) groups are optionally
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 N-linked and O-linked oligosaccharides 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, in one
aspect, confers 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). In other aspects, such
sites are glycosylated by synthetic or semi-synthetic procedures
known in the art.
[0039] Alternatively, the compounds are made by synthetic methods
using, for example, solid phase synthesis techniques. 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.
Fragments, Variants and Analogs of VWF
[0040] Methods for preparing polypeptide fragments, variants or
analogs are well-known in the art.
[0041] 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.
[0042] 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, for example and without limitation,
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 or both termini of a
protein and include, for example, fusion proteins. Combinations of
the aforementioned analogs are also contemplated.
[0043] 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 complete loss of other functions or
properties. In one aspect, substitutions are conservative
substitutions. "Conservative amino acid substitution" is
substitution of an amino acid with an amino acid having a side
chain or 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).
[0044] In one aspect, analogs are substantially homologous or
substantially identical to the recombinant VWF from which they are
derived. Analogs include those which retain at least some of the
biological activity of the wild-type polypeptide, e.g. blood
clotting activity.
[0045] Polypeptide variants contemplated include, without
limitation, 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.
[0046] Preparing pegylated polypeptide analogs will in one aspect
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 are 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
has a single PEG moiety at the N-terminus. Polyethylene glycol
(PEG) may be attached to the blood clotting factor to, for example,
provide a longer half-life in vivo. The PEG group may be of any
convenient molecular weight and is 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. In certain aspects, 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.
[0047] 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 (CA) containing 0.1 M NaIO4 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 are optionally be separated from the
rVWF-polysialic acid conjugate by, for example,
ultrafiltration/diafiltration. Conjugation of rVWF with polysialic
acid is achieved using glutaraldehyde as cross-linking reagent
(Migneault et al., Biotechniques 37: 790-796, 2004).
[0048] It is further contemplated in another aspect that a
polypeptide of the invention is 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.
[0049] It is also contemplated in another aspect that prepro-VWF
and pro-VWF polypeptides will 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 generation in
vitro. In addition to recombinant, biologically active fragments,
variants, or other 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.
[0050] 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. In various aspects, these
polynucleotides are 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).
Lyophilization
[0051] In one aspect, the formulations comprising a VWF polypeptide
of the invention are 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)].
[0052] 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.
[0053] The lyophilization cycle not only determines the final
physical state of 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 the
structure may 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.
Formulations and Excipients in General
[0054] Excipients are additives that either impart or enhance the
stability and delivery of a drug product (e.g., protein).
Regardless of the reason for their inclusion, excipients are an
integral component of a formulation 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.
[0055] A lyophilized formulation is, in one aspect, at least
comprised of one or more of a buffer, a bulking agent, and a
stabilizer. In this aspect, the utility of a surfactant is
evaluated and selected in cases where aggregation during the
lyophilization step or during reconstitution becomes an issue. An
appropriate buffering agent is included to maintain the formulation
within stable zones of pH during lyophilization. A comparison of
the excipient components contemplated for liquid and lyophilized
protein formulations is provided in Table A.
TABLE-US-00001 TABLE A Excipient components of lyophilized protein
formulations Excipient component Function in lyophilized
formulation Buffer Maintain pH of formulation during lyophilization
and upon reconstitution Tonicity agent/ Stabilizers include cryo
and lyoprotectants stabilizer Examples include Polyols, sugars and
polymers Cryoprotectants protect proteins from freezing stresses
Lyoprotectants stabilize proteins in the freeze-dried state Bulking
agent Used to enhance product elegance and to prevent blowout
Provides structural strength to the lyo cake Examples include
mannitol and glycine Surfactant Employed if aggregation during the
lyophilization process is an issue May serve to reduce
reconstitution times Examples include polysorbate 20 and 80
Anti-oxidant Usually not employed, molecular reactions in the lyo
cake are greatly retarded Metal ions/ May be included if a specific
metal ion is included only chelating agent as a co-factor or where
the metal is required for protease activity Chelating agents are
generally not needed in lyo formu- lations Preservative For
multi-dose formulations only Provides protection against microbial
growth in formu- lation Is usually included in the reconstitution
diluent (e.g. bWFI)
[0056] The principal challenge in developing formulations for
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 are 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.
[0057] 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 protein).
For example, the amount and type of a salt to be included in a
biopharmaceutical formulation of the invention is 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.
[0058] 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.
[0059] 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.
[0060] 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 protein 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.
Buffers and Buffering Agents
[0061] The stability of a pharmacologically active protein
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,
are useful in this endeavor (Remmele R. L. Jr., et al.,
Biochemistry, 38(16): 5241-7 (1999)). Once a formulation is
finalized, the protein must be manufactured and maintained
throughout its shelf-life. Hence, buffering agents are almost
always employed to control pH in the formulation.
[0062] 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.
[0063] 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 protein 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)).
[0064] Buffers for lyophilized formulations need additional
consideration. Some buffers like sodium phosphate can crystallize
out of the protein amorphous phase during freezing resulting in
shifts in pH. Other common buffers such as acetate and imidazole
may sublime or evaporate during the lyophilization process, thereby
shifting the pH of formulation during lyophilization or after
reconstitution.
[0065] 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.
[0066] 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, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500
mM.
[0067] Exemplary pH buffering agents used to buffer the formulation
as set out herein include, but are not limited to organic acids,
glycine, histidine, glutamate, succinate, phosphate, acetate,
citrate, Tris, HEPES, and amino acids or mixtures of amino acids,
including, but not limited to aspartate, histidine, and glycine. In
one embodiment of the present invention, the buffering agent is
citrate.
Stabilizers and Bulking Agents
[0068] In one aspect of the present pharmaceutical formulations, a
stabilizer (or a combination of stabilizers) is 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 physical
degradation, including chemical degradation (for example,
autolysis, deamidation, oxidation, etc.) in an aqueous state.
Stabilizers contemplated 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-methyl pyrollidene, 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. In one embodiment f the
present invention, mannitol and trehalose are used as stabilizing
agents.
[0069] If desired, the formulations also include appropriate
amounts of bulking and osmolarity regulating agents. Bulking agents
include, for example and without limitation, 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.
Surfactants
[0070] Proteins 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.
[0071] 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. Surfactants contemplated herein include, without
limitation, 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.
[0072] 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.
[0073] 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.
[0074] Surfactants are also added in appropriate amounts to prevent
surface related aggregation phenomenon during freezing and drying
[Chang, B, J. Pharm. Sci. 85:1325, (1996)]. Thus, exemplary
surfactants include, without limitation, 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 one embodiment of the present invention, the surfactant is
TWEEN-80. In the present formulations, the surfactant is
incorporated in a concentration of about 0.01 to about 0.5 g/L. In
formulations provided, the surfactant concentration is 0.005, 0.01,
0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9 or 1.0 g/L.
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.
Other Common Excipient Components
Amino Acids
[0076] Amino acids have found versatile use in protein formulations
as buffers, bulking agents, stabilizers and antioxidants. Thus, in
one aspect 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. Histidine is commonly found in marketed protein
formulations, 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, 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 was also observed by others to
reduce the viscosity of a high protein concentration formulation.
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. 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)).
[0077] In various aspects, formulations are provided which include
one or more of the amino acids glycine, proline, serine, arginine
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. Arginine has been shown to be an
effective agent in inhibiting aggregation and has been used in both
liquid and lyophilized formulations.
[0078] In formulations provided, the amino acid concentration is
between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300,
and 500 mM. In one embodiment of the present invention, the amino
acid is glycine.
Antioxidants
[0079] 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 invention therefore
contemplates the use of the pharmaceutical antioxidants including,
without limitation, reducing agents, oxygen/free-radical
scavengers, or chelating agents. Antioxidants in therapeutic
protein formulations are, in one aspect, 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.
[0080] 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. Antioxidants contemplated in certain
aspects include, without limitation, reducing agents and
oxygen/free-radical scavengers, EDTA, and sodium thiosulfate.
Metal Ions
[0081] 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).
[0082] 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.
Preservatives
[0083] 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, without limitation, 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)).
[0084] 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.
[0085] 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)).
[0086] Development of liquid formulations containing preservatives
are more challenging than lyophilized formulations. Freeze-dried
products can be lyophilized without the preservative and
reconstituted with a preservative containing diluent at the time of
use. This shortens the time for which a preservative is in contact
with the protein significantly minimizing the associated stability
risks. With liquid formulations, preservative effectiveness and
stability have to be maintained over the entire product shelf-life
(.about.18-24 months). An important point to note is that
preservative effectiveness has to be demonstrated in the final
formulation containing the active drug and all excipient
components.
[0087] 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)). In various aspects the use of
preservatives provide a benefit that outweighs any side
effects.
Methods of Preparation
[0088] The present invention further contemplates methods for the
preparation of pharmaceutical formulations.
[0089] The present methods further comprise one or more of the
following steps: adding a stabilizing agent as described herein to
said mixture prior to lyophilizing, adding at least one agent
selected from a bulking agent, an osmolarity regulating agent, and
a surfactant, each of which as described herein, to said mixture
prior to lyophilization.
[0090] The standard reconstitution practice for lyophilized
material is to add back a volume of pure water or sterile water for
injection (WFI) (typically equivalent to the volume removed during
lyophilization), although dilute solutions of antibacterial agents
are sometimes used in the production of pharmaceuticals for
parenteral administration [Chen, Drug Development and Industrial
Pharmacy, 18:1311-1354 (1992)]. Accordingly, methods are provided
for preparation of reconstituted rVWF compositions comprising the
step of adding a diluent to a lyophilized rVWFcomposition of the
invention.
[0091] The lyophilized material may be reconstituted as an aqueous
solution. 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, an aqueous
suspension that contains the active compound in admixture with
excipients suitable for the manufacture of aqueous suspensions). In
various aspects, such excipients are suspending agents, for example
and without limitation, sodium carboxymethylcellulose,
methylcellulose, hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents are a naturally-occurring phosphatide, for example
and without limitation, lecithin, or condensation products of an
alkylene oxide with fatty acids, for example and without
limitation, polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example and
without limitation, 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 and without
limitation, polyethylene sorbitan monooleate. In various aspects,
the aqueous suspensions also contain one or more preservatives, for
example and without limitation, ethyl, or n-propyl,
p-hydroxybenzoate.
Administration
[0092] 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.
[0093] The pharmaceutical formulations are 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,
intramusclar, 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.
[0094] Single or multiple administrations of the compositions are
carried out with the dose levels and pattern being selected by the
treating physician. For the prevention or treatment of disease, the
appropriate dosage depends 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.
Kits
[0095] As an additional aspect, the invention includes kits which
comprise one or more lyophilized compositions 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.
Dosages
[0096] 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.
[0097] In one aspect, formulations of the invention are
administered by an initial bolus followed by a continuous infusion
to maintain therapeutic circulating levels of drug product. As
another example, the inventive compound is 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 depends on the
pharmacokinetic parameters of the agents and the route of
administration. The optimal pharmaceutical formulation is
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 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 is 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 is 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.
[0098] The following examples are not intended to be limiting but
only exemplary of specific embodiments of the invention.
Example 1
Shaking Experiments
[0099] In order to determine the amount of precipitation of rVWF in
various formulations, the extent of aggregation of rVWF following
turbulent shaking was tested under a variety of conditions.
[0100] As shown in Table 1 below, various rVWF formulations were
assessed in a 20 mM citrate buffer, pH 7.3. Shaking experiments
were designed to simulate mechanical stress conditions. 1-2 ml of
each formulation was shaken with a laboratory shaker for 10 minutes
at 1200 rpm.
TABLE-US-00002 TABLE 1 PEG Tween Lyo 25 Lysine Histidine Glycine
Serine Mannitol 1500 80 Sucrose Trehalose Raffinose 18 30 mM 5 g/L
19 30 mM 5 g/L 20 30 mM 5 g/L 21 30 mM 5 g/L 22 30 mM 5 g/L 23 30
mM 5 g/L 24 30 mM 5 g/L 25 30 mM 5 g/L 26 30 mM 0.1 g/L 27 30 mM
0.1 g/L 28 30 mM 0.1 g/L 29 30 mM 0.1 g/L 30 30 mM 5 g/L 5 g/L 31
30 mM 5 g/L 5 g/L 32 30 mM 5 g/L 5 g/L 33 30 mM 5 g/L 5 g/L 34 30
mM 5 g/L 0.1 g/L 35 30 mM 5 g/L 0.1 g/L 36 30 mM 5 g/L 0.1 g/L 37
30 mM 5 g/L 0.1 g/L 38 30 mM 5 g/L 5 g/L 0.1 g/L 39 30 mM 5 g/L 5
g/L 0.1 g/L 40 30 mM 5 g/L 5 g/L 0.1 g/L 41 30 mM 5 g/L 5 g/L 0.1
g/L 42 5 g/L 43 5 g/L 44 5 g/L 45 5 g/L 46 5 g/L 5 g/L 47 5 g/L 5
g/L 48 5 g/L 5 g/L 49 5 g/L 5 g/L 50 5 g/L 0.1 g/L 51 0.1 g/L 5 g/L
52 0.1 g/L 5 g/L 53 0.1 g/L 5 g/L
[0101] The assessment of the visible VWF aggregates was done
according to the scheme shown below. "Visible aggregates," in most
cases, are gelatinous fibers ranging in size from about 100 nm to
1-2 cm.
TABLE-US-00003 SCHEME Particles A no particles B several particles,
rarely visible (dots) B1 many particles, rarely visible (dots) C
several particles, easily visible (fibers) D many particles, easily
visible (fibers) E visible particles (>1 mm fibers) E1 fluffy
white precipitate (swims on the surface) E2 jellyfish
[0102] The results of the shaking experiments are shown in Table 2,
below.
TABLE-US-00004 TABLE 2 Shaking 1-2 mL Lyo 25 PEG Tween 1200 rpm
Samples Lysine Histidine Glycine Serine Mannitol 1500 80 30 min 18
30 mM 5 g/L E1 19 30 mM 5 g/L E1 20 30 mM 5 g/L E1 21 30 mM 5 g/L
E1 22 30 mM 5 g/L E1 23 30 mM 5 g/L E1 24 30 mM 5 g/L E1 25 30 mM 5
g/L E1 26 30 mM 0.1 g/L E2 big 27 30 mM 0.1 g/L E2 big 28 30 mM 0.1
g/L E2 ~6 mm 29 30 mM 0.1 g/L E2 ~3 mm 30 30 mM 5 g/L 5 g/L E1 31
30 mM 5 g/L 5 g/L E1 32 30 mM 5 g/L 5 g/L E1 33 30 mM 5 g/L 5 g/L
E1 34 30 mM 5 g/L 0.1 g/L B1 35 30 mM 5 g/L 0.1 g/L B 36 30 mM 5
g/L 0.1 g/L E2 big 37 30 mM 5 g/L 0.1 g/L E2 big 38 30 mM 5 g/L 5
g/L 0.1 g/L D 39 30 mM 5 g/L 5 g/L 0.1 g/L D 40 30 mM 5 g/L 5 g/L
0.1 g/L B 41 30 mM 5 g/L 5 g/L 0.1 g/L B
[0103] In summary, the shaking experiments described above indicate
that formulations containing Tween-80 and Mannitol provide the best
results (i.e., the least amount of aggregation).
Example 2
Freeze-Thaw Experiments
[0104] Freeze-thaw experiments were designed to assess the impact
of stress caused by repeated freezing and thawing. In addition to
the formulations described above for the shaking experiments (Table
1), the following formulations were assessed (Table 3 and Table
4):
TABLE-US-00005 TABLE 3 Lyo 25 PEG Tween Samples Mannitol 1500 80
Sucrose Trehalose Raffinose 42 5 g/L 43 5 g/L 44 5 g/L 45 5 g/L 46
5 g/L 5 g/L 47 5 g/L 48 5 g/L 49 5 g/L 50 5 g/L 0.1 g/L 51 0.1 g/L
5 g/L 52 0.1 g/L 5 g/L 53 0.1 g/L 5 g/L
TABLE-US-00006 TABLE 4 Lyo 25 PEG Tween- Samples Lysine Histidine
Glycine Serine Mannitol 1500 80 Sucrose Trehalose Raffinose 76 20
g/L 0.2 g/L 20 g/L 77 20 g/L 0.2 g/L 10 g/L 78 20 g/L 0.2 g/L 10
g/L 79 15 mM 20 g/L 0.2 g/L 20 g/L 80 15 mM 20 g/L 0.2 g/L 10 g/L
81 15 mM 20 g/L 0.2 g/L 10 g/L 82 15 mM 15 mM 20 g/L 0.2 g/L 20 g/L
83 15 mM 15 mM 20 g/L 0.2 g/L 10 g/L 84 15 mM 15 mM 20 g/L 0.2 g/L
10 g/L 85 15 mM 15 mM 20 g/L 5 g/L 0.2 g/L 86 15 mM 15 mM 20 g/L 5
g/L 0.2 g/L 10 g/L 87 15 mM 15 mM 20 g/L 15 g/L 0.2 g/L 10 g/L 88
15 mM 15 mM 20 g/L 0.2 g/L 5 g/L 89 15 mM 15 mM 20 g/L 0.2 g/L 15
g/L 90 15 mM 20 g/L 0.2 g/L 15 g/L 92 15 mM 15 mM 20 g/L 0.2 g/L 10
g/L 93 30 mM 20 g/L 0.2 g/L 10 g/L 94 30 mM 20 g/L 0.2 g/L 10 g/L
95 30 mM 20 g/L 0.2 g/L 10 g/L 96 30 mM 20 g/L 0.2 g/L 10 g/L 97 15
mM 20 g/L 0.2 g/L 10 g/L 98 15 mM 15 mM 20 g/L 0.2 g/L 10 g/L 99 15
mM 15 mM 20 g/L 0.2 g/L 5 g/L 100 15 mM 20 g/L 0.2 g/L 10 g/L 15 mM
20 g/L 0.2 g/L 10 g/L
[0105] All formulations were frozen at -20.degree. C. in a freezer
for approximately 1 hour and then thawed at room temperature. The
results are shown in Table 5 below.
TABLE-US-00007 TABLE 5 Freeze/ Freeze/ Thaw Thaw Lyo 25 PEG Tween-
(4 (~10 Samples Lysine Histidine Glycine Serine Mannitol 1500 80
Sucrose Trehalose Raffinose times) times) 18 30 mM 5 g/L E1 15 19
30 mM 5 g/L E1 19 20 30 mM 5 g/L C 20 21 30 mM 5 g/L C 21 22 30 mM
5 g/L C 22 23 30 mM 5 g/L C/B1 23 24 30 mM 5 g/L C 24 25 30 mM 5
g/L C 25 26 30 mM 0.1 g/L B 26 27 30 mM 0.1 g/L B-B1 27 28 30 mM
0.1 g/L B 28 29 30 mM 0.1 g/L E 29 30 30 mM 5 g/L 5 g/L E 30 31 30
mM 5 g/L 5 g/L D 31 32 30 mM 5 g/L 5 g/L B1-C 32 33 30 mM 5 g/L 5
g/L C/D 33 34 30 mM 5 g/L 0.1 g/L E2 (rest B) 34 35 30 mM 5 g/L 0.1
g/L E 35 36 30 mM 5 g/L 0.1 g/L E 36 37 30 mM 5 g/L 0.1 g/L B 37 38
30 mM 5 g/L 5 g/L 0.1 g/L B 38 39 30 mM 5 g/L 5 g/L 0.1 g/L B 39 40
30 mM 5 g/L 5 g/L 0.1 g/L B 40 41 30 mM 5 g/L 5 g/L 0.1 g/L A 41 42
5 g/L D 42 43 5 g/L D 43 44 5 g/L E1 44 45 5 g/L E1 45 46 5 g/L 5
g/L D 46 47 5 g/L 5 g/L D 47 48 5 g/L 5 g/L D-E 48 49 5 g/L 5 g/L E
49 50 5 g/L 0.1 g/L B1 50 51 0.1 g/L 5 g/L C-D 51 52 0.1 g/L 5 g/L
B1 52 53 0.1 g/L 5 g/L B1 53
[0106] As shown above, Trehalose provided the best results (i.e.,
the least amount of aggregation).
Example 3
Lyophilization Experiments
[0107] Lyophilization experiments were designed to assess the
ability of various formulations to allow the formation of a
lyo-cake which dissolves in less than 10 minutes and results in a
clear solution. An accelerated stability study was also performed
to demonstrate that no significant loss of biological activity.
[0108] The formulations shown in Table 6 below were lyophilized
with a nitrogen lyophilizer TS20002 according to the manufacturer's
instructions. The total time for lyophilization was approximately
72 hours. Each of the formulations below also contained 20 g/L
Mannitol and 0.1 g/L Tween-80.
TABLE-US-00008 TABLE 6 Lyo 26 Citrate HEPES Glycine Histidine
Acetate Tris Phosphate Lysine Histidine Glycine Trehalose Raffinose
1 15 mM 10 g/L 2 15 mM 15 mM 10 g/L 3 15 mM 15 mM 10 g/L 4 15 mM 15
mM 10 g/L 5 15 mM 15 mM 15 mM 10 g/L 6 15 mM 30 mM 10 g/L 7 15 mM
10 g/L 8 15 mM 15 mM 10 g/L 9 15 mM 10 g/L 10 15 mM 15 mM 10 g/L 11
15 mM 15 mM 10 g/L 12 15 mM 15 mM 10 g/L 13 15 mM 15 mM 15 mM 10
g/L 14 15 mM 15 mM 15 mM 10 g/L 15 15 mM 10 g/L 16 15 mM 15 mM 10
g/L 17 15 mM 10 g/L 18 15 mM 15 mM 10 g/L 19 15 mM 15 mM 15 mM 10
g/L 20 15 mM 15 mM 15 mM 10 g/L 21 15 mM 15 mM 15 mM 10 g/L
[0109] The results of the lyophilzation experiments are shown in
Table 7 below.
TABLE-US-00009 TABLE 7 Lyo Buffer -- Excipents -- -- -- 26 Citrate
HEPES Glycine Histidine Acetate Tris Phosphate Lysine Histidine
Glycine Trehalose Raffinose 1 15 mM 10 g/L 2 15 mM 15 mM 10 g/L 3
15 mM 15 mM 10 g/L 4 15 mM 15 mM 10 g/L 5 15 mM 15 mM 10 g/L 6 15
mM 30 mM 10 g/L 7 15 mM 10 g/L 8 15 mM 15 mM 10 g/L 9 15 mM 10 g/L
10 15 mM 15 mM 10 g/L 11 15 mM 15 mM 10 g/L 12 15 mM 15 mM 10 g/L
13 15 mM 15 mM 15 mM 10 g/L 14 15 mM 15 mM 15 mM 10 g/L 15 15 mM 10
g/L 16 15 mM 15 mM 10 g/L 17 15 mM 10 g/L 18 15 mM 15 mM 10 g/L 19
15 mM 15 mM 15 mM 10 g/L 20 15 mM 15 mM 15 mM 10 g/L 21 15 mM 15 mM
15 mM 10 g/L
[0110] As shown above, either a Citrate or HEPES buffer in
combination with an amino acid provided the clearest solution.
[0111] In order to assess stability of the reconstituted
lyophilized rVWF, VWF:Ag and VWF:RCo tests were performed. 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. Samples were
stored at 40.degree. C. Assuming applicability of the Arrhenius
equation, one month stability at 40.degree. C. is equivalent to
approximately one year at 4.degree. C. The results of the stability
experiments are shown in Table 8 and Table 9 below.
TABLE-US-00010 TABLE 8 rVWF: Ag Weeks at 40.degree. formulation 0 4
5 8 1 121.1 89.8 113.0 106.6 2 121.8 102.0 114.0 112.8 3 119.9
102.0 105.,0 112.7 4 117.3 100.0 108.0 114.4 5 121.2 98.2 117.0
114.9 6 123.8 96.6 107.0 -- 7 135.2 96.6 112.0 112.4 8 130.6 82.2
108.0 115.7 9 112.0 89.5 109.0 107.0 10 122.4 87.1 106.0 107.7 11
119.3 97.5 115.0 114.2 12 124.2 109.0 109.0 103.4 13 110.2 92.3
106.0 112.4 14 108.9 107.0 103.0 109.0
TABLE-US-00011 TABLE 9 rVWF: RCo Weeks at 40.degree. formulation 0
4 5 8 1 86 102 97.0 93.0 2 84 97 88.0 89.0 3 85 100 87.0 93.0 4 102
81.0 98.0 5 85 89 88.0 98.0 6 83 102 88.0 7 92 97.0 95.0 8 88 94
90.0 104.0 9 93 91 97.0 100.0 10 95 87 87.0 87.0 11 86 93 89.0 99.0
12 84 91 89.0 95.0 13 88 87 96.0 89.0 14 90 91 86.0 92.0
[0112] The standard deviation for the ELISA is in the range of
10-20%. The results above indicate that all of the formulations
tested provide good stability over 8 weeks at 40.degree. C.
[0113] Additional stability experiments were performed where
different amino acids were used in the formulations (e.g., glycine,
lysine or histidine at 15 mM or 20 m), and where the citrate buffer
was varied (e.g., 15, 20 or 25 mM). As described above, stability
of rVWF was monitored using the VWF:RCo activity assay. Even after
13 months no significant differences were observed for VWF:RCo
activity values of rVWF stability samples stored at 40.degree. C.
the significance of the measurements were tested with a t-Test. The
intermediate precision of the assay was determined by calculating
the Coefficient of Variance. In all series of the stability data
the CV was below 20% and met the validation criteria of a
CV<20%. Based on the above, it can be concluded that rVWF is
stable in all citrate buffer systems tested, independent of buffer
molarity and amino acids added. rVWF remains stable for at least 13
months even when stored at 40.degree. C. The potency determination
using the VWF:RCo activity assay shows good intermediate precision
with CV values below 20%.
[0114] Thus, in view of the data presented herein, a formulation
was proposed for rVWF including 15 mM citrate
(Na.sub.3Citrate.times.2H.sub.2O), 15 mM glycine, 10 g/L Trehalose,
20 g/L Mannitol, 0.1 g/L Tween-80, pH 7.3.
Example 4
Long Term Stability
[0115] Accelerated and Long-Term Stability Testing
[0116] Studies were conducted to evaluate the stability of the rVWF
final drug product (FDP) stored at both the recommended and
elevated storage conditions. Data from the elevated storage
conditions provides assurance that deviations in the temperature
will not impact the quality of the rVWF FDP and will be used to
extrapolate the acceptable expiry condition of the material in the
absence of real-time, real-condition stability data.
[0117] The current specification is .ltoreq.3.0% residual moisture
(as determined using the Karl Fischer Method). Lots rVWFF#4FC,
rVWFF#5FC, rVWFF#6FC and rVWFF#7FC were released with moisture
levels of 1.2%, 1.3%, 1.2%, and 1.5% respectively. Based on the
past experience with other products with similar vial and stopper
configurations, it is expected that any rVWF lots released with
approximately 1.3% residual moisture will meet the specification
limit of .ltoreq.3.0% at the end of the proposed shelf life (i.e.
24 months at the intended storage temperature of 5.degree.
C..+-.3.degree. C.).
[0118] Long-term stability studies at the recommended storage
condition (i.e. 5.degree. C..+-.3.degree. C.) and elevated
temperatures (i.e. 40.degree. C..+-.2.degree. C.) were conducted
with four rVWF FDP lots that have been manufactured. These studies
have provided sufficient data to compare the stability behavior of
the individual clinical lots.
[0119] The stability protocol, including a description of the
stability-indicating assays and stability-acceptance criteria, can
be found in Table 10 which also contains information related to the
rVWF FDP lots evaluated in the stability studies.
TABLE-US-00012 TABLE 10 Storage Conditions Completed (and Proposed)
(.degree. C.) Batch Number Test Intervals 5.degree. C. .+-.
3.degree. C. rVWF#1FC 0, 1, 2, 3, 6, 9, 12, 18, 24 months
30.degree. C. rVWF#1FC 0, 1, 3, 6 months 5.degree. C. .+-.
3.degree. C. rVWF#2FC 0, 1, 2, 3, 6, months 5.degree. C. .+-.
3.degree. C. rVWF#3FC 0, 1, 2, 3, 6, 9, 12, 18, 24 months
30.degree. C. rVWF#3FC 0, 0.5, 1, 2, 3, 6 months 40.degree. C.
rVWF#3FC 0, 0.5, 1, 2, 3 months 5.degree. C. .+-. 3.degree. C.
rVWF#4FC 0, 1, 2, 3, 6, 9, 12, 18, 24, (30) months 40.degree. C.
rVWF#4FC 0, 1, 2, 3, 6, 9 months 5.degree. C. .+-. 3.degree. C.
rVWF#5FC 0, 1, 2, 3, 6, 9, 12, 18, (24, 30) months 40.degree. C.
rVWF#5FC 0, 1, 2, 3 6, 9 months 5.degree. C. .+-. 3.degree. C.
rVWF#6FC 0, 1, 2, 3, 6, 9, 12, (18, 24, 30) months 40.degree. C.
rVWF#6FC 0, 1, 2, 3, 6, 9 months 5.degree. C. .+-. 3.degree. C.
rVWF#7FC 0, 1, 2, 3, 6, 9, 12, (18, 24, 30) months 40.degree. C.
rVWF#7FC 0, 1, 2, 3, 6, 9 months
[0120] Summary and Discussion of Overall Stability (24 Months)
[0121] The rVWF FDP stability data presented is comprised of the
following:
[0122] 1. 24 months data of long-term studies at 5.degree.
C..+-.3.degree. C. (complete testing) and 6 months intermediate
data at 30.degree. C..+-.2.degree. C. (complete testing) for lot
rVWF#1FC;
[0123] 2. 6 months data at 5.degree. C..+-.3.degree. C. (complete
testing) for lot rVWF#2FC;
[0124] 3. 24 months data of long-term studies at 2-8.degree.
(complete testing), 6 months data at 30.degree. C..+-.2.degree. C.
and 3 months data at 40.degree. C..+-.2.degree. C. (complete
testing) for lot rVWF#3FC;
[0125] 4. 24 months stability data at 5.degree. C..+-.3.degree. C.
and 9 months data at 40.degree. C..+-.2.degree. C. for lot
rVWFF#4FC;
[0126] 5. 24 months stability data at 5.degree. C..+-.3.degree. C.
and 9 months data at 40.degree. C..+-.2.degree. C. for lot
rVWFF#5FC;
[0127] 6. 12 months stability data at 5.degree. C..+-.3.degree. C.
and 9 months data at 40.degree. C..+-.2.degree. C. for lot
rVWFF#6FC; and
[0128] 7. 12 months stability data at 5.degree. C..+-.3.degree. C.
and 9 months data at 40.degree. C..+-.2.degree. C. for lot
rVWFF#7FC
[0129] The variation observed in residual moisture for lots
rVWFF#4FC, rVWFF#5FC, rVWFF#6FC and rVWFF#7FC has remained well
below the acceptance criterion .ltoreq.3%, and has not impacted the
functional activity (VWF:RCo). There was no observable change in
the stability results for qualitative analytical techniques (i.e.
appearance, SDS-PAGE analysis, etc.) for the lots manufactured to
be suitable for use in the non-clinical and clinical studies.
Similarly, there was no trend in decreasing stability for the total
protein analysis, the VWF:Ag analysis or the observed number of VWF
multimers during storage.
[0130] Variation in both the ratio of VWF:RCo activity to VWF:Ag
activity and the VWF:RCo data presented for lots rVWF#1FC, rVWF#2FC
and rVWF#3FC was likely the result of variation of the test method,
the fact that the individual VWF:RCo stability test results
consisted of data from a single determination of one stability
sample, and/or data from the non-Ph. Eur.-conforming method assay
methodology. All testing time points for the non-clinical lots
subsequent to the modification of the assay methodology to the Ph.
Eur.-conforming assay were tested using both the original and new
assay methodology.
[0131] The rVWF FDP manufactured at a large-scale exhibited similar
stability characteristics to the rVWF FDP lots manufactured at an
experimental scale. These rVWF FDP lots maintained VWF:RCo activity
for up to 24 months of storage at 5.degree. C..+-.3.degree. C.
There was no change in the VWF multimer pattern in samples of the
large-scale lots currently on stability, even after 6 months of
storage at 30.degree. C..+-.2.degree. C. or 9 months storage at
40.degree. C..+-.2.degree. C. Table 11 shows results for VWF:RCo,
VWF:Ag and VWF multimer pattern of the batches rVWF#4FC, rVWF#5FC,
rVWF#6FC and rVWF#7FC stored under stress condition at 40.degree.
C..+-.2.degree. C. The results indicate stability at elevated
temperature storage conditions for 9 months which can be
extrapolated into a shelf life of more than 3 years at ambient
temperatures or even more under refrigerated conditions.
TABLE-US-00013 TABLE 11 Results at Time (Months) Attribute
Specification 0 1 2 3 6 9 Stability Data for rVWF#4FC at 40.degree.
C. .+-. 2.degree. C. VWF:RCo 70-150 130 117 118 127 132 142
Activity [U/ml].sup.1) VWF:Ag Report result 86 87 79 81 79 86 ELISA
(U/ml) VWF multimer Report result 21 20 20 20 21 18 analysis
Stability Data for rVWF#5FC at 40.degree. C. .+-. 2.degree. C.
VWF:RCo 70-150 107 119 120 116 132 134 Activity [U/ml].sup.1)
VWF:Ag Report result 94 86 84 91 90 79 ELISA (U/ml) VWF multimer
Report result 20 20 18 19 20 19 analysis Stability Data for
rVWF#6FC at 40.degree. C. .+-. 2.degree. C. VWF:RCo 70-150 118 111
126 129 130 119 Activity [U/ml].sup.1) VWF:Ag Report result 85 95
86.3 73.5 80.8 70.3 ELISA (U/ml) VWF multimer Report result 20 19
20 20 20 n.t. analysis Stability Data for rVWF#7FC at 40.degree. C.
.+-. 2.degree. C. VWF:RCo 70-150 111 115 122 105 99 112 Activity
[U/ml].sup.1) VWF:Ag Report result 87.3 85.3 77.5 68.8 75 73.8
ELISA (U/ml) VWF multimer Report result 21 20 20 19 19 19
analysis
[0132] An analysis of covariance (ANCOVA analysis) demonstrated
that the difference in slopes of the regression lines (lots
rVWFF#4FC, rVWFF#5FC, rVWFF#6FC and rVWFF#7FC stored at 5.degree.
C..+-.3.degree. C.) is not significant (p=0.906), allowing the
VWF:RCo activity data to be pooled as described in ICH Q1A (R2).
The difference in elevation of the trend lines of the individual
lots is also not significant. Extrapolation of the pooled worse
case slope, as shown in FIG. 1, shows that the confidence intervals
are well within the acceptance criteria for a minimum of 24 months.
The lower confidence interval for the mean curve decreases to 80%
of initial activity at 51 months (80% is also the maximum
difference between estimated potency and stated potency for Human
von Willebrand Factor in Ph. Eur). The pooled worse case slope
shows a decrease of 0.0344 U VWF:RCo per month. This comparison
shows that stability characteristics of the rVWF FDP, specifically
the VWF:RCo activity, did not change as a result of the changes in
the production process. The above extrapolation supports the
extension of the provisional shelf life of rVWF FDP to 24 months
when stored at the recommended storage temperature.
[0133] The transfer of moisture from the stopper to the lyophilized
product is dependent on the stopper material and is influenced by
the residual moisture of the stopper after sterilization, the
humidity at which the sample is stored and the intrinsic moisture
transfer rate of the stopper. The residual moisture in the lots
rVWFF#4FC, rVWFF#5FC, rVWFF#6FC and rVWFF#7FC stored at 5.degree.
C..+-.3.degree. C. was comparable (the difference in comparison of
slopes being not significant, with p=0.734), as shown in FIG. 2.
Lots stored at the elevated temperature condition 40.degree.
C..+-.2.degree. C. also showed a comparable increase in residual
moisture over 9 months (FIG. 3). ANCOVA analysis demonstrates here
that the difference in slope of the regression lines is comparable
(p=0.546). FIG. 3 shows the extrapolation of the worse case pooled
slope up to 24 months.
[0134] These are sufficient data to support the use of lots
rVWFF#6FC and rVWFF#7FC for the duration of the described expiry
period of 24 months when stored at 5.degree. C..+-.3.degree. C.
[0135] Proposed Storage Conditions and Shelf Life
[0136] The recommended storage condition for the rVWF FDP is
5.degree. C..+-.3.degree. C. A provisional shelf life of 24 months
for the rVWF FDP is therefore proposed when stored at the
recommended storage condition. The shelf life for the rVWF FDP lots
likely can be further extended based on additional data to be
generated for longer storage periods.
Sequence CWU 1
1
318833DNAHomo sapiens 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 883322783PRTHomo sapiens
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
Cys 20 25 30Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly Ser
Met Tyr 35 40 45Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly
Cys Gln Lys 50 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 Asn 85 90 95Gly Thr Val Thr Gln Gly Asp Gln Arg
Val Ser Met Pro Tyr Ala Ser 100 105 110Lys Leu Glu Thr Glu Ala Gly
Tyr Tyr Lys Leu Ser Gly Glu Ala Tyr 115 120 125Gly Phe Val Ala Arg
Ile Asp Gly Ser Gly Asn Phe Gln Val Leu Leu 130 135 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 Ser
165 170 175Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala Leu Ser Ser Gly
Glu Gln 180 185 190Trp Cys Glu Arg Pro Ser Ser Ser Cys Asn Ile Ser
Ser Gly Glu Met 195 200 205Gln Lys Gly Leu Trp Glu Gln Cys Gln Leu
Leu Lys Ser Thr Ser Val 210 215 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 Leu 245 250 255Leu Glu Tyr
Ala Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly 260 265 270Trp
Thr Asp His Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu 275 280
285Tyr Arg Gln Cys Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His
290 295 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 Cys 325 330 335Pro Cys Val His Ser Gly Lys Arg Tyr
Pro Pro Gly Thr Ser Leu Ser 340 345 350Arg Asp Cys Asn Thr Cys Ile
Cys Arg Asn Ser Gln Trp Ile Cys Ser 355 360 365Asn Glu Glu Cys Pro
Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe 370 375 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 Glu
405 410 415Thr Val Gln Cys Ala Asp Asp Arg Asp Ala Val Cys Thr Arg
Ser Val 420 425 430Thr Val Arg Leu Pro Gly Leu His Asn Ser Leu Val
Lys Leu Lys His 435 440 445Gly Ala Gly Val Ala Met Asp Gly Gln Asp
Val Gln Leu Pro Leu Leu 450 455 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 Leu 485 490 495Leu Val Lys
Leu Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys 500 505 510Gly
Asn Tyr Asn Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly 515 520
525Leu Ala Glu Pro Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His
530 535 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 Thr 565 570 575Ser Pro Thr Phe Glu Ala Cys His Arg
Ala Val Ser Pro Leu Pro Tyr 580 585 590Leu Arg Asn Cys Arg Tyr Asp
Val Cys Ser Cys Ser Asp Gly Arg Glu 595 600 605Cys Leu Cys Gly Ser
Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg 610 615 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 Ser
645 650 655Leu Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu
Gly Cys 660 665 670Phe Cys Pro Pro Met Asp Glu Arg Gly Asp Cys Val
Pro Lys Ala Gln 675 680 685Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe
Gln Pro Glu Asp Ile Phe 690 695 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 Ser 725 730 735Ser Pro Leu
Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg Pro Pro 740 745 750Met
Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu Gly Leu 755 760
765Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met Ser Met
770 775 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 Ala 805 810 815Pro Gly Glu Thr Val Lys Ile Gly Cys
Asn Thr Cys Val Cys Arg Asp 820 825 830Arg Lys Trp Asn Cys Thr Asp
His Val Cys Asp Ala Thr Cys Ser Thr 835 840 845Ile Gly Met Ala His
Tyr Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe 850 855 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 Pro
885 890 895Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu Val Glu Gly
Gly Glu 900 905 910Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys Arg
Pro Met Lys Asp 915 920 925Glu Thr His Phe Glu Val Val Glu Ser Gly
Arg Tyr Ile Ile Leu Leu 930 935 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 Gly 965 970 975Asn Phe Asp
Gly Ile Gln Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln 980 985 990Val
Glu Glu Asp Pro Val Asp Phe Gly Asn Ser Trp Lys Val Ser Ser 995
1000
1005Gln Cys Ala Asp Thr Arg Lys Val Pro Leu Asp Ser Ser Pro Ala
1010 1015 1020Thr Cys His Asn Asn Ile Met Lys Gln Thr Met Val Asp
Ser Ser 1025 1030 1035Cys Arg Ile Leu Thr Ser Asp Val Phe Gln Asp
Cys Asn Lys Leu 1040 1045 1050Val Asp Pro Glu Pro Tyr Leu Asp Val
Cys Ile Tyr Asp Thr Cys 1055 1060 1065Ser Cys Glu Ser Ile Gly Asp
Cys Ala Cys Phe Cys Asp Thr Ile 1070 1075 1080Ala Ala Tyr Ala His
Val Cys Ala Gln His Gly Lys Val Val Thr 1085 1090 1095Trp Arg Thr
Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg Asn 1100 1105 1110Leu
Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn Ser Cys 1115 1120
1125Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu Ala
1130 1135 1140Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys
Pro Pro 1145 1150 1155Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys
Val Asp Pro Glu 1160 1165 1170Asp Cys Pro Val Cys Glu Val Ala Gly
Arg Arg Phe Ala Ser Gly 1175 1180 1185Lys Lys Val Thr Leu Asn Pro
Ser Asp Pro Glu His Cys Gln Ile 1190 1195 1200Cys His Cys Asp Val
Val Asn Leu Thr Cys Glu Ala Cys Gln Glu 1205 1210 1215Pro Gly Gly
Leu Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro 1220 1225 1230Thr
Thr Leu Tyr Val Glu Asp Ile Ser Glu Pro Pro Leu His Asp 1235 1240
1245Phe Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu Asp Gly
1250 1255 1260Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu Lys
Ala Phe 1265 1270 1275Val Val Asp Met Met Glu Arg Leu Arg Ile Ser
Gln Lys Trp Val 1280 1285 1290Arg Val Ala Val Val Glu Tyr His Asp
Gly Ser His Ala Tyr Ile 1295 1300 1305Gly Leu Lys Asp Arg Lys Arg
Pro Ser Glu Leu Arg Arg Ile Ala 1310 1315 1320Ser Gln Val Lys Tyr
Ala Gly Ser Gln Val Ala Ser Thr Ser Glu 1325 1330 1335Val Leu Lys
Tyr Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp Arg 1340 1345 1350Pro
Glu Ala Ser Arg Ile Thr Leu Leu Leu Met Ala Ser Gln Glu 1355 1360
1365Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly Leu
1370 1375 1380Lys Lys Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly
Pro His 1385 1390 1395Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys
Gln Ala Pro Glu 1400 1405 1410Asn Lys Ala Phe Val Leu Ser Ser Val
Asp Glu Leu Glu Gln Gln 1415 1420 1425Arg Asp Glu Ile Val Ser Tyr
Leu Cys Asp Leu Ala Pro Glu Ala 1430 1435 1440Pro Pro Pro Thr Leu
Pro Pro Asp Met Ala Gln Val Thr Val Gly 1445 1450 1455Pro Gly Leu
Leu Gly Val Ser Thr Leu Gly Pro Lys Arg Asn Ser 1460 1465 1470Met
Val Leu Asp Val Ala Phe Val Leu Glu Gly Ser Asp Lys Ile 1475 1480
1485Gly Glu Ala Asp Phe Asn Arg Ser Lys Glu Phe Met Glu Glu Val
1490 1495 1500Ile Gln Arg Met Asp Val Gly Gln Asp Ser Ile His Val
Thr Val 1505 1510 1515Leu Gln Tyr Ser Tyr Met Val Thr Val Glu Tyr
Pro Phe Ser Glu 1520 1525 1530Ala Gln Ser Lys Gly Asp Ile Leu Gln
Arg Val Arg Glu Ile Arg 1535 1540 1545Tyr Gln Gly Gly Asn Arg Thr
Asn Thr Gly Leu Ala Leu Arg Tyr 1550 1555 1560Leu Ser Asp His Ser
Phe Leu Val Ser Gln Gly Asp Arg Glu Gln 1565 1570 1575Ala Pro Asn
Leu Val Tyr Met Val Thr Gly Asn Pro Ala Ser Asp 1580 1585 1590Glu
Ile Lys Arg Leu Pro Gly Asp Ile Gln Val Val Pro Ile Gly 1595 1600
1605Val Gly Pro Asn Ala Asn Val Gln Glu Leu Glu Arg Ile Gly Trp
1610 1615 1620Pro Asn Ala Pro Ile Leu Ile Gln Asp Phe Glu Thr Leu
Pro Arg 1625 1630 1635Glu Ala Pro Asp Leu Val Leu Gln Arg Cys Cys
Ser Gly Glu Gly 1640 1645 1650Leu Gln Ile Pro Thr Leu Ser Pro Ala
Pro Asp Cys Ser Gln Pro 1655 1660 1665Leu Asp Val Ile Leu Leu Leu
Asp Gly Ser Ser Ser Phe Pro Ala 1670 1675 1680Ser Tyr Phe Asp Glu
Met Lys Ser Phe Ala Lys Ala Phe Ile Ser 1685 1690 1695Lys Ala Asn
Ile Gly Pro Arg Leu Thr Gln Val Ser Val Leu Gln 1700 1705 1710Tyr
Gly Ser Ile Thr Thr Ile Asp Val Pro Trp Asn Val Val Pro 1715 1720
1725Glu Lys Ala His Leu Leu Ser Leu Val Asp Val Met Gln Arg Glu
1730 1735 1740Gly Gly Pro Ser Gln Ile Gly Asp Ala Leu Gly Phe Ala
Val Arg 1745 1750 1755Tyr Leu Thr Ser Glu Met His Gly Ala Arg Pro
Gly Ala Ser Lys 1760 1765 1770Ala Val Val Ile Leu Val Thr Asp Val
Ser Val Asp Ser Val Asp 1775 1780 1785Ala Ala Ala Asp Ala Ala Arg
Ser Asn Arg Val Thr Val Phe Pro 1790 1795 1800Ile Gly Ile Gly Asp
Arg Tyr Asp Ala Ala Gln Leu Arg Ile Leu 1805 1810 1815Ala Gly Pro
Ala Gly Asp Ser Asn Val Val Lys Leu Gln Arg Ile 1820 1825 1830Glu
Asp Leu Pro Thr Met Val Thr Leu Gly Asn Ser Phe Leu His 1835 1840
1845Lys Leu Cys Ser Gly Phe Val Arg Ile Cys Met Asp Glu Asp Gly
1850 1855 1860Asn Glu Lys Arg Pro Gly Asp Val Trp Thr Leu Pro Asp
Gln Cys 1865 1870 1875His Thr Val Thr Cys Gln Pro Asp Gly Gln Thr
Leu Leu Lys Ser 1880 1885 1890His Arg Val Asn Cys Asp Arg Gly Leu
Arg Pro Ser Cys Pro Asn 1895 1900 1905Ser Gln Ser Pro Val Lys Val
Glu Glu Thr Cys Gly Cys Arg Trp 1910 1915 1920Thr Cys Pro Cys Val
Cys Thr Gly Ser Ser Thr Arg His Ile Val 1925 1930 1935Thr Phe Asp
Gly Gln Asn Phe Lys Leu Thr Gly Ser Cys Ser Tyr 1940 1945 1950Val
Leu Phe Gln Asn Lys Glu Gln Asp Leu Glu Val Ile Leu His 1955 1960
1965Asn Gly Ala Cys Ser Pro Gly Ala Arg Gln Gly Cys Met Lys Ser
1970 1975 1980Ile Glu Val Lys His Ser Ala Leu Ser Val Glu Leu His
Ser Asp 1985 1990 1995Met Glu Val Thr Val Asn Gly Arg Leu Val Ser
Val Pro Tyr Val 2000 2005 2010Gly Gly Asn Met Glu Val Asn Val Tyr
Gly Ala Ile Met His Glu 2015 2020 2025Val Arg Phe Asn His Leu Gly
His Ile Phe Thr Phe Thr Pro Gln 2030 2035 2040Asn Asn Glu Phe Gln
Leu Gln Leu Ser Pro Lys Thr Phe Ala Ser 2045 2050 2055Lys Thr Tyr
Gly Leu Cys Gly Ile Cys Asp Glu Asn Gly Ala Asn 2060 2065 2070Asp
Phe Met Leu Arg Asp Gly Thr Val Thr Thr Asp Trp Lys Thr 2075 2080
2085Leu Val Gln Glu Trp Thr Val Gln Arg Pro Gly Gln Thr Cys Gln
2090 2095 2100Pro Glu Gln Cys Leu Val Pro Asp Ser Ser His Cys Gln
Val Leu 2105 2110 2115Leu Leu Pro Leu Phe Ala Glu Cys His Lys Val
Leu Ala Pro Ala 2120 2125 2130Thr Phe Tyr Ala Ile Cys Gln Gln Asp
Ser Cys His Gln Glu Gln 2135 2140 2145Val Cys Glu Val Ile Ala Ser
Tyr Ala His Leu Cys Arg Thr Asn 2150 2155 2160Gly Val Cys Val Asp
Trp Arg Thr Pro Asp Phe Cys Ala Met Ser 2165 2170 2175Cys Pro Pro
Ser Leu Val Tyr Asn His Cys Glu His Gly Cys Pro 2180 2185 2190Arg
His Cys Asp Gly Asn Val Ser Ser Cys Gly Asp His Pro Ser 2195 2200
2205Glu Gly Cys Phe Cys Pro Pro Asp Lys Val Met Leu Glu Gly Ser
2210 2215 2220Cys Val Pro Glu Glu Ala Cys Thr Gln Cys Ile Gly Glu
Asp Gly 2225 2230 2235Val Gln His Gln Phe Leu Glu Ala Trp Val Pro
Asp His Gln Pro 2240 2245 2250Cys Gln Ile Cys Thr Cys Leu Ser Gly
Arg Lys Val Asn Cys Thr 2255 2260 2265Thr Gln Pro Cys Pro Thr Ala
Lys Ala Pro Thr Cys Gly Leu Cys 2270 2275 2280Glu Val Ala Arg Leu
Arg Gln Asn Ala Asp Gln Cys Cys Pro Glu 2285 2290 2295Tyr Glu Cys
Val Cys Asp Pro Val Ser Cys Asp Leu Pro Pro Val 2300 2305 2310Pro
His Cys Glu Arg Gly Leu Gln Pro Thr Leu Thr Asn Pro Gly 2315 2320
2325Glu Cys Arg Pro Asn Phe Thr Cys Ala Cys Arg Lys Glu Glu Cys
2330 2335 2340Lys Arg Val Ser Pro Pro Ser Cys Pro Pro His Arg Leu
Pro Thr 2345 2350 2355Leu Arg Lys Thr Gln Cys Cys Asp Glu Tyr Glu
Cys Ala Cys Asn 2360 2365 2370Cys Val Asn Ser Thr Val Ser Cys Pro
Leu Gly Tyr Leu Ala Ser 2375 2380 2385Thr Ala Thr Asn Asp Cys Gly
Cys Thr Thr Thr Thr Cys Leu Pro 2390 2395 2400Asp Lys Val Cys Val
His Arg Ser Thr Ile Tyr Pro Val Gly Gln 2405 2410 2415Phe Trp Glu
Glu Gly Cys Asp Val Cys Thr Cys Thr Asp Met Glu 2420 2425 2430Asp
Ala Val Met Gly Leu Arg Val Ala Gln Cys Ser Gln Lys Pro 2435 2440
2445Cys Glu Asp Ser Cys Arg Ser Gly Phe Thr Tyr Val Leu His Glu
2450 2455 2460Gly Glu Cys Cys Gly Arg Cys Leu Pro Ser Ala Cys Glu
Val Val 2465 2470 2475Thr Gly Ser Pro Arg Gly Asp Ser Gln Ser Ser
Trp Lys Ser Val 2480 2485 2490Gly Ser Gln Trp Glu Asn Pro Cys Leu
Ile Asn Glu Cys Val Arg 2495 2500 2505Val Lys Glu Glu Val Phe Ile
Gln Gln Arg Asn Val Ser Cys Pro 2510 2515 2520Gln Leu Glu Val Pro
Val Cys Pro Ser Gly Phe Gln Leu Ser Cys 2525 2530 2535Lys Thr Ser
Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu 2540 2545 2550Ala
Cys Met Leu Asn Gly Thr Val Ile Gly Pro Gly Lys Thr Val 2555 2560
2565Met Ile Asp Val Cys Thr Thr Cys Arg Cys Met Val Gln Val Gly
2570 2575 2580Val Ile Ser Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr
Cys Asn 2585 2590 2595Pro Cys Pro Leu Gly Tyr Lys Glu Glu Asn Asn
Thr Gly Glu Cys 2600 2605 2610Cys Gly Arg Cys Leu Pro Thr Ala Cys
Thr Ile Gln Leu Arg Gly 2615 2620 2625Gly Gln Ile Met Thr Leu Lys
Arg Asp Glu Thr Leu Gln Asp Gly 2630 2635 2640Cys Asp Thr His Phe
Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe 2645 2650 2655Trp Glu Lys
Arg Val Thr Gly Cys Pro Pro Phe Asp Glu His Lys 2660 2665 2670Cys
Leu Ala Glu Gly Gly Lys Ile Met Lys Ile Pro Gly Thr Cys 2675 2680
2685Cys Asp Thr Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg
2690 2695 2700Leu Gln Tyr Val Lys Val Gly Ser Cys Lys Ser Glu Val
Glu Val 2705 2710 2715Asp Ile His Tyr Cys Gln Gly Lys Cys Ala Ser
Lys Ala Met Tyr 2720 2725 2730Ser Ile Asp Ile Asn Asp Val Gln Asp
Gln Cys Ser Cys Cys Ser 2735 2740 2745Pro Thr Arg Thr Glu Pro Met
Gln His Cys Thr Asn Gly Ser Val 2750 2755 2760Val Tyr His Glu Val
Leu Asn Ala Met Glu Cys Lys Cys Ser Pro 2765 2770 2775Arg Lys Cys
Ser Lys 278032050PRTHomo sapiens 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 Tyr 20 25 30Asp Leu Glu Cys Met Ser
Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45Pro Gly Met Val Arg
His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 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 Thr 85 90 95Asp
His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr 100 105
110Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr
115 120 125Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe
Arg Ile 130 135 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 Gly 165 170 175Glu Val Asn Val Lys Arg Pro
Met Lys Asp Glu Thr His Phe Glu Val 180 185 190Val Glu Ser Gly Arg
Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205Val Val Trp
Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215 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
Val 245 250 255Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala
Asp Thr Arg 260 265 270Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys
His Asn Asn Ile Met 275 280 285Lys Gln Thr Met Val Asp Ser Ser Cys
Arg Ile Leu Thr Ser Asp Val 290 295 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 Cys 325 330 335Phe Cys
Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly 340 345
350Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu
355 360 365Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg
Tyr Asn 370 375 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 Pro 405 410 415Gly Lys Ile Leu Asp Glu Leu
Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430Cys Pro Val Cys Glu
Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440 445Val Thr Leu
Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys 450 455 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
Val 485 490 495Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr Cys
Ser Arg Leu 500 505 510Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser
Arg Leu Ser Glu Ala 515 520 525Glu Phe Glu Val Leu Lys Ala Phe Val
Val Asp Met Met Glu Arg Leu 530 535 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 Glu 565 570 575Leu Arg
Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala 580 585
590Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys
595 600 605Ile Asp Arg Pro Glu Ala Ser Arg Ile Thr Leu Leu Leu Met
Ala Ser 610 615 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 His 645 650 655Ala Asn Leu Lys Gln Ile Arg
Leu Ile Glu Lys Gln Ala Pro Glu Asn 660
665 670Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg
Asp 675 680 685Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala
Pro Pro Pro 690 695 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 Val 725 730 735Ala Phe Val Leu Glu Gly
Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn 740 745 750Arg Ser Lys Glu
Phe Met Glu Glu Val Ile Gln Arg Met Asp Val Gly 755 760 765Gln Asp
Ser Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr 770 775
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 Gly 805 810 815Leu Ala Leu Arg Tyr Leu Ser Asp His Ser
Phe Leu Val Ser Gln Gly 820 825 830Asp Arg Glu Gln Ala Pro Asn Leu
Val Tyr Met Val Thr Gly Asn Pro 835 840 845Ala Ser Asp Glu Ile Lys
Arg Leu Pro Gly Asp Ile Gln Val Val Pro 850 855 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 Arg 885 890
895Glu Ala Pro Asp Leu Val Leu Gln Arg Cys Cys Ser Gly Glu Gly Leu
900 905 910Gln Ile Pro Thr Leu Ser Pro Ala Pro Asp Cys Ser Gln Pro
Leu Asp 915 920 925Val Ile Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro
Ala Ser Tyr Phe 930 935 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 Thr 965 970 975Thr Ile Asp Val Pro
Trp Asn Val Val Pro Glu Lys Ala His Leu Leu 980 985 990Ser Leu Val
Asp Val Met Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly 995 1000
1005Asp Ala Leu Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His
1010 1015 1020Gly Ala Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu
Val Thr 1025 1030 1035Asp Val Ser Val Asp Ser Val Asp Ala Ala Ala
Asp Ala Ala Arg 1040 1045 1050Ser Asn Arg Val Thr Val Phe Pro Ile
Gly Ile Gly Asp Arg Tyr 1055 1060 1065Asp Ala Ala Gln Leu Arg Ile
Leu Ala Gly Pro Ala Gly Asp Ser 1070 1075 1080Asn Val Val Lys Leu
Gln Arg Ile Glu Asp Leu Pro Thr Met Val 1085 1090 1095Thr Leu Gly
Asn Ser Phe Leu His Lys Leu Cys Ser Gly Phe Val 1100 1105 1110Arg
Ile Cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp 1115 1120
1125Val Trp Thr Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro
1130 1135 1140Asp Gly Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys
Asp Arg 1145 1150 1155Gly Leu Arg Pro Ser Cys Pro Asn Ser Gln Ser
Pro Val Lys Val 1160 1165 1170Glu Glu Thr Cys Gly Cys Arg Trp Thr
Cys Pro Cys Val Cys Thr 1175 1180 1185Gly Ser Ser Thr Arg His Ile
Val Thr Phe Asp Gly Gln Asn Phe 1190 1195 1200Lys Leu Thr Gly Ser
Cys Ser Tyr Val Leu Phe Gln Asn Lys Glu 1205 1210 1215Gln Asp Leu
Glu Val Ile Leu His Asn Gly Ala Cys Ser Pro Gly 1220 1225 1230Ala
Arg Gln Gly Cys Met Lys Ser Ile Glu Val Lys His Ser Ala 1235 1240
1245Leu Ser Val Glu Leu His Ser Asp Met Glu Val Thr Val Asn Gly
1250 1255 1260Arg Leu Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu
Val Asn 1265 1270 1275Val Tyr Gly Ala Ile Met His Glu Val Arg Phe
Asn His Leu Gly 1280 1285 1290His Ile Phe Thr Phe Thr Pro Gln Asn
Asn Glu Phe Gln Leu Gln 1295 1300 1305Leu Ser Pro Lys Thr Phe Ala
Ser Lys Thr Tyr Gly Leu Cys Gly 1310 1315 1320Ile Cys Asp Glu Asn
Gly Ala Asn Asp Phe Met Leu Arg Asp Gly 1325 1330 1335Thr Val Thr
Thr Asp Trp Lys Thr Leu Val Gln Glu Trp Thr Val 1340 1345 1350Gln
Arg Pro Gly Gln Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys 1355 1360
1365Leu Val Pro Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu
1370 1375 1380Phe Ala Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe
Tyr Ala 1385 1390 1395Ile Cys Gln Gln Asp Ser Cys His Gln Glu Gln
Val Cys Glu Val 1400 1405 1410Ile Ala Ser Tyr Ala His Leu Cys Arg
Thr Asn Gly Val Cys Val 1415 1420 1425Asp Trp Arg Thr Pro Asp Phe
Cys Ala Met Ser Cys Pro Pro Ser 1430 1435 1440Leu Val Tyr Asn His
Cys Glu His Gly Cys Pro Arg His Cys Asp 1445 1450 1455Gly Asn Val
Ser Ser Cys Gly Asp His Pro Ser Glu Gly Cys Phe 1460 1465 1470Cys
Pro Pro Asp Lys Val Met Leu Glu Gly Ser Cys Val Pro Glu 1475 1480
1485Glu Ala Cys Thr Gln Cys Ile Gly Glu Asp Gly Val Gln His Gln
1490 1495 1500Phe Leu Glu Ala Trp Val Pro Asp His Gln Pro Cys Gln
Ile Cys 1505 1510 1515Thr Cys Leu Ser Gly Arg Lys Val Asn Cys Thr
Thr Gln Pro Cys 1520 1525 1530Pro Thr Ala Lys Ala Pro Thr Cys Gly
Leu Cys Glu Val Ala Arg 1535 1540 1545Leu Arg Gln Asn Ala Asp Gln
Cys Cys Pro Glu Tyr Glu Cys Val 1550 1555 1560Cys Asp Pro Val Ser
Cys Asp Leu Pro Pro Val Pro His Cys Glu 1565 1570 1575Arg Gly Leu
Gln Pro Thr Leu Thr Asn Pro Gly Glu Cys Arg Pro 1580 1585 1590Asn
Phe Thr Cys Ala Cys Arg Lys Glu Glu Cys Lys Arg Val Ser 1595 1600
1605Pro Pro Ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr
1610 1615 1620Gln Cys Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys Val
Asn Ser 1625 1630 1635Thr Val Ser Cys Pro Leu Gly Tyr Leu Ala Ser
Thr Ala Thr Asn 1640 1645 1650Asp Cys Gly Cys Thr Thr Thr Thr Cys
Leu Pro Asp Lys Val Cys 1655 1660 1665Val His Arg Ser Thr Ile Tyr
Pro Val Gly Gln Phe Trp Glu Glu 1670 1675 1680Gly Cys Asp Val Cys
Thr Cys Thr Asp Met Glu Asp Ala Val Met 1685 1690 1695Gly Leu Arg
Val Ala Gln Cys Ser Gln Lys Pro Cys Glu Asp Ser 1700 1705 1710Cys
Arg Ser Gly Phe Thr Tyr Val Leu His Glu Gly Glu Cys Cys 1715 1720
1725Gly Arg Cys Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro
1730 1735 1740Arg Gly Asp Ser Gln Ser Ser Trp Lys Ser Val Gly Ser
Gln Trp 1745 1750 1755Ala Ser Pro Glu Asn Pro Cys Leu Ile Asn Glu
Cys Val Arg Val 1760 1765 1770Lys Glu Glu Val Phe Ile Gln Gln Arg
Asn Val Ser Cys Pro Gln 1775 1780 1785Leu Glu Val Pro Val Cys Pro
Ser Gly Phe Gln Leu Ser Cys Lys 1790 1795 1800Thr Ser Ala Cys Cys
Pro Ser Cys Arg Cys Glu Arg Met Glu Ala 1805 1810 1815Cys Met Leu
Asn Gly Thr Val Ile Gly Pro Gly Lys Thr Val Met 1820 1825 1830Ile
Asp Val Cys Thr Thr Cys Arg Cys Met Val Gln Val Gly Val 1835 1840
1845Ile Ser Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro
1850 1855 1860Cys Pro Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu
Cys Cys 1865 1870 1875Gly Arg Cys Leu Pro Thr Ala Cys Thr Ile Gln
Leu Arg Gly Gly 1880 1885 1890Gln Ile Met Thr Leu Lys Arg Asp Glu
Thr Leu Gln Asp Gly Cys 1895 1900 1905Asp Thr His Phe Cys Lys Val
Asn Glu Arg Gly Glu Tyr Phe Trp 1910 1915 1920Glu Lys Arg Val Thr
Gly Cys Pro Pro Phe Asp Glu His Lys Cys 1925 1930 1935Leu Ala Glu
Gly Gly Lys Ile Met Lys Ile Pro Gly Thr Cys Cys 1940 1945 1950Asp
Thr Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg Leu 1955 1960
1965Gln Tyr Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp
1970 1975 1980Ile His Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met
Tyr Ser 1985 1990 1995Ile Asp Ile Asn Asp Val Gln Asp Gln Cys Ser
Cys Cys Ser Pro 2000 2005 2010Thr Arg Thr Glu Pro Met Gln Val Ala
Leu His Cys Thr Asn Gly 2015 2020 2025Ser Val Val Tyr His Glu Val
Leu Asn Ala Met Glu Cys Lys Cys 2030 2035 2040Ser Pro Arg Lys Cys
Ser Lys 2045 2050
* * * * *