U.S. patent application number 11/815259 was filed with the patent office on 2009-09-24 for anti-madcam antibody compositions.
Invention is credited to Corey M. Allan, Tapan Kanti Das, Scott Steven Ganser, Sandeep Nema, Satish K. Singh, David Li Zeng.
Application Number | 20090238820 11/815259 |
Document ID | / |
Family ID | 36646098 |
Filed Date | 2009-09-24 |
United States Patent
Application |
20090238820 |
Kind Code |
A1 |
Allan; Corey M. ; et
al. |
September 24, 2009 |
ANTI-MAdCAM ANTIBODY COMPOSITIONS
Abstract
The present invention relates to anti-MAdCAM antibody
compositions comprising a chelating agent, and methods of treating
inflammatory disease in a subject.
Inventors: |
Allan; Corey M.;
(Manchester, MO) ; Das; Tapan Kanti; (Ballwin,
MO) ; Ganser; Scott Steven; (O'Fallon, MO) ;
Nema; Sandeep; (St. Louis, MO) ; Singh; Satish
K.; (Wildwood, MO) ; Zeng; David Li;
(Broomfield, CO) |
Correspondence
Address: |
PHARMACIA CORPORATION;GLOBAL PATENT DEPARTMENT
POST OFFICE BOX 1027
ST. LOUIS
MO
63006
US
|
Family ID: |
36646098 |
Appl. No.: |
11/815259 |
Filed: |
March 2, 2006 |
PCT Filed: |
March 2, 2006 |
PCT NO: |
PCT/US06/07554 |
371 Date: |
May 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60659766 |
Mar 8, 2005 |
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60728165 |
Oct 19, 2005 |
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60752712 |
Dec 20, 2005 |
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60762456 |
Jan 26, 2006 |
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Current U.S.
Class: |
424/133.1 ;
424/152.1; 424/172.1 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 5/50 20180101; A61K 9/19 20130101; A61P 9/10 20180101; A61P
9/14 20180101; A61P 11/06 20180101; A61P 17/02 20180101; A61P 21/04
20180101; A61P 1/16 20180101; A61P 3/02 20180101; A61P 19/10
20180101; A61P 35/02 20180101; A61K 2039/505 20130101; A61P 3/10
20180101; A61P 11/00 20180101; A61P 1/04 20180101; A61K 39/39591
20130101; A61K 39/3955 20130101; A61P 1/02 20180101; A61P 37/08
20180101; A61K 47/183 20130101; A61P 7/06 20180101; A61P 1/14
20180101; A61P 27/02 20180101; A61P 19/02 20180101; A61P 37/00
20180101; A61P 1/18 20180101; A61K 47/26 20130101; A61P 21/02
20180101; A61P 19/08 20180101; A61P 31/12 20180101; A61P 35/00
20180101; A61P 43/00 20180101; A61P 7/02 20180101; A61K 9/08
20130101; A61P 11/04 20180101; A61P 15/00 20180101; A61P 31/04
20180101; C07K 2317/21 20130101; A61P 35/04 20180101; C07K 16/243
20130101; A61P 17/00 20180101; A61P 31/18 20180101; A61P 37/02
20180101; A61P 5/14 20180101; A61P 25/00 20180101; A61P 17/06
20180101; A61P 21/00 20180101; A61P 37/06 20180101; A61P 41/00
20180101; A61P 13/10 20180101; A61P 31/00 20180101; A61K 39/3955
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/133.1 ;
424/172.1; 424/152.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 29/00 20060101 A61P029/00 |
Claims
1. A composition comprising: at least one chelating agent; and at
least one antibody comprising: an amino acid sequence that is at
least 90% identical to a heavy chain amino acid sequence shown in
SEQ ID NO: 2; and an amino acid sequence that is at least 90%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4; wherein the antibody binds to human MAdCAM.
2. The composition according to claim 1, wherein the composition is
a liquid composition and the antibody is a human IgG2 antibody and
the antibody does not comprise a signal sequence.
3. The composition according to claim 1, wherein the antibody
comprises a heavy chain amino acid sequence with at least 99%
sequence identity to SEQ ID NO: 2 and a light chain amino acid
sequence with at least 99% sequence identity to SEQ ID NO: 4.
4. The composition according to claim 1, wherein the antibody
comprises a heavy chain amino acid sequence comprising SEQ ID NO: 2
and a light chain amino acid sequence comprising SEQ ID NO: 4.
5. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA.
6. The composition according to claim 1, wherein the composition
further comprises a buffer.
7. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA, and further
comprises histidine.
8. The composition according to claim 1, wherein the composition
further comprises a buffer and a surfactant.
9. The composition according to claim 1, wherein the composition
further comprises a buffer, a surfactant, and a tonicity agent.
10. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA, and further
comprises a buffer, a surfactant, and a tonicity agent.
11. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA, and further
comprises histidine, a surfactant, and a tonicity agent.
12. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA, and further
comprises histidine, polysorbate 80, and a tonicity agent.
13. The composition according to claim 1, wherein the composition
comprises at least one chelating agent that is EDTA, and further
comprises histidine, polysorbate 80, and trehalose.
14. The composition according to claim 1, wherein the composition
comprises: from about 1 mg/ml to about 200 mg/ml of antibody; from
about 0.01 millimolar to about 5.0 millimolar of a chelating agent;
and from about 1 mM to about 100 mM of histidine.
15. The composition according to claim 1, wherein the composition
comprises: from about 1 mg/ml to about 200 mg/ml of antibody; from
about 0.01 millimolar to about 5.0 millimolar of EDTA; and from
about 1 mM to about 100 mM of histidine.
16. The composition according to claim 1, wherein the composition
comprises: from about 1 mg/ml to about 200 mg/ml of antibody; from
about 0.01 millimolar to about 5.0 millimolar of a chelating agent;
from about 1 mM to about 100 mM of a buffer; from about 0.005
millimolar to about 10 millimolar of a surfactant; and from about
100 millimolar to about 400 millimolar of a tonicity agent.
17. The composition according to claim 1, wherein the composition
comprises: from about 1 mg/ml to about 200 mg/ml of antibody; from
about 0.01 millimolar to about 5.0 millimolar of EDTA; from about 1
mM to about 100 mM of histidine; from about 0.005 millimolar to
about 10 millimolar of polysorbate 80; and from about 100
millimolar to about 400 millimolar of a tonicity agent.
18. The composition according to claim 1, wherein the composition
comprises: from about 1 mg/ml to about 200 mg/ml of antibody; from
about 0.01 millimolar to about 5.0 millimolar of EDTA; from about 1
mM to about 100 mM of histidine; from about 0.005 millimolar to
about 10 millimolar of polysorbate 80; and from about 100
millimolar to about 400 millimolar of trehalose.
19. The composition according to claim 1, wherein the composition
comprises: from about 0.1 mg/ml to about 100 mg/ml of antibody;
from about 0.001 mg/ml to about 1.0 mg/ml of EDTA; from about 1 mM
to about 50 mM of histidine; from about 0.01 mg/ml to about 10
mg/ml of polysorbate 80; and from about 10 mg/ml to about 100 mg/ml
of trehalose.
20. A stable composition comprising at least one monoclonal
anti-MAdCAM antibody and a chelating agent, wherein the composition
comprises an amount of the chelating agent sufficient to stabilize
the composition when maintained at a temperature of about
40.degree. C. for a period of at least about 26 weeks.
21. A liquid pharmaceutical composition comprising at least one
monoclonal anti-MAdCAM antibody and a pharmaceutically acceptable
chelating agent, wherein the molar concentration of the antibody
ranges from about 0.0006 millimolar to about 1.35 millimolar and
the molar concentration of the chelating agent ranges from about
0.003 millimolar to about 50 millimolar, and wherein the molar
ratio of antibody to chelating agent ranges from about 0.00001 to
about 450.
22. A liquid pharmaceutical composition comprising: at least one
antibody comprising an amino acid sequence that is at least 95%
identical to a heavy chain amino acid sequence shown in SEQ ID NO:
2, and further comprising an amino acid sequence that is at least
95% identical to a light chain amino acid sequence shown in SEQ ID
NO: 4, wherein the antibody binds to human MAdCAM; and a
pharmaceutically acceptable excipient, wherein the composition
contains a concentration of antibody that is at least about 50
mg/ml.
23. A process for preparing a liquid pharmaceutical composition
comprising mixing at least one anti-MAdCAM antibody having the
heavy chain amino acid sequence of SEQ ID NO:2 and light chain
amino acid sequence of SEQ ID NO:4, in solution, with at least one
chelating agent.
24. A method for the treatment of an inflammatory disease in a
subject, comprising administering to the subject a liquid
pharmaceutical composition comprising: a) a therapeutically
effective amount of at least one anti-MAdCAM antibody; and b) a
pharmaceutically acceptable chelating agent.
Description
CROSS-REFERENCE TO RELATED PATENTS AND PATENT APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/752,712, filed Mar. 8, 2005; U.S.
Provisional Patent Application Ser. No. 60/762,456, filed Jan. 26,
2006; U.S. Provisional Patent Application Ser. No. 60/659,766,
filed Mar. 8, 2005; U.S. Provisional Patent Application No.
60/728,165, filed Oct. 19, 2005, all of which are incorporated by
reference herein in their entireties.
BACKGROUND
[0002] The invention relates to antibody compositions and methods
of stabilizing antibodies, and to pharmaceutically acceptable
compositions comprising anti-MAdCAM antibodies, and methods of
reducing anti-MAdCAM antibody instability.
[0003] Mucosal addressin cell adhesion molecule (MAdCAM) is a
member of the immunoglobulin superfamily of cell adhesion
receptors. While MAdCAM plays a physiological role in gut immune
surveillance, it appears to facilitate excessive lymphocyte
extravasation in inflammatory bowel disease under conditions of
chronic gastrointestinal tract inflammation. Antibodies that
inhibit the binding of .alpha..sub.4.beta..sub.7.sup.+ lymphocytes
to MAdCAM have been shown to reduce lymphocyte recruitment, tissue
extravasation, inflammation and disease severity in animal
models.
[0004] Antibodies that bind to and inhibit the activity of MAdCAM
have been reported in the literature. For example, International
Patent Application Number PCT/US2005/000370 reports several human
monoclonal antibodies to MAdCAM, including an antibody having the
heavy and light chain amino acid sequences of antibody 7.16.6. A
hybridoma cell line producing antibody 7.16.6 was deposited in the
European Collection of Cell Cultures (ECACC), H.P.A. at CAMR,
Porton Down, Salisbury, Wiltshire SP4 0JG on 9 Sep. 2003 with
Deposit No. 03090909.
[0005] One possible mode of administering such MAdCAM antibodies is
parenteral. Anti-MAdCAM compositions, as with all protein
compositions, are subject to concerns regarding chemical and
physical degradation of the antibody in the composition over time.
In general, anti-MAdCAM antibody compositions must exhibit
acceptable chemical and physical stability under the expected range
of storage and use conditions, i.e., the anti-MAdCAM antibody
compositions must have a sufficient shelf life and yet remain
biologically active. The present application discloses novel
anti-MAdCAM antibody compositions that exhibit improved chemical
and/or physical stability relative to anti-MAdCAM compositions
previously disclosed in the literature.
SUMMARY
[0006] In one aspect, the present invention provides a liquid
pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a chelating
agent.
[0007] The present invention also provides a method for preparing a
stable liquid pharmaceutical composition comprising mixing at least
one monoclonal anti-MAdCAM antibody with a pharmaceutically
acceptable chelating agent in an amount which reduces instability
of the antibody, wherein when the composition is stored for a
period of about 26 weeks at a temperature of about 40.degree. C.;
the decrease between an aggregate chromatogram peak area for the
stable liquid pharmaceutical composition comprising monoclonal
anti-MAdCAM antibodies and the chelating agent; and an aggregate
chromatogram peak area for an otherwise identical composition
lacking the chelating agent that is stored for a period of about 26
weeks at a temperature of about 40.degree. C. is at least about
2%.
[0008] The present invention also provides a method for stabilizing
at least one monoclonal anti-MAdCAM antibody in a liquid
pharmaceutical composition comprising forming a liquid composition
comprising the at least one antibody and a pharmaceutically
acceptable chelating agent, wherein when the composition is stored
for a period of about 26 weeks at a temperature of about 40.degree.
C.; the decrease between an aggregate chromatogram peak area for
the stable liquid pharmaceutical composition comprising at least
one monoclonal anti-MAdCAM antibody and the chelating agent; and an
aggregate chromatogram peak area for an otherwise identical
composition lacking the chelating agent that is stored for a period
of about 26 weeks at a temperature of about 40.degree. C. is at
least about 2%.
[0009] The present invention also provides a method for the
treatment of an inflammatory disease in a subject, comprising
administering to the subject a liquid pharmaceutical composition
comprising: a therapeutically effective amount of monoclonal
anti-MAdCAM antibody 7.16.6; and a pharmaceutically acceptable
chelating agent.
[0010] The present invention also provides a kit for preparing a
liquid composition of a stabilized antibody comprising: a first
container comprising at least one monoclonal anti-MAdCAM antibody
7.16.6 in solution, and a second container comprising a
pharmaceutically acceptable chelating agent.
[0011] The present invention also provides an article of
manufacture comprising a container which holds a mixture of at
least one monoclonal anti-MAdCAM antibody 7.16.6 and a
pharmaceutically acceptable chelating agent.
[0012] The present invention also provides a liquid pharmaceutical
composition comprising at least one monoclonal anti-MAdCAM antibody
and a pharmaceutically acceptable chelating agent, wherein the
molar concentration of the antibody ranges from about 0.0006
millimolar to about 1.35 millimolar and the molar concentration of
the chelating agent ranges from about 0.003 millimolar to about 50
millimolar, and wherein the molar ratio of antibodies to chelating
agent ranges from about 0.00001 to about 450.
[0013] The present invention also provides a liquid pharmaceutical
composition comprising at least one human monoclonal anti-MAdCAM
antibody, wherein the antibody binds to human MAdCAM; and a
chelating agent.
[0014] The present invention also provides a liquid pharmaceutical
composition comprising at least one antibody comprising an amino
acid sequence that is at least 95% identical to a heavy chain amino
acid sequence shown in SEQ ID NO: 2, and further comprising an
amino acid sequence that is at least 95% identical to a light chain
amino acid sequence shown in SEQ ID NO: 4, wherein the antibody
binds to human MAdCAM; and a pharmaceutically acceptable excipient,
wherein the composition contains a concentration of antibody that
is at least about 10 mg/ml.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a graph that illustrates the percent aggregation
in various test compositions differing in mAb concentration after
storage at 40.degree. C. for up to 26 weeks by size exclusion
chromatography (SEC);
[0016] FIG. 2 is a graph that illustrates the percent aggregation
in various test compositions differing in EDTA concentration after
storage at 40.degree. C. for up to 26 weeks, by SEC;
[0017] FIG. 3 is a graph that illustrates the percent aggregation
in various test compositions differing in PS80 concentration after
storage at 40.degree. C. for up to 26 weeks by SEC;
[0018] FIG. 4 is a graph that illustrates the percent aggregation
in various test compositions differing in buffer species after
storage at 40.degree. C. for up to 26 weeks by SEC;
[0019] FIG. 5 is a graph that illustrates the percent aggregation
in various test compositions differing in stabilizer/tonicifier
species after storage at 40.degree. C. for up to 26 weeks by
SEC;
[0020] FIG. 6 is a graph that illustrates the percent aggregation
in various test compositions differing in surfactant species after
storage at 40.degree. C. for up to 26 weeks by SEC.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The methods and techniques of the present invention are
generally performed according to conventional methods well known in
the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification unless otherwise indicated. See, e.g., Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and
Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates (1992), and Harlow and Lane Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1990). Enzymatic reactions and purification
techniques are performed according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The nomenclatures used in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of subjects.
DEFINITIONS
[0022] In order to aid the reader in understanding the following
detailed description, the following definitions are provided:
[0023] As used herein, the term "composition" as it relates to an
anti-MAdCAM antibody is meant to describe the antibody in
combination with a pharmaceutically acceptable excipient comprising
a chelating agent. For example, the compositions of the invention
have an improved shelf life and/or stability as compared to art
recognized compositions comprising an anti-MAdCAM antibody.
[0024] As used herein, the term "antibody" refers to an intact
antibody or an antigen-binding portion that competes with the
intact antibody for specific binding. See generally, Fundamental
Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).
Antigen-binding portions may be produced by recombinant DNA
techniques or by enzymatic or chemical cleavage of intact
antibodies. In some embodiments, antigen-binding portions include
Fab, Fab', F(ab').sub.2, Fd, Fv, dAb, and complementarity
determining region (CDR) fragments, single-chain antibodies (scFv),
chimeric antibodies, diabodies and polypeptides that contain at
least a portion of an antibody that is sufficient to confer
specific antigen binding to the polypeptide. From N-terminus to
C-terminus, both the mature light and heavy chain variable domains
comprise the regions FR1, CDR1, FR2, CDR2, FR3, --CDR3 and FR4. The
assignment of amino acids to each domain is in accordance with the
definitions of Kabat, Sequences of Proteins of Immunological
Interest (National Institutes of Health, Bethesda, Md., (1987 and
1991)), Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987), or
Chothia et al., Nature 342:878-883 (1989).
[0025] As used herein, an antibody that is referred to by number
has the same heavy and light chain amino acid sequences as a
monoclonal antibody that is obtained from the hybridoma of the same
number. For example, monoclonal antibody 7.16.6 has the same heavy
and light chain amino acid sequences as one obtained from hybridoma
7.16.6. Thus, reference to antibody 7.16.6 includes the antibody
which has the heavy and light chain amino acid sequences shown in
SEQ ID NOS. 2 and 4. It also includes an antibody lacking a
terminal lysine on the heavy chain, as this is normally lost in a
proportion of antibodies during manufacture.
[0026] As used herein, the term "polypeptide" encompasses native or
artificial proteins, protein fragments and polypeptide analogs of a
protein sequence. A polypeptide may be monomeric or polymeric.
[0027] As used herein, an Fd fragment means an antibody fragment
that consists of the V.sub.H and C.sub.H 1 domains; an Fv fragment
consists of the V.sub.L and V.sub.H domains of a single arm of an
antibody; and a dAb fragment (Ward et al., Nature 341:544-546
(1989)) consists of a V.sub.H domain.
[0028] The term "or an antigen-binding portion thereof" when used
with the term "antibody" refers to a polypeptide that has an
amino-terminal and/or carboxy-terminal deletion, but where the
remaining amino acid sequence is identical to the corresponding
positions in the naturally-occurring sequence. In some embodiments,
fragments are at least 5, 6, 8 or 10 amino acids long. In other
embodiments, the fragments are at least 14, at least 20, at least
50, or at least 70, 80, 90, 100, 150 or 200 amino acids long.
[0029] As used herein, the term "monoclonal antibody" refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts or lacking a
C-terminal lysine. Monoclonal antibodies are highly specific, being
directed against a single antigenic site. Furthermore, in contrast
to conventional (polyclonal) antibody preparations, which typically
include different antibodies, directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler, et al., Nature 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson, et al., Nature 352:624-628 (1991) and Marks,
et al., J. Mol. Biol. 222:581-597 (-1991), for example.
[0030] As used herein, the terms "isolated antibody" or "purified
antibody" refers to an antibody that by virtue of its origin or
source of derivation has one to four of the following: (1) is not
associated with naturally associated components that accompany it
in its native state, (2) is free of other proteins from the same
species, (3) is expressed by a cell from a different species, or
(4) does not occur in nature. Thus, an antibody that is chemically
synthesized or synthesized in a cellular system different from the
cell from which it naturally originates is isolated and purified
from its naturally associated components. An antibody may also be
rendered substantially free of naturally associated components by
isolation and purification, using protein purification techniques
well known in the art. Examples of isolated/purified antibodies
include an anti-MAdCAM antibody that has been affinity purified
using MAdCAM, an anti-MAdCAM antibody that has been synthesized by
a hybridoma or other cell line in vitro, and a human anti-MAdCAM
antibody derived from a transgenic mouse.
[0031] An antibody is "substantially pure," "substantially
homogeneous," or "substantially purified" when at least about 60 to
75% of a sample exhibits a single species of antibody. The antibody
may be monomeric or multimeric. A substantially pure antibody will
typically comprise about 50%, 60%, 70%, 80% or 90% w/w of an
antibody sample, more usually about 95%, and preferably will be
over 99% pure. Antibody purity or homogeneity may be indicated by a
number of means well known in the art, such as polyacrylamide gel
electrophoresis of an antibody sample, followed by visualizing a
single polypeptide band upon staining the gel with a stain well
known in the art. For certain purposes, higher resolution may be
achieved by using HPLC or other means well known in the art for
purification.
[0032] As used herein, the term "human antibody" is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo), for example in the CDRs and in particular CDR3.
However, the term "human antibody", as used herein, is not intended
to include antibodies in which CDR sequences derived from the
germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.
[0033] As used herein, the term "recombinant human antibody" is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies expressed using a recombinant expression vector
transfected into a host cell, antibodies isolated from a
recombinant, combinatorial human antibody library, antibodies
isolated from an animal (e.g., a mouse) that is transgenic for
human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992)
Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed,
created or isolated by any other means that involves splicing of
human immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies have variable and constant regions
derived from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies are
subjected to in vitro mutagenesis (or, when an animal transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and
thus the amino acid sequences of the VH and VL regions of the
recombinant antibodies are sequences that, while derived from and
related to human germline VH and VL sequences, may not naturally
exist within the human antibody germline repertoire in vivo.
[0034] As used herein, the term "polynucleotide" means a polymeric
form of nucleotides of at least 10 bases in length, either
ribonucleotides or deoxynucleotides or a modified form of either
type of nucleotide. The term includes single and double stranded
forms.
[0035] As used herein, the term "isolated polynucleotide" means a
polynucleotide of genomic, cDNA, or synthetic origin or some
combination thereof, which by virtue of its origin or source of
derivation, the "isolated polynucleotide" has one to three of the
following: (1) is not associated with all or a portion of a
polynucleotide with which the "isolated polynucleotide" is found in
nature, (2) is operably linked to a polynucleotide to which it is
not linked in nature, or (3) does not occur in nature as part of a
larger sequence.
[0036] As used herein, the term "naturally occurring nucleotides"
includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" as used herein includes nucleotides with
modified or substituted sugar groups and the like. The term
"oligonucleotide linkages" referred to herein includes
oligonucleotides linkages such as phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the
like. See e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986);
Stec et al., J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucl.
Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design
6:539 (1991); Zon et al., Oligonucleotides and Analogues: A
Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University
Press, Oxford England (1991)); U.S. Pat. No. 5,151,510; Uhlmann and
Peyman, Chemical Reviews 90:543 (1990), the disclosures of which
are hereby incorporated by reference. An oligonucleotide can
include a label for detection, if desired.
[0037] "Operably linked" sequences include both expression control
sequences that are contiguous with the gene of interest and
expression control sequences that act in trans or at a distance to
control the gene of interest. The term "expression control
sequence" as used herein means polynucleotide sequences that are
necessary to effect the expression and processing of coding
sequences to which they are ligated. Expression control sequences
include appropriate transcription initiation, termination, promoter
and enhancer sequences; efficient RNA processing signals such as
splicing and polyadenylation signals; sequences that stabilize
cytoplasmic mRNA; sequences that enhance translation efficiency
(i.e., Kozak consensus sequence); sequences that enhance protein
stability; and when desired, sequences that enhance protein
secretion. The nature of such control sequences differs depending
upon the host organism; in prokaryotes, such control sequences
generally include promoter, ribosomal binding site, and
transcription termination sequence; in eukaryotes, generally, such
control sequences include promoters and transcription termination
sequence. The term "control sequences" is intended to include, at a
minimum, all components whose presence is essential for expression
and processing, and can also include additional components whose
presence is advantageous, for example, leader sequences and fusion
partner sequences.
[0038] As used herein, the term "vector" means a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. In some embodiments, the vector is a plasmid,
i.e., a circular double stranded DNA loop into which additional DNA
segments may be ligated. In some embodiments, the vector is a viral
vector, wherein additional DNA segments may be ligated into the
viral genome. In some embodiments, the vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). In other embodiments,
the vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "expression vectors").
[0039] As used herein, the terms "recombinant host cell" (or simply
"host cell") means a cell into which a recombinant expression
vector has been introduced. It should be understood that
"recombinant host cell" and "host cell" mean not only the
particular subject cell but also the progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term "host cell" as used
herein.
[0040] As used herein, the terms "is capable of specifically
binding" refers to when an antibody binds to an antigen with a
dissociation constant that is .ltoreq.1 .mu.M, preferably .ltoreq.1
nM and most preferably .ltoreq.10 pM.
[0041] As used herein, the terms "selectively hybridize" mean to
detectably and specifically bind. Polynucleotides, oligonucleotides
and fragments thereof in accordance with the invention selectively
hybridize to nucleic acid strands under hybridization and wash
conditions that minimize appreciable amounts of detectable binding
to nonspecific nucleic acids. "High stringency" or "highly
stringent" conditions can be used to achieve selective
hybridization conditions as known in the art and discussed herein.
One example of "high stringency" or "highly stringent" conditions
is the incubation of a polynucleotide with another polynucleotide,
wherein one polynucleotide may be affixed to a solid surface such
as a membrane, in a hybridization buffer of 6.times.SSPE or SSC,
50% formamide, 5.times.Denhardt's reagent, 0.5% SDS, 100 .mu.g/ml
denatured, fragmented salmon sperm DNA at a hybridization
temperature of 42.degree. C. for 12-16 hours, followed by twice
washing at 55.degree. C. using a wash buffer of 1.times.SSC, 0.5%
SDS. See also Sambrook et al., supra, pp. 9.50-9.55.
[0042] The term "percent sequence identity" in the context of
nucleic acid sequences means the percent of residues when a first
contiguous sequence is compared and aligned for maximum
correspondence to a second contiguous sequence. The length of
sequence identity comparison may be over a stretch of at least
about nine nucleotides, usually at least about 18 nucleotides, more
usually at least about 24 nucleotides, typically at least about 28
nucleotides, more typically at least about 32 nucleotides, and
preferably at least about 36, 48 or more nucleotides. There are a
number of different algorithms known in the art that can be used to
measure nucleotide sequence identity. For instance, polynucleotide
sequences can be compared using FASTA, Gap or Bestfit, which are
programs in Wisconsin Package Version 10.0, Genetics Computer Group
(GCG), Madison, Wis. FASTA, which includes, e.g., the programs
FASTA2 and FASTA3, provides alignments and percent sequence
identity of the regions of the best overlap between the query and
search sequences (Pearson, Methods Enzymol. 183:63-98 (1990);
Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods
Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84
(1998); herein incorporated by reference). Unless otherwise
specified, default parameters for a particular program or algorithm
are used. For instance, percent sequence identity between nucleic
acid sequences can be determined using FASTA with its default
parameters (a word size of 6 and the NOPAM factor for the scoring
matrix) or using Gap with its default parameters as provided in GCG
Version 6.1, herein incorporated by reference.
[0043] A reference to a "polynucleotide" or a "nucleic acid"
sequence encompasses its complement unless otherwise specified.
Thus, a reference to a nucleic acid having a particular sequence
should be understood to encompass its complementary strand, with
its complementary sequence.
[0044] The term "substantial similarity" or "substantial sequence
similarity," when referring to a nucleic acid or fragment thereof,
means that when optimally aligned with appropriate nucleotide
insertions or deletions with another nucleic acid (or its
complementary strand), there is nucleotide sequence identity in at
least about 85%, preferably at least about 90%, and more preferably
at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases,
as measured by any well-known algorithm of sequence identity, such
as FASTA, BLAST or Gap, as discussed above.
[0045] As applied to polypeptides, the term "substantial identity",
"percent identity" or "% identical" means that two peptide
sequences, when optimally aligned, such as by the programs GAP or
BESTFIT using default gap weights, as supplied with the programs,
share at least 70%, 75% or 80% sequence identity, preferably at
least 90% or 95% sequence identity, and more preferably at least
96%, 97%, 98% or 99% sequence identity. In certain embodiments,
residue positions that are not identical differ by conservative
amino acid substitutions. A "conservative amino acid substitution"
is one in which an amino acid residue is substituted by another
amino acid residue having a side chain R group with similar
chemical properties (e.g., charge or hydrophobicity). In general, a
conservative amino acid substitution will not substantially change
the functional properties of a protein. In cases where two or more
amino acid sequences differ from each other by conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well-known to those of skill
in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31
(1994). Examples of groups of amino acids that have side chains
with similar chemical properties include 1) aliphatic side chains:
glycine, alanine, valine, leucine, and isoleucine; 2)
aliphatic-hydroxyl side chains: serine and threonine; 3)
amide-containing side chains: asparagine and glutamine; 4) aromatic
side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side
chains: lysine, arginine, and histidine; 6) acidic side chains:
aspartic acid and glutamic acid; and 7) sulfur-containing side
chains: cysteine and methionine. Conservative amino acids
substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine,
glutamate-aspartate, and asparagine-glutamine.
[0046] Sequence identity for polypeptides, is typically measured
using sequence analysis software. Protein analysis software matches
sequences using measures of similarity assigned to various
substitutions, deletions and other modifications, including
conservative amino acid substitutions. For instance, GCG contains
programs such as "Gap" and "Bestfit" which can be used with default
parameters, as specified with the programs, to determine sequence
homology or sequence identity between closely related polypeptides,
such as homologous polypeptides from different species of organisms
or between a wild type protein and a mutant thereof. See, e.g., GCG
Version 6.1. Polypeptide sequences also can be compared using FASTA
using default or recommended parameters, see GCG Version 6.1.
(University of Wisconsin WI) FASTA (e.g., FASTA2 and FASTA3)
provides alignments and percent sequence identity of the regions of
the best overlap between the query and search sequences (Pearson,
Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.
132:185-219 (2000)). Another preferred algorithm when comparing a
sequence of the invention to a database containing a large number
of sequences from different organisms is the computer program
BLAST, especially blastp or tblastn, using default parameters, as
supplied with the programs. See, e.g., Altschul et al., J. Mol.
Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res.
25:3389-402 (1997). The length of polypeptide sequences compared
for homology will generally be at least about 16 amino acid
residues, usually at least about 20 residues, more usually at least
about 24 residues, typically at least about 28 residues, and
preferably more than about 35 residues. When searching a database
containing sequences from a large number of different organisms, it
is preferable to compare amino acid sequences.
[0047] A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result, which includes treatment or
prophylactic prevention of inflammatory diseases. It is to be noted
that dosage values may vary with the severity of the condition to
be alleviated. It is to be further understood that for any
particular subject, specific dosage regimens should be adjusted
over time according to the individual need and the professional
judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set
forth herein are exemplary only and are not intended to limit the
scope or practice of the claimed composition. Likewise, a
therapeutically effective amount of the antibody or antibody
portion may vary according to factors such as the disease state,
age, sex, and weight of the individual, the ability of the antibody
or antibody portion to elicit a desired response in the individual,
and the desired route of administration of the antibody
composition. A therapeutically effective amount is also one in
which any toxic or detrimental effects of the antibody or antibody
portion are outweighed by the therapeutically beneficial
effects.
[0048] As used herein, the term "saccharide" refers to a class of
molecules that are derivatives of polyhydric alcohols.
[0049] As used herein, the term "subject" for purposes of treatment
includes any subject, and preferably is a subject who is in need of
the treatment of an inflammatory disease. For purposes of
prevention, the subject is any subject, and preferably is a subject
that is at risk for, or is predisposed to, developing an
inflammatory disease. The term "subject" is intended to include
living organisms, e.g., prokaryotes and eukaryotes. Examples of
subjects include mammals, e.g., humans, dogs, cows, horses, pigs,
sheep, goats, cats, mice, rabbits, rats, and transgenic non-human
animals. In specific embodiments of the invention, the subject is a
human.
[0050] As used herein, the term "treatment" refers to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic disease or condition. Those in need of treatment include
those already with the disease as well as those prone to have the
disease or those in whom the disease is to be prevented.
[0051] When introducing elements of the present invention or the
preferred embodiment(s) thereof, the articles "a", "an", "the" and
"said" are intended to mean that there are one or more of the
elements. The terms "comprising", "comprise", "comprises",
"including" and "having" are intended to be inclusive and mean that
there may be additional elements other than the listed
elements.
Anti-MAdCAM Antibodies:
[0052] In accordance with the present invention, it has been
discovered that the stability of certain monoclonal anti-MAdCAM
antibodies that are described herein can be improved in solution by
mixing the anti-MAdCAM antibodies with a pharmaceutically
acceptable chelating agent, such as ethylenediaminetetraacetic acid
("EDTA").
[0053] While not wishing to be bound by theory, it is believed that
the presence of a chelating agent in the composition of the present
invention help to improve stability of the antibody polypeptide by
reducing the incidence of one or more of the following: anti-MAdCAM
antibody aggregation, fragmentation, oxidation, freeze/thaw
instability, discoloration, and/or deamidation. The present
invention comprises anti-MAdCAM antibody compositions having
improved chemical and/or physical stability as compared to
previously disclosed antibody compositions.
[0054] Therefore, in certain aspects, the present invention
provides a liquid pharmaceutical composition comprising a
pharmaceutically acceptable chelating agent, such as EDTA and at
lease one monoclonal anti-MAdCAM antibody or an antigen-binding
portion thereof. In still other aspects, the aforementioned liquid
anti-MAdCAM antibody compositions comprising a chelating agent can
include additional pharmaceutically acceptable excipients,
including, but not limited to, one or more excipients that are
chosen from buffers, tonicity agents, surfactants, and mixtures
thereof.
[0055] The present invention provides novel compositions comprising
anti-MAdCAM antibodies. As used herein, the phrase "anti-MAdCAM
antibody" refers to any antibody, or any portion thereof, that is
capable of binding to any portion of a MAdCAM polypeptide that may
be present within or isolated from any animal. In certain
embodiments, the MAdCAM polypeptide is a human MAdCAM
polypeptide.
[0056] Suitable anti-MAdCAM antibodies for use with the present
invention may be chosen from polyclonal or monoclonal antibodies.
In certain aspects, the monoclonal anti-MAdCAM antibody can be a
murine, chimeric, humanized or human antibody. In further
embodiments, the monoclonal anti-MAdCAM antibody is a human
monoclonal anti-MAdCAM antibody.
[0057] In certain embodiments, the anti-MAdCAM antibodies which are
suitable for use with the present invention include those
anti-MAdCAM antibodies and methods to prepare them that are
described in International Application Number PCT/US2005/000370,
filed 7 Jan. 2005 and published 28 Jul. 2005. In other embodiments,
the anti-MAdCAM antibodies which are suitable for use with the
present invention include those anti-MAdCAM monoclonal antibodies
having the heavy and light chain amino acid sequences of the
antibody designated 7.16.6 in International Application Number
PCT/US2005/000370.
[0058] In addition, such anti-MAdCAM antibodies may be chosen based
on differences in the amino acid sequences in the constant region
of their heavy chains. For example, the anti-MAdCAM antibodies may
be chosen from the IgG class, which have "gamma" type heavy chains.
The class and subclass of anti-MAdCAM antibodies may be determined
by any method known in the art. In general, the class and subclass
of an antibody may be determined using antibodies that are specific
for a particular class and subclass of antibody. Such antibodies
are commercially available. The class and subclass can be
determined by ELISA, or Western Blot as well as other techniques.
Alternatively, the class and subclass may be determined by
sequencing all or a portion of the constant domains of the heavy
and/or light chains of the antibodies, comparing their amino acid
sequences to the known amino acid sequences of various class and
subclasses of immunoglobulins, and determining the class and
subclass of the antibodies.
[0059] The anti-MAdCAM antibody can be an IgG, an IgM, an IgE, an
IgA, or an IgD molecule. In further embodiments, the anti-MAdCAM
antibody is an IgG and is an IgG1, IgG2, IgG3 or IgG4 subclass.
However, as it will be appreciated; it is generally not desirable
to kill MAdCAM expressing cells. Rather, one generally desires to
simply inhibit MAdCAM binding with its ligands to mitigate T cell
down regulation. One of the major mechanisms through which
antibodies kill cells is through fixation of complement and
participation in CDC. The constant region of an antibody plays an
important role in connection with an antibody's ability to fix
complement and participate in CDC. Thus, generally one selects the
isotype of an antibody to either provide the ability of complement
fixation, or not. In the case of the present invention, generally,
as mentioned above, it is generally not preferred to utilize an
antibody that kills the cells. There are a number of isotypes of
antibodies that are capable of complement fixation and CDC,
including, without limitation, the following: murine IgM, murine
IgG2a, murine IgG2b, murine IgG3, human IgM, human IgG1, and human
IgG3. In contrast, preferred isotypes which are not capable of
complement fixation and CDC include, without limitation, human IgG2
and human IgG4. In addition to heavy chain sequence differences,
the IgG antibodies differ within their subclass based on the number
of disulfide bonds and length of the hinge region. For example, the
IgG2 subclass has several differences distinct from the other
subclasses. The IgG2 and IgG4 subclasses are known to have 4
disulfide bonds within their hinge region, while IgG1 has 2 and
IgG3 has 11 disulfide bonds. Other differences for IgG2 antibodies
include their reduced ability to cross the placenta and the
inability of IgG2 antibodies to bind to lymphocyte Fc receptors.
Thus, in certain embodiments, the anti-MAdCAM antibody is subclass
IgG2 or IgG4. In another preferred embodiment, the anti-MAdCAM
antibody is subclass IgG2.
[0060] In some embodiments, the antibody is a single-chain antibody
(scFv) in which a V.sub.L and V.sub.H domains are paired to form a
monovalent molecules via a synthetic linker that enables them to be
made as a single protein chain. (Bird et al., Science 242:423-426
(1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883
(1988)). In some embodiments, the antibodies are diabodies, i.e.,
are bivalent antibodies in which V.sub.H and V.sub.L domains are
expressed on a single polypeptide chain, but using a linker that is
too short to allow for pairing between the two domains on the same
chain, thereby forcing the domains to pair with complementary
domains of another chain and creating two antigen binding sites.
(See e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993), and Poljak R. J. et al., Structure 2:1121-1123
(1994)). In some embodiments, one or more CDRs from an antibody of
the invention may be incorporated into a molecule either covalently
or noncovalently to make it an immunoadhesin that specifically
binds to MAdCAM. In such embodiments, the CDR(s) may be
incorporated as part of a larger polypeptide chain, may be
covalently linked to another polypeptide chain, or may be
incorporated noncovalently.
[0061] In another embodiment, the anti-MAdCAM antibody has
selectivity (or specificity) for MAdCAM that is at least 100 times
greater than its selectivity for any other polypeptide. In some
embodiments, the anti-MAdCAM antibody does not exhibit any
appreciable specific binding to any other protein other than
MAdCAM. One can determine the selectivity of the anti-MAdCAM
antibody for MAdCAM using methods well known in the art following
the teachings of the specification. For instance, one can determine
the selectivity using Western blot, FACS, ELISA, or RIA. Thus, in
some embodiments, the monoclonal anti-MAdCAM antibody is capable of
specifically binding to MAdCAM.
[0062] In some embodiments, the C-terminal lysine of the heavy
chain of the anti-MAdCAM antibody of the invention is not present.
In various embodiments of the invention, the heavy and light chains
of the anti-MAdCAM antibodies may optionally include a signal
sequence.
[0063] Table 2 lists the sequence identifiers (SEQ ID NOS) of the
nucleic acids that encode the heavy and light chains and the
corresponding predicted amino acid sequences for the anti-MAdCAM
monoclonal antibody 7.16.6. While DNA sequences encoding a signal
polypeptide are shown in the sequence identifiers (SEQ ID NOS), the
antibody typically does not comprise a signal polypeptide because
the signal polypeptide is generally eliminated during
post-translational modifications. In various embodiments of the
invention, one or both of the heavy and light chains of the
anti-MAdCAM antibodies includes a signal sequence (or a portion of
the signal sequence). In other embodiments of the invention,
neither the heavy nor light chain of the anti-MAdCAM antibodies
includes a signal sequence.
TABLE-US-00001 TABLE 1 HUMAN ANTI-MAdCAM ANTIBODY 7.16.6 SEQUENCE
IDENTIFIER (SEQ ID NOS:) Heavy Amino Light MAb DNA Acid DNA Amino
Acid 7.16.6 1 2 3 4
[0064] In some embodiments, the nucleic acid molecule comprises a
nucleotide sequence that encodes the V.sub.L amino acid sequence of
monoclonal antibody 7.16.6 (SEQ ID NO: 4), or a portion thereof. In
some embodiments, said portion comprises at least the CDR3 region.
In some embodiments, the nucleic acid encodes the amino acid
sequence of the light chain CDRs of said antibody. In some
embodiments, said portion is a contiguous portion comprising
CDR1-CDR3.
[0065] In still other embodiments, the nucleic acid molecule
encodes a V.sub.L amino acid sequence that is at least 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a
V.sub.L amino acid sequence of antibody 7.16.6, or an amino acid
sequence of SEQ ID NO: 4. Nucleic acid molecules of the invention
include nucleic acids that hybridize under highly stringent
conditions, such as those described above, to a nucleic acid
sequence encoding the light chain amino acid sequence of SEQ ID NO:
4.
[0066] In further embodiments, the nucleic acid molecule comprises
a nucleotide sequence that encodes at least a portion of the
V.sub.H amino acid sequence of 7.16.6 (SEQ ID NO: 2) or said
sequence having conservative amino acid mutations and/or a total of
three or fewer non-conservative amino acid substitutions. In
various embodiments the sequence encodes one or more CDR regions,
preferably a CDR3 region, all three CDR regions, a contiguous
portion including CDR1-CDR3, or the entire V.sub.H region.
[0067] In some embodiments, the nucleic acid molecule encodes a
V.sub.H amino acid sequence that is at least 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the V.sub.H amino
acid sequence of SEQ ID NO: 2. Nucleic acid molecules of the
invention include nucleic acids that hybridize under highly
stringent conditions, such as those described above, to a
nucleotide sequence encoding the heavy chain amino acid sequence of
SEQ ID NO: 2.
[0068] In other aspects, the present invention provides a liquid
pharmaceutical composition comprising at least one purified human
antibody that binds to MAdCAM, wherein the antibody comprises a
heavy chain amino acid sequence with at least 95% sequence identity
to SEQ ID NO: 2 and a light chain amino acid sequence with at least
95% sequence identity to SEQ ID NO: 4. In other aspects, the
antibody comprises a heavy chain amino acid sequence with at least
99% sequence identity to SEQ ID NO: 2 and a light chain amino acid
sequence with at least 99% sequence identity to SEQ ID NO: 4. In
still other aspects, the antibody comprises a heavy chain amino
acid sequence that comprises the variable region of SEQ ID NO: 2
and a light chain amino acid sequence that comprises the variable
region SEQ ID NO: 4. In further aspects, the antibody comprises a
heavy chain amino acid sequence comprising SEQ ID NO: 2 and a light
chain amino acid sequence comprising SEQ ID NO: 4.
[0069] In one embodiment of the present invention, the anti-MAdCAM
antibody specifically binds to a conformational epitope on human
MAdCAM.
Preparation of the Monoclonal Anti-MAdCAM Antibody
Compositions:
[0070] The anti-MAdCAM antibody typically is formulated as a
pharmaceutical composition for parenteral administration to a
subject. In one embodiment, the pharmaceutical composition is a
liquid composition. In another embodiment, the pharmaceutical
composition is an liquid composition.
[0071] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising at least one anti-MAdCAM
antibody and a pharmaceutically acceptable chelating agent. In
another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising at least one anti-MAdCAM
antibody and EDTA. In another embodiment, the invention is directed
to a liquid pharmaceutical composition comprising at least one
anti-MAdCAM antibody, a pharmaceutically acceptable chelating
agent, and a pharmaceutically acceptable buffer. In another
embodiment, the invention is directed to a liquid pharmaceutical
composition comprising at least one anti-MAdCAM antibody, a
pharmaceutically acceptable chelating agent, and histidine. In
another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising an anti-MAdCAM antibody,
EDTA, and histidine. In another embodiment, the invention is
directed to a liquid pharmaceutical composition comprising an
anti-MAdCAM antibody, DTPA, and histidine.
[0072] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising an anti-MAdCAM antibody, a
pharmaceutically acceptable chelating agent, and a pharmaceutically
acceptable tonicity agent. In another embodiment, the invention is
directed to a liquid pharmaceutical composition comprising an
anti-MAdCAM antibody, a pharmaceutically acceptable chelating
agent, and trehalose. In another embodiment, the invention is
directed to a liquid pharmaceutical composition comprising an
anti-MAdCAM antibody, EDTA, and trehalose. In another embodiment,
the invention is directed to a liquid pharmaceutical composition
comprising an anti-MAdCAM antibody, DTPA, and trehalose.
[0073] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising an anti-MAdCAM antibody, a
pharmaceutically acceptable chelating agent, and a pharmaceutically
acceptable surfactant. In another embodiment, the invention is
directed to a liquid pharmaceutical composition comprising an
anti-MAdCAM antibody, EDTA, and a pharmaceutically acceptable
surfactant. In another embodiment, the invention is directed to a
liquid pharmaceutical composition comprising an anti-MAdCAM
antibody, DTPA, and a pharmaceutically acceptable surfactant. In
another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising an anti-MAdCAM antibody, a
pharmaceutically acceptable chelating agent selected from the group
consisting of EDTA and DTPA, and polysorbate 80.
[0074] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising an anti-MAdCAM antibody, a
pharmaceutically acceptable buffer, and a pharmaceutically
acceptable surfactant. In another embodiment, the invention is
directed to a liquid pharmaceutical composition comprising an
anti-MAdCAM antibody, histidine, and a pharmaceutically acceptable
surfactant. In another embodiment, the invention is directed to a
liquid pharmaceutical composition comprising anti-MAdCAM antibody,
histidine, and polysorbate 80.
[0075] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising an anti-MAdCAM antibody, a
pharmaceutically acceptable chelating agent, a pharmaceutically
acceptable buffer, and a pharmaceutically acceptable
surfactant.
[0076] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising an anti-MAdCAM antibody, a
pharmaceutically acceptable chelating agent, a pharmaceutically
acceptable buffer, and a pharmaceutically acceptable tonicity
agent.
[0077] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising an anti-MAdCAM antibody, a
pharmaceutically acceptable chelating agent, a pharmaceutically
acceptable buffer, a pharmaceutically acceptable surfactant, and a
pharmaceutically acceptable tonicity agent.
[0078] In another embodiment, the invention is directed to a liquid
pharmaceutical composition comprising an anti-MAdCAM antibody and
histidine.
[0079] The term "pharmaceutical composition" refers to preparations
which are in such form as to permit the biological activity of the
active ingredients to be effective. "Pharmaceutically acceptable
excipients" (vehicles, additives) are those, which can reasonably
(i.e., safely) be administered to a subject to provide an effective
dose of the active ingredient employed. The term "excipient" or
"carrier" as used herein refers to an inert substance, which is
commonly used as a diluent, vehicle, preservative, binder or
stabilizing agent for drugs. As used herein, the term "diluent"
refers to a pharmaceutically acceptable (safe and non-toxic for
administration to a human) solvent and is useful for the
preparation of the liquid compositions herein. Exemplary diluents
include, but are not limited to, sterile water and bacteriostatic
water for injection (BWFI).
[0080] The anti-MAdCAM antibody present in the liquid
pharmaceutical composition can be as previously described in this
application. In one embodiment, the liquid pharmaceutical
composition comprises an anti-MAdCAM antibody comprising a V.sub.L
amino acid sequence that is 90%, 95%, or 99% identical to a V.sub.L
amino acid sequence shown in SEQ ID NO: 4, and further comprises a
V.sub.H amino acid sequence that is 90%, 95%, or 99% identical to a
V.sub.H amino acid sequence shown in SEQ ID NO: 2. In another
embodiment, the liquid pharmaceutical composition comprises an
anti-MAdCAM antibody that is monoclonal anti-MAdCAM antibody
7.16.6.
[0081] The concentration of the anti-MAdCAM antibody in the liquid
pharmaceutical compositions of the present invention is generally
at least about 0.1 milligram per milliliter (mg/ml) or higher, at
least about 1.0 mg/ml or higher, at least about 10 mg/ml or higher,
at least about 50 mg/ml or higher, at least about 75 mg/ml or
higher, at least about 100 mg/ml or higher, or at least about 200
mg/ml or higher. In certain embodiments, the concentration of the
anti-MAdCAM antibody generally ranges from about 1 mg/ml to about
200 mg/ml, from about 2 mg/ml to about 100 mg/ml, from about 5
mg/ml to about 90 mg/ml, from about 10 mg/ml to about 80 mg/ml,
from about 50 mg/ml to about 90 mg/ml, from about 60 mg/ml to about
80 mg/ml, from about 65 mg/ml to about 85 mg/ml, or is about 75
mg/ml. In one embodiment, the concentration of the anti-MAdCAM4
antibody in the liquid pharmaceutical composition ranges from about
50 mg/ml to about 100 mg/ml.
[0082] As used herein, the terms "chelating agent" generally refers
to an excipient that can form at least one bond (e.g., covalent,
ionic, or otherwise) to a metal ion. A chelating agent is typically
a multidentate ligand that can be used in selected liquid
compositions as a stabilizer to complex with species, which might
promote instability. Often, compounds that can act as a chelating
agent will have electron-rich functional groups. Suitable
electron-rich functional groups include carboxylic acid groups,
hydroxy groups and amino groups. Arrangement of these groups in
aminopolycarboxylic acids, hydroxypolycarboxylic acids,
hydroxyaminocarboxylic acids, and the like, result in moieties that
have the capacity to bind metal.
[0083] However, the present invention is not intended to be limited
to chelating agents that enhance antibody stability primarily by
the chelating agent's ability to form bonds with a metal ion.
Therefore, the present invention is not intended to be limited by
the specific mechanism by which the chelating agent stabilizes the
compositions of the present invention and the excipients termed
chelating agents herein may achieve their antibody stability
enhancing properties primarily through mechanisms that are
altogether unrelated to the chelating agent's ability to form bonds
with a metal ion.
[0084] Chelating agents that are suitable for use in the present
invention, include, but are not limited to, aminopolycarboxylic
acids, hydroxyaminocarboxylic acids, N-substituted glycines,
2-(2-amino-2-oxoethyl) aminoethane sulfonic acid (BES),
deferoxamine (DEF), citric acid, niacinamide, and desoxycholates.
Examples of suitable aminopolycarboxylic acids include
ethylenediaminetetraacetic acid (EDTA), diethylenetriamine
pentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA),
N-2-acetamido-2-iminodiacetic acid (ADA),
bis(aminoethyl)glycolether, N,N,N',N'-tetraacetic acid-(EGTA),
trans-diaminocyclohexane tetraacetic acid (DCTA), glutamic acid,
and aspartic acid. Examples of suitable hydroxyaminocarboxylic
acids include N-hydroxyethyliminodiacetic acid (HIMDA),
N,N-bis-hydroxyethylglycine (bicine) and
N-(trishydroxymethylmethyl) 10 glycine (tricine). An example of a
suitable N-substituted glycine is glycylglycine. An example of a
suitable desoxycholate is sodium desoxycholate.
[0085] Chelating agents used in the invention can be present, where
possible, as the free acid or free base form of the compound (e.g.,
referred to interchangeably herein as "EDTA" or "edetate") or as a
corresponding salt form (e.g., the corresponding acid addition salt
or base addition salt, such as disodium edetate). Suitable acid
addition salts, e.g., include alkali metal salts (e.g., sodium or
potassium salts), alkaline earth metal salts (e.g., calcium), and
salts can be prepared using other weakly bound metal ions. As is
known in the art, the nature of the salt and the number of charges
to be neutralized will depend on the number of carboxyl groups
present and the pH at which the stabilizing chelating agent is
supplied. As is also known in the art, chelating agents have
varying strengths with which particular target ions are bound. By
way of further illustration, suitable salts of EDTA include
dipotassium edetate, disodium edetate, edetate calcium disodium,
sodium edetate, trisodium edetate, and potassium edetate; and a
suitable salt of deferoxamine (DEF) is deferoxamine mesylate
(DFM).
[0086] Chelating agents used in the invention can be present as an
anhydrous, solvated or hydrated form of the compound or
corresponding salt. Where the chelating agent is in a solvated or
hydrated form, it can be present in varying states of solvation or
hydration (including, e.g., anhydrous, hydrated, dihydrated, and
trihydrated forms). By way of further illustration, a suitable
hydrate of EDTA is disodium EDTA dihydrate; and suitable forms of
citric acid include anhydrous citric acid, citric acid monohydrate,
and trisodium citrate-dihydrate.
[0087] Suitable chelating agents used in an antibody composition of
the present invention also include, for example, those that bind to
metal ions in solution to render them unable to react with
available O.sub.2, thereby minimizing or preventing generation of
hydroxyl radicals which are free to react with and degrade the
antibody. Other chelating agents such as DFM can lower the
formation of reduced oxygen species, reduce acidic species (e.g.,
deamidation) formation and/or reduce antibody fragmentation. In
still other embodiments, the chelating agent can reduce or prevent
aggregation of the antibodies in the compositions described herein.
Such chelating agents can reduce or prevent degradation of an
antibody that is formulated without the protection of a chelating
agent.
[0088] When a concentration of a chelating agent is referred to, it
is intended that the recited concentration represents the molar
concentration of the free acid or free base form of the chelating
agent. For example, the concentration of chelating agent in certain
liquid pharmaceutical compositions generally ranges from about 0.3
micromolar to about 50 millimolar, from about 3.0 micromolar to
about 10.0 millimolar, from about 30 micromolar to about 5.0
millimolar, from about 0.1 millimolar to about 1 millimolar. In
specific embodiments, the concentration of chelating-agent in the
liquid pharmaceutical composition can be about 3 micromolar, about
13 micromolar, about 27 micromolar, about 0.27 millimolar, about 1
millimolar or about 2.7 millimolar. In one embodiment, the
concentration of chelating agent is about 0.27 millimolar. Unless
stated otherwise, the concentrations listed herein are those
concentrations at ambient conditions, (i.e., at 25.degree. C. and
atmospheric pressure).
[0089] In one embodiment, the chelating agent is selected from the
group consisting of EDTA, DTPA, DFM, and mixtures thereof. In
another embodiment, the chelating is agent is DFM. In another
embodiment, the chelating agent is EDTA. In another embodiment, the
chelating agent is DTPA. In another embodiment, the liquid
pharmaceutical composition comprises EDTA in an amount ranging from
about 0.3 micromolar to about 50 millimolar, and in some
embodiments, from about 0.1 millimolar to about 1.0 millimolar. In
another embodiment, the liquid pharmaceutical composition comprises
EDTA in an amount of about 0.27 millimolar.
[0090] As noted above, the liquid pharmaceutical compositions of
the present invention optionally may further comprise a
pharmaceutically acceptable buffer in addition to a chelating
agent. As used herein, the term "buffer" refers to an added
composition that allows a liquid antibody composition to resist
changes in pH. In certain embodiments, the added buffer allows a
liquid antibody composition to resist changes in pH by the action
of its acid-base conjugate components.
[0091] Examples of suitable buffers include, but are not limited
to, acetate (e.g., sodium acetate), succinate (e.g., sodium
succinate), gluconate, citrate, and other organic acid buffers,
including, but not limited to, buffers such as amino acids (e.g.,
histidine), acetic acid, phosphoric acid and phosphates, ascorbate,
tartartic acid, maleic acid, glycine, lactate, lactic acid,
ascorbic acid, imidazoles, carbonic acid and bicarbonates, succinic
acid, sodium benzoic acid and benzoates, gluconate, edetate (EDTA),
acetate, malate, imidazole, tris, phosphate, and mixtures
thereof.
[0092] In one embodiment, the buffer is histidine. The histidine
starting material used to prepare the liquid pharmaceutical
compositions of the present invention can exist in different forms.
For example, the histidine can be an enantiomeric (e.g., L- or
D-enantiomer) or racemic form of histidine, a free acid or free
base form of histidine, a salt form (e.g., a monohydrochloride,
dihydrochloride, hydrobromide, sulfate, or acetate salt) of
histidine, a solvated form of histidine, a hydrated form (e.g.,
monohydrate) of histidine, or an anhydrous form of histidine. The
purity of histidine base and/or salt used to prepare the liquid
pharmaceutical compositions generally can be at least about 98%, at
least about 99%, or at least about 99.5%. As used herein, the term
"purity" in the context of histidine refers to chemical purity of
histidine as understood in the art, e.g., as described in The Merck
Index, 13th ed., O'Neil et al. ed. (Merck & Co., 2001).
[0093] When a concentration of a buffer is referred to, it is
intended that the recited concentration represent the molar
concentration of the free acid or free base form of the buffer. For
example, the concentration of the buffer when present in certain
liquid pharmaceutical compositions can range from about 0.1
millimolar (mM) to about 100 mM. In one embodiment, the
concentration of the buffer is from about 1 mM to about 50 mM. In
another embodiment, the concentration of the buffer is from about 5
mM to about 20 mM. In various embodiments, the concentration of the
buffer is about 1 mM, about 5 mM, about 10 mM, about 15 mM, about
20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45
mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70
mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM
or about 100 mM. In one embodiment, the concentration of histidine
in the pharmaceutical composition is about 10 mM. In one
embodiment, the pharmaceutical composition contains about 10 mM of
L-histidine (in base form).
[0094] In general, the buffer is used to maintain an acceptable pH
level (which can affect antibody stability) in the liquid
pharmaceutical composition. The liquid pharmaceutical composition
typically is buffered to maintain a pH in the range of from about 4
to about 8; from about 4.5 to about 7; or from about 5.2 to about
5.8. Ranges intermediate to the above-recited pH's are also
intended to be part of this invention. For example, ranges of
values using a combination of any of the above-recited values as
upper and/or lower limits are intended to be included. In one
embodiment, the liquid pharmaceutical composition is buffered to
maintain a pH of about 5.5.
[0095] As noted above, the liquid pharmaceutical compositions of
the present invention optionally may further comprise a
pharmaceutically acceptable tonicity agent in addition to a
chelating agent. As used herein, the terms "tonicity agent" or
"tonicifier" refers to an excipient that can adjust the osmotic
pressure of a liquid antibody composition. In certain embodiments,
the tonicity agent can adjust the osmotic pressure of a liquid
antibody composition to isotonic so that the antibody composition
is physiologically compatible with the cells of the body tissue of
the subject. In still other embodiments, the "tonicity agent" may
contribute to an improvement in stability of any of the anti-MAdCAM
antibodies described herein. An "isotonic" composition is one that
has essentially the same osmotic pressure as human blood. Isotonic
compositions generally have an osmotic pressure from about 250 to
350 mOsm. The term "hypotonic" describes a composition with an
osmotic pressure below that of human blood. Correspondingly, the
term "hypertonic" is used to describe a composition with an osmotic
pressure above that of human blood. Isotonicity can be measured
using a vapor pressure or ice-freezing type osmometer, for
example.
[0096] The tonicity agent used to prepare the liquid pharmaceutical
compositions of the present invention can exist in different forms.
For example, the tonicity agent can be in an enantiomeric (e.g., L-
or D-enantiomer) or racemic form; isomers such as alpha or beta,
including alpha, alpha or beta, beta, or alpha, beta or beta,
alpha; a free acid or free base form; a hydrated form (e.g.,
monohydrate), or an anhydrous form.
[0097] In one embodiment, the tonicity agent is a saccharide.
Saccharides are commonly referred to as carbohydrates and may
contain different amounts of sugar (saccharide) units, e.g.,
monosaccharides, disaccharides and polysaccharides. Saccharides
that are suitable for use as a tonicity agent in the present
invention, include, but are not limited to, saccharides selected
from the group consisting of fructose, glucose, mannose, sorbose,
xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin,
cyclodextrins, soluble starch, hydroxyethyl starch, water-soluble
glucans, and mixtures thereof. In one embodiment, the tonicity
agent is sucrose.
[0098] In another embodiment, the tonicity agent is a polyol. As
used herein, the term "polyol" refers an excipient with multiple
hydroxyl groups, and includes sugars (reducing and nonreducing
sugars), sugar alcohols and sugar acids. In one embodiment, the
polyol has a molecular weight that is less than about 600 kD (e.g.,
in the range from about 120 to about 400 kD). A "reducing sugar" is
one which contains a hemiacetal group that can reduce metal ions or
react covalently with lysine and other amino groups in proteins and
a "nonreducing sugar" is one which does not have these properties
of a reducing sugar. Polyols that are suitable for use as a
tonicity agent in the present invention, include, but are not
limited to, polyols selected from the group consisting of mannitol,
trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol,
xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol,
inositol, and mixtures thereof. In one embodiment, the tonicity
agent is a non-reducing sugar selected from the group consisting of
sucrose, trehalose, sorbose, melezitose, raffinose, and mixtures
thereof. In another embodiment, the tonicity agent is a
non-reducing sugar selected from the group consisting of trehalose
and sucrose, and mixtures thereof. In another embodiment, the
tonicity agent is mannitol. In another embodiment, the tonicity
agent is D-mannitol. In another embodiment, the tonicity agent is
trehalose. In another embodiment, the tonicity agent is a
.alpha.-trehalose dihydrate. In another embodiment, the tonicity
agent is a salt, such as sodium chloride.
[0099] The concentration of the tonicity agent when present in the
liquid pharmaceutical composition can range from about 1.0
millimolar to about 600 millimolar, from about 10 millimolar to
about 400 millimolar, or from about 100 millimolar to about 300
millimolar. In one embodiment, the concentration of the tonicity
agent ranges from about 200 millimolar to about 250 millimolar. In
another embodiment, the concentration of the tonicity agent in the
liquid pharmaceutical composition is about 222 millimolar, about
238 millimolar, or about 247 millimolar.
[0100] In yet another embodiment, the tonicity agent is mannitol
and is present in the liquid pharmaceutical composition at a
concentration of 247 millimolar. In another embodiment, the
tonicity agent is trehalose and is present in the liquid
pharmaceutical composition at a concentration of 222 millimolar. In
another embodiment, the tonicity agent is trehalose and is present
in the liquid pharmaceutical composition at a concentration of 238
millimolar.
[0101] As noted above, the liquid pharmaceutical compositions of
the present invention optionally may further comprise a
pharmaceutically acceptable surfactant in addition to a chelating
agent. As used herein, the term "surfactant" refers to an excipient
that can alter the surface tension of a liquid antibody
composition. In certain embodiments, the surfactant reduces the
surface tension of a liquid antibody composition. In still other
embodiments, the "surfactant" may contribute to an improvement in
stability of any of the anti-MAdCAM antibodies described herein.
For example, the surfactant may reduce aggregation of the
formulated antibody and/or minimize the formation of particulates
in the composition and/or reduces adsorption. The surfactant may
also improve stability of the antibody during and after a
freeze/thaw cycle.
[0102] Suitable surfactants include polysorbate surfactants,
poloxamer 18, triton surfactants such as Triton X-100.RTM.,
polysorbate surfactants such as Tween 20.RTM. and Tween 80.RTM.,
sodium dodecyl sulfate, sodium laurel sulfate, sodium octyl
glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine,
linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine,
myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine,
linoleyl-betaine, myristyl-betaine, cetyl-betaine,
lauroamidopropyl-betaine, cocamidopropyl-betaine,
linoleamidopropyl-betaine, myristamidopropyl-betaine,
palmidopropyl-betaine, isostearamidopropyl-betaine,
myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine,
isostearamidopropyl-dimethylamine, sodium methyl cocoyl-taurate,
disodium methyl oleyl-taurate, dihydroxypropyl peg 5 linoleammonium
chloride, polyethylene glycol, polypropylene glycol, and mixtures
thereof.
[0103] In one embodiment, the surfactant is a polysorbate
surfactant comprising at least one excipient that is selected from
the group consisting of polysorbate 20, polysorbate 21, polysorbate
40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80,
polysorbate 81, polysorbate 85, and mixtures thereof. In another
embodiment, the liquid pharmaceutical composition comprises
polysorbate 80.
[0104] The concentration of the surfactant when present in the
liquid pharmaceutical composition can range from about 0.005
millimolar to about 10 millimolar, from about 0.007 millimolar to
about 5.0 millimolar, or from about 0.01 millimolar to about 1.0
millimolar. In another embodiment, the concentration of the
surfactant is from about 0.05 millimolar to about 1.0 millimolar.
In another embodiment, the liquid pharmaceutical composition
contains about 0.30 millimolar polysorbate 80.
[0105] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a chelating
agent.
[0106] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a chelating
agent; and wherein the composition is substantially free of
chloride ions or acetate ions or both.
[0107] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one human
monoclonal anti-MAdCAM antibody, wherein the antibody binds to
human MAdCAM; and a chelating agent.
[0108] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one human
monoclonal anti-MAdCAM antibody, wherein the antibody binds to
human MAdCAM; and a chelating agent; and wherein the composition is
substantially free of chloride ions or acetate ions or both.
[0109] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a
pharmaceutically acceptable excipient, wherein the composition
contains a concentration of antibody that is at least about 50
mg/ml, at least about 60 mg/ml, at least about 70 mg/ml, at least
about 80 mg/ml, at least about 90 mg/ml, or at least about 100
mg/ml.
[0110] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a
pharmaceutically acceptable excipient, wherein the composition
contains a concentration of antibody that ranges from about 10
mg/ml to about 200 mg/ml.
[0111] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a
pharmaceutically acceptable excipient, wherein the composition
contains a concentration of antibody that ranges from about 15
mg/ml to about 200 mg/ml.
[0112] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a
pharmaceutically acceptable excipient, wherein the composition
contains a concentration of antibody that ranges from about 20
mg/ml to about 200 mg/ml.
[0113] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a
pharmaceutically acceptable excipient, wherein the composition
contains a concentration of antibody that ranges from about 50
mg/ml to about 200 mg/ml.
[0114] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a
pharmaceutically acceptable excipient, wherein the composition
contains a concentration of antibody that ranges from about 100
mg/ml to about 200 mg/ml.
[0115] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MAdCAM; and a
pharmaceutically acceptable excipient, wherein the composition
contains a concentration of antibody that ranges from about 65
mg/ml to about 85 mg/ml.
[0116] In one embodiment, the present invention encompasses a
liquid pharmaceutical composition comprising at least one antibody
comprising an amino acid sequence that is at least 95% identical to
a heavy chain amino acid sequence shown in SEQ ID NO: 2, and
further comprising an amino acid sequence that is at least 95%
identical to a light chain amino acid sequence shown in SEQ ID NO:
4, wherein the antibody binds to human MADCAM; and a
pharmaceutically acceptable excipient, wherein the composition
contains a concentration of antibody that is about 75 mg/ml.
[0117] In one embodiment, the liquid pharmaceutical composition
comprises from about 0.01 mg/ml to about 200 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and from about 0.3 micromolar to about
50 millimolar of chelating agent.
[0118] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and from about 30 micromolar to about
5.0 millimolar of chelating agent.
[0119] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and about 0.27 millimolar of chelating
agent.
[0120] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and from about 0.3 micromolar to about
50 millimolar of EDTA.
[0121] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and from about 30 micromolar to about
10.0 millimolar of EDTA.
[0122] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and from about 0.1 millimolar to about
1.0 millimolar of EDTA.
[0123] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and about 0.27 millimolar of EDTA.
[0124] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and from about 30 micromolar to about
5.0 millimolar of DTPA.
[0125] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 100 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and from about 30 micromolar to about
5.0 millimolar of deferoxamine.
[0126] In one embodiment, the liquid pharmaceutical composition
comprises from about 0.01 mg/ml to about 200 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; and from about 1 mM to about 100 mM of
histidine.
[0127] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; from about 30 micromolar to about 5.0
millimolar of chelating agent; and from about 1 mM to about 100 mM
of histidine.
[0128] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; from about 30 micromolar to about 5.0
millimolar of chelating agent; and from about 10 millimolar to
about 400 millimolar of trehalose.
[0129] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; from about 30 micromolar to about 5.0
millimolar of chelating agent; from about 10 millimolar to about
400 millimolar of trehalose; and from about 1 mM to about 100 mM of
histidine.
[0130] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; from about 30 micromolar to about 5.0
millimolar of chelating agent; from about 10 millimolar to about
400 millimolar of trehalose; from about 1 mM to about 100 mM of
histidine; and from about 0.005 millimolar to about 10 millimolar
of polysorbate 80.
[0131] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; from about 30 micromolar to about 5.0
millimolar of EDTA; from about 10 millimolar to about 400
millimolar of a tonicity agent; from about 1 mM to about 100 mM of
a buffer; and from about 0.005 millimolar to about 10 millimolar of
a surfactant.
[0132] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; from about 30 micromolar to about 5.0
millimolar of EDTA; from about 10 millimolar to about 400
millimolar of a tonicity agent; from about 1 mM to about 100 mM of
histidine; and from about 0.005 millimolar to about 10 millimolar
of a surfactant.
[0133] In another embodiment, the liquid pharmaceutical composition
comprises from about 0.1 mg/ml to about 200 mg/ml of monoclonal
anti-MAdCAM antibody 7.16.6; from about 30 micromolar to about 5.0
millimolar of EDTA; from about 10 millimolar to about 400
millimolar of trehalose; from about 1 mM to about 100 mM of
histidine; and from about 0.005 millimolar to about 10 millimolar
of a surfactant.
[0134] In certain aspects of the present invention, the liquid
anti-MAdCAM antibody compositions comprise from about 10 mg/ml to
about 200 mg/ml of monoclonal anti-MAdCAM antibody 7.16.6; from
about 1 mM to about 100 mM of histidine; from about 0.005
millimolar to about 10 millimolar of polysorbate 80; from about 30
micromolar to about 5.0 millimolar of EDTA; and from about 10
millimolar to about 400 millimolar of trehalose.
[0135] In other aspects of the present invention, the liquid
anti-MAdCAM antibody compositions comprise from about 50 mg/ml to
about 100 mg/ml of monoclonal anti-MAdCAM antibody 7.16.6; from
about 5 mM to about 30 mM of histidine; from about 0.01 millimolar
to about 1.0 millimolar of polysorbate 80; from about 30 micromolar
to about 5.0 millimolar of EDTA; and from about 100 millimolar to
about 300 millimolar of trehalose.
[0136] In other aspects of the present invention, the liquid
anti-MAdCAM antibody compositions comprise from about 65 mg/ml to
about 85 mg/ml of monoclonal anti-MAdCAM antibody 7.16.6; from
about 5 mM to about 15 mM of histidine; from about 0.05 millimolar
to about 0.5 millimolar of polysorbate 80; from about 0.1
millimolar to about 1 millimolar of EDTA; and from about 200
millimolar to about 250 millimolar of trehalose.
[0137] In other aspects of the present invention, the liquid
anti-MAdCAM antibody compositions comprise about 75 mg/ml of
monoclonal anti-MAdCAM antibody 7.16.6; about 20 mM of histidine;
about 0.3 millimolar of polysorbate 80; about 0.27 millimolar of
EDTA; and about 238 millimolar of trehalose.
[0138] In another embodiment, the invention is directed to a stable
liquid pharmaceutical composition comprising an anti-MAdCAM
antibody and a pharmaceutically acceptable chelating agent, wherein
the molar concentration of the antibody ranges from about 0.0006
millimolar to about 1.35 millimolar and the molar concentration of
the chelating agent ranges from about 0.003 millimolar to about 50
millimolar, and wherein the molar ratio of antibody to chelating
agent ranges from about 0.00001 to about 450; from about 0.0001 to
about 100; from about 0.005 to about 50; from about 0.001 to about
10; from about 0.01 to about 5; from about 0.1 to about 3; or
is about 1.9.
[0139] In another embodiment, the invention is directed to a stable
liquid pharmaceutical composition comprising anti-MAdCAM antibody
7.16.6 and a pharmaceutically acceptable chelating agent, wherein
the molar concentration of the antibody ranges from about 0.0006
millimolar to about 1.35 millimolar and the molar concentration of
the chelating agent ranges from about 0.003 millimolar to about 50
millimolar, and wherein the molar ratio of antibody to chelating
agent ranges from about 0.00001 to about 450; from about 0.0001 to
about 100; from about 0.005 to about 50; from about 0.001 to about
10; from about 0.01 to about 5; from about 0.1 to about 3; or is
about 1.9.
[0140] In another embodiment, the invention is directed to a stable
liquid pharmaceutical composition comprising anti-MAdCAM antibody
7.16.6, a pharmaceutically acceptable chelating agent, and
histidine; wherein the molar concentration of the antibody ranges
from about 0.0006 millimolar to about 1.35 millimolar, the molar
concentration of the chelating agent ranges from about 0.003
millimolar to about 50 millimolar, and the molar concentration of
histidine ranges from about 1 millimolar to about 100 millimolar;
and wherein the molar ratio of antibody to chelating agent ranges
from about 0.00001 to about 450; from about 0.0001 to about 100;
from about 0.005 to about 50; from about 0.001 to about 10; from
about 0.01 to about 5; from about 0.1 to about 1; or is about
0.5.
[0141] In another embodiment, the invention is directed to a stable
liquid pharmaceutical composition comprising anti-MAdCAM antibody
7.16.6, a pharmaceutically acceptable chelating agent, and
histidine; wherein the molar concentration of the antibody ranges
from about 0.0006 millimolar to about 1.35 millimolar, the molar
concentration of the chelating agent ranges from about 0.003
millimolar to about 50 millimolar, and the molar concentration of
histidine ranges from about 5 millimolar to about 30 millimolar;
and wherein the molar ratio of antibody to chelating agent ranges
from about 0.0001 to about 100; from about 0.005 to about 50; from
about 0.001 to about 10; from about 0.01 to about 5; from about 0.1
to about 3; or is about 1.9.
[0142] In another embodiment, the invention is directed to a stable
liquid pharmaceutical composition comprising anti-MAdCAM antibody
7.16.6, a pharmaceutically acceptable chelating agent, and
histidine; wherein the molar concentration of the antibody ranges
from about 0.0006 millimolar to about 1.35 millimolar, the molar
concentration of the chelating agent ranges from about 0.003
millimolar to about 50 millimolar, and the molar concentration of
histidine ranges from about 5 millimolar to about 20 millimolar;
and wherein the molar ratio of antibody to chelating agent ranges
from about 0.005 to about 50; from about 0.001 to about 10; from
about 0.01 to about 5; from about 0.1 to about 3; or is about
1.9.
[0143] In another embodiment, the invention is directed to a stable
liquid pharmaceutical composition comprising anti-MAdCAM antibody
7.16.6, a pharmaceutically acceptable chelating agent, and
histidine; wherein the molar concentration of the antibody ranges
from about 0.0006 millimolar to about 1.35 millimolar, the molar
concentration of the chelating agent ranges from about 0.003
millimolar to about 50 millimolar, and the molar concentration of
histidine ranges from about 5 millimolar to about 20 millimolar;
and wherein the molar ratio of antibody to chelating agent ranges
from about 0.001 to about 10; from about 0.01 to about 5; from
about 0.1 to about 3; or is about 1.9.
[0144] In another embodiment, the invention is directed to a stable
liquid pharmaceutical composition comprising anti-MAdCAM antibody
7.16.6, a pharmaceutically acceptable chelating agent, and
histidine; wherein the molar concentration of the antibody ranges
from about 0.0006 millimolar to about 1.35 millimolar, the molar
concentration of the chelating agent ranges from about 0.003
millimolar to about 50 millimolar, and the molar concentration of
histidine is about 20 millimolar; and wherein the molar ratio of
antibody to chelating agent ranges from about 0.001 to about 10;
from about 0.01 to about 5; from about 0.1 to about 3; or is about
1.9.
Methods of Producing Anti-MAdCAM Antibodies and Antibody Producing
Cell Lines:
[0145] Antibodies in accordance with the invention can be prepared
through the utilization of a transgenic mouse that has a
substantial portion of the human antibody producing genome
inserted, but that is rendered deficient in the production of
endogenous, murine, antibodies. Such mice, then, are capable of
producing human immunoglobulin molecules and antibodies and are
deficient in the production of murine immunoglobulin molecules and
antibodies. Technologies utilized for achieving the same are
discussed below.
[0146] It is possible to produce transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. In particular, however, one embodiment of transgenic
production of mice and antibodies therefrom is disclosed in
International Application Number PCT/US2005/000370. Through use of
such technology, antibodies that bind to MAdCAM and hybridomas
producing such antibodies can be prepared.
[0147] Human antibodies avoid certain of the problems associated
with antibodies that possess murine or rat variable and/or constant
regions. The presence of such murine or rat derived proteins can
lead to the rapid clearance of the antibodies or can lead to the
generation of an immune response against the antibody by a subject
that receives administration of such antibodies.
[0148] For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (J.sub.H) gene
in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array in such germ-line mutant mice
will result in the production of human antibodies upon antigen
(e.g., MAdCAM) challenge. See, e.g., Jakobovits et al, Proc. Natl.
Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,
362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33
(1993); and Duchosal et al., Nature 355:258 (1992). Human
antibodies can also be derived from phage-display libraries
(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J.
Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech
14:309 (1996)).
[0149] In some embodiments, human anti-MAdCAM antibodies can be
produced by immunizing a non-human transgenic animal, e.g.,
XENOMOUSE.TM. mice, whose genome comprises human immunoglobulin
genes so that the recombinant mouse produces human antibodies.
XENOMOUSE.TM. mice are engineered mouse strains that comprise large
fragments of human immunoglobulin heavy chain and light chain loci
and are deficient in mouse antibody production. XENOMOUSE.TM. mice
produce an adult-like human repertoire of fully human antibodies
and generate antigen-specific human antibodies. In some
embodiments, the XENOMOUSE.TM. mice contain approximately 80% of
the human antibody V gene repertoire through introduction of
megabase sized, germline configuration yeast artificial chromosome
(YAC) fragments of the human heavy chain loci and kappa light chain
loci. In other embodiments, XENOMOUSE.TM. mice further contain
approximately all of the lambda light chain locus. See, e.g., Green
et al., Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos.
5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001,
6,114,598, 6,130,364, 6,162,963 and 6,150,584. See also WO
91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO
98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00/09560, and
WO 00/037504.
[0150] In some embodiments, the non-human animal comprising human
immunoglobulin genes are animals that have a human immunoglobulin
"minilocus". In the minilocus approach, an exogenous Ig locus is
mimicked through the inclusion of individual genes from the Ig
locus. Thus, one or more V.sub.H genes, one or more D.sub.H genes,
one or more J.sub.H genes, a mu constant domain, and a second
constant domain (preferably a gamma constant domain) are formed
into a construct for insertion into an animal. This approach is
described, inter alia, in U.S. Pat. Nos. 5,545,807, 5,545,806,
5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650,
5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, and
5,643,763.
[0151] Therefore, in some embodiments, human antibodies can be
produced by immunizing a non-human animal comprising in its genome
some or all of human immunoglobulin heavy chain and light chain
loci with a MAdCAM antigen.
[0152] In some embodiments, the MAdCAM antigen is isolated and/or
purified MAdCAM. In a preferred embodiment, the MAdCAM antigen is
human MAdCAM. In some embodiments, the MAdCAM antigen is a fragment
of MAdCAM. In some embodiments, the MAdCAM fragment comprises at
least one epitope of MAdCAM. In other embodiments, the MAdCAM
antigen is a cell that expresses or overexpresses MAdCAM or an
immunogenic fragment thereof on its surface. In still other
embodiments, the MAdCAM antigen is a MAdCAM fusion protein. MAdCAM
can be purified from natural sources using known techniques. In
addition, recombinant MAdCAM protein is commercially available.
[0153] In a preferred embodiment, the non-human animal is a
XENOMOUSE.TM. animal (Abgenix Inc., Fremont, Calif.). Another
non-human animal that may be used is a transgenic mouse produced by
Medarex (Medarex, Inc., Princeton, N.J.).
[0154] Immunization of animals can be by any method known in the
art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,
New York: Cold Spring Harbor Press, 1990. Methods for immunizing
non-human animals such as mice, rats, sheep, goats, pigs, cattle
and horses are well known in the art. See, e.g., Harlow and Lane,
supra, and U.S. Pat. No. 5,994,619. In a preferred embodiment, the
MAdCAM antigen is administered with an adjuvant to stimulate the
immune response. Exemplary adjuvants include complete or incomplete
Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM
(immunostimulating complexes). Such adjuvants may protect the
polypeptide from rapid dispersal by sequestering it in a local
deposit, or they may contain substances that stimulate the host to
secrete factors that are chemotactic for macrophages and other
components of the immune system. Preferably, if a polypeptide is
being administered, the immunization schedule can involve two or
more administrations of the polypeptide, spread out over several
weeks.
[0155] After immunization of an animal with a MAdCAM antigen,
antibodies and/or antibody-producing cells can be obtained from the
animal. In some embodiments, anti-MAdCAM antibody-containing serum
is obtained from the animal by bleeding or sacrificing the animal.
The serum may be used as it is obtained from the animal, an
immunoglobulin fraction may be obtained from the serum, or the
anti-MAdCAM antibodies may be purified from the serum.
[0156] In some embodiments, antibody-producing immortalized cell
lines are prepared from cells isolated from the immunized animal.
After immunization, the animal is sacrificed and lymph node and/or
splenic B cells are immortalized. Methods of immortalizing cells
include, but are not limited to, transfecting them with oncogenes,
infecting them with an oncogenic virus, cultivating them under
conditions that select for immortalized cells, subjecting them to
carcinogenic or mutating compounds, fusing them with an
immortalized cell, e.g., a myeloma cell, and inactivating a tumor
suppressor gene. See, e.g., Harlow and Lane, supra. In a preferred
embodiment, the immunized animal is a non-human animal that
expresses human immunoglobulin genes and the splenic B cells are
fused to a myeloma cell line from the same species as the non-human
animal. In a more preferred embodiment, the immunized animal is a
XENOMOUSE.TM. animal and the myeloma cell line is a non-secretory
mouse myeloma. If fusion with myeloma cells is used, the myeloma
cells preferably do not secrete immunoglobulin polypeptides (a
non-secretory cell line). Immortalized cells are screened using
MAdCAM, a portion thereof, or a cell expressing MAdCAM. In a
preferred embodiment, the initial screening is performed using an
enzyme-linked immunoassay (ELISA) or a radioimmunoassay. An example
of ELISA screening is provided in WO 00/37504.
[0157] Anti-MAdCAM antibody-producing cells, e.g., hybridomas, are
selected, cloned and further screened for desirable
characteristics, including robust growth, high antibody production
and desirable antibody characteristics, as discussed further below.
Hybridomas can be expanded in vivo in syngeneic animals, in animals
that lack an immune system, e.g., nude mice, or in cell culture in
vitro. Methods of selecting, cloning and expanding hybridomas are
well known to those of ordinary skill in the art.
[0158] As will be appreciated, antibodies in accordance with the
present invention can be recombinantly expressed in cell lines
other than hybridoma cell lines. Nucleic acid sequences encoding
the cDNAs or genomic clones for the particular antibodies can be
used for transformation of a suitable mammalian or nonmammalian
host cells.
[0159] The present invention also encompasses nucleic acid
molecules encoding anti-MAdCAM antibodies. In some embodiments,
different nucleic acid molecules encode a heavy chain and a light
chain of an anti-MAdCAM immunoglobulin. In other embodiments, the
same nucleic acid molecule encodes a heavy chain and a light chain
of an anti-MAdCAM immunoglobulin. In one embodiment, the nucleic
acid encodes an anti-MAdCAM antibody of the invention.
[0160] A nucleic acid molecule encoding the heavy or entire light
chain of an anti-MAdCAM antibody or portions thereof can be
isolated from any source that produces such antibody. In various
embodiments, the nucleic acid molecules are isolated from a B cell
isolated from an animal immunized with anti-MAdCAM or from an
immortalized cell derived from such a B cell that expresses an
anti-MAdCAM antibody. Methods of isolating mRNA encoding an
antibody are well-known in the art. See, e.g., Sambrook. et al.,
Molecular Cloning 3rd Ed. Vol. 3 (1989). The mRNA may be used to
produce cDNA for use in the polymerase chain reaction (PCR) or cDNA
cloning of antibody genes. In a preferred embodiment, the nucleic
acid molecule is isolated from a hybridoma that has as one of its
fusion partners a human immunoglobulin-producing cell from a
non-human transgenic animal. In an even more preferred embodiment,
the human immunoglobulin producing cell is isolated from a
XENOMOUSE.TM. animal. In another embodiment, the human
immunoglobulin-producing cell is from a non-human, non-mouse
transgenic animal, as described above. In another embodiment, the
nucleic acid is isolated from a non-human, non-transgenic animal.
The nucleic acid molecules isolated from a non-human animal may be
used, e.g., for humanized antibodies.
[0161] In some embodiments, a nucleic acid encoding a heavy chain
of an anti-MAdCAM antibody of the invention can comprise a
nucleotide sequence encoding a V.sub.H domain of the invention
joined in-frame to a nucleotide sequence encoding a heavy chain
constant domain from any source. Similarly, a nucleic acid molecule
encoding a light chain of an anti-MAdCAM antibody of the invention
can comprise a nucleotide sequence encoding a V.sub.L domain of the
invention joined in-frame to a nucleotide sequence encoding a light
chain constant domain from any source.
[0162] In a further aspect of the invention, nucleic acid molecules
encoding the variable domain of the heavy (V.sub.H) and light
(V.sub.L) chains are "converted" to full-length antibody genes. In
one embodiment, nucleic acid molecules encoding the V.sub.H or
V.sub.L domains are converted to full-length antibody genes by
insertion into an expression vector already encoding heavy chain
constant (C.sub.H) or light chain (C.sub.L) constant domains,
respectively, such that the V.sub.H segment is operatively linked
to the C.sub.H segment(s) within the vector, and the V.sub.L
segment is operatively linked to the C.sub.L segment within the
vector. In another embodiment, nucleic acid molecules encoding the
V.sub.H and/or V.sub.L domains are converted into full-length
antibody genes by linking, e.g., ligating, a nucleic acid molecule
encoding a V.sub.H and/or V.sub.L domains to a nucleic acid
molecule encoding a C.sub.H and/or C.sub.L domain using standard
molecular biological techniques. Nucleic acid sequences of human
heavy and light chain immunoglobulin constant domain genes are
known in the art. See, e.g., Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed., NIH Publ. No. 91-3242, 1991.
Nucleic acid molecules encoding the full-length heavy and/or light
chains may then be expressed from a cell into which they have been
introduced and the anti-MAdCAM antibody isolated.
[0163] The present invention also provides vectors comprising
nucleic acid molecules that encode the heavy chain of an
anti-MAdCAM antibody of the invention or an antigen-binding portion
thereof. The invention also provides vectors comprising nucleic
acid molecules that encode the light chain of such antibodies or
antigen-binding portion thereof. The invention further provides
vectors comprising nucleic acid molecules encoding fusion proteins,
modified antibodies, antibody fragments, and probes thereof.
[0164] In some embodiments, the anti-MAdCAM antibodies, or
antigen-binding portions of the invention are expressed by
inserting DNAs encoding partial or full-length light and heavy
chains, obtained as described above, into expression vectors such
that the genes are operatively linked to necessary expression
control sequences such as transcriptional and translational control
sequences. Expression vectors include plasmids, retroviruses,
adenoviruses, adeno-associated viruses (AAV), plant viruses such as
cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV
derived episomes, and the like. The antibody gene is ligated into a
vector such that transcriptional and translational control
sequences within the vector serve their intended function of
regulating the transcription and translation of the antibody gene.
The expression vector and expression control sequences are chosen
to be compatible with the expression host cell used. The antibody
light chain gene and the antibody heavy chain gene can be inserted
into separate vectors. In a preferred embodiment, both genes are
inserted into the same expression vector. The antibody genes are
inserted into the expression vector by standard methods (e.g.,
ligation of complementary restriction sites on the antibody gene
fragment and vector, or blunt end ligation if no restriction sites
are present).
[0165] A convenient vector is one that encodes a functionally
complete human C.sub.H or C.sub.L immunoglobulin sequence, with
appropriate restriction sites engineered so that any V.sub.H or
V.sub.L sequence can easily be inserted and expressed, as described
above. In such vectors, splicing usually occurs between the splice
donor site in the inserted J region and the splice acceptor site
preceding the human C domain, and also at the splice regions that
occur within the human C.sub.H exons. Polyadenylation and
transcription termination occur at native chromosomal sites
downstream of the coding regions. The recombinant expression vector
also can encode a signal peptide that facilitates secretion of the
antibody chain from a host cell. The antibody chain gene may be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the immunoglobulin chain. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0166] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
It will be appreciated by those skilled in the art that the design
of the expression vector, including the selection of regulatory
sequences may depend on such factors as the choice of the host cell
to be transformed, the level of expression of protein desired, etc.
Preferred regulatory sequences for mammalian host cell expression
include viral elements that direct high levels of protein
expression in mammalian cells, such as promoters and/or enhancers
derived from retroviruses (such as retroviral LTRs),
cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian
Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus,
(e.g., the adenovirus major late promoter (AdMLP)), polyoma and
strong mammalian promoters such as native immunoglobulin and actin
promoters. For further description of viral regulatory elements,
and sequences thereof, see e.g., U.S. Pat. No. 5,168,062, U.S. Pat.
No. 4,510,245 and U.S. Pat. No. 4,968,615. Methods for expressing
antibodies in plants, including a description of promoters and
vectors, as well as transformation of plants is known in the art.
See, e.g., U.S. Pat. Nos. 6,517,529, herein incorporated by
reference. Methods of expressing polypeptides in bacterial cells or
fungal cells, e.g., yeast cells, are also well known in the
art.
[0167] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification), the neomycin resistance
gene (for G418 selection), and the glutamate synthetase gene.
[0168] Nucleic acid molecules encoding anti-MAdCAM antibodies and
vectors comprising these nucleic acid molecules can be used for
transformation of a suitable mammalian, plant, bacterial or yeast
host cell. Antibodies of the invention can be produced
transgenically through the generation of a mammal or plant that is
transgenic for the immunoglobulin heavy and light chain sequences
of interest and production of the antibody in a recoverable form
therefrom.
[0169] Transformation can be by any known method for introducing
polynucleotides into a host cell, including, for example packaging
the polynucleotide in a virus (or into a viral vector) and
transducing a host cell with the virus (or vector) or by
transfection procedures known in the art, as exemplified by U.S.
Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. The
transformation procedure used depends upon the host to be
transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are well known in the art and
include, but are not limited to, dextran-mediated transfection,
calcium phosphate precipitation, polybrene mediated transfection,
protoplast fusion, electroporation, particle bombardment,
encapsulation of the polynucleotide(s) in liposomes, peptide
conjugates, dendrimers, and direct microinjection of the DNA into
nuclei.
[0170] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells, NS0
cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney
cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),
and a number of other cell lines. Non-mammalian cells including but
not limited to bacterial, yeast, insect, and plants can also be
used to express recombinant antibodies. Site directed mutagenesis
of the antibody CH2 domain to eliminate glycosylation may be
preferred in order to prevent changes in either the immunogenicity,
pharmacokinetic, and/or effector functions resulting from non-human
glycosylation. The expression methods are selected by determining
which system generates the highest expression levels and produce
antibodies with constitutive MAdCAM binding properties.
[0171] Further, expression of antibodies of the invention (or other
moieties therefrom) from production cell lines can be enhanced
using a number of known techniques. For example, the glutamine
synthetase and DHFR gene expression systems are common approaches
for enhancing expression under certain conditions. High expressing
cell clones can be identified using conventional techniques, such
as limited dilution cloning and Microdrop technology. The Glutamine
Synthetase system is discussed in whole or part in connection with
European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and
European Patent Application No. 89303964.4.
[0172] In connection with the transgenic production in mammals,
antibodies can also be produced in, and recovered from, the milk of
goats, cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690,
5,756,687, 5,750,172, and 5,741,957.
[0173] The anti-MAdCAM antibodies expressed in cell lines as
described above may be purified and/or isolated from the associated
cellular material. The antibodies may be present in whole cells, in
a cell lysate, or in a partially purified or substantially pure
form. Purification is performed in order to eliminate other
cellular components or other contaminants, e.g. other cellular
nucleic acids or proteins, by standard techniques, including
alkaline/SDS treatment, column chromatography and others well known
in the art. See Ausubel, F., et al., ed. Current Protocols in
Molecular Biology, Greene Publishing and Wiley Interscience, New
York (1987).
[0174] In the present invention, it is possible that the
anti-MAdCAM antibodies of the present invention expressed by
different cell lines or in transgenic animals will have different
glycosylation patterns from each other. However, all of the
anti-MAdCAM antibodies encoded by the nucleic acids and amino acids
provided herein are considered part of the instant invention,
regardless of their glycosylation pattern or modification or
deletion thereof.
[0175] As used herein, the term "glycosylation" means the pattern
of carbohydrate units that are covalently attached to an antibody.
When it is said that the anti-MAdCAM antibodies herein have a
particular glycosylation pattern, it is meant that the majority of
the referenced anti-MAdCAM antibodies have that particular
glycosylation pattern. In other aspects, when it is said that the
anti-MAdCAM antibodies herein have a particular glycosylation
pattern, it is meant that greater than or equal to 50%, 75%, 90%,
95%, 99% or 100% of the referenced anti-MAdCAM antibodies have that
particular glycosylation pattern.
[0176] The anti-MAdCAM antibodies of the present invention also
encompass glycosylation variants thereof (e.g., by insertion of a
glycosylation site or deletion of any glycosylation site by
deletion, insertion or substitution of suitable amino acid
residues). For purposes of the present invention, the anti-MAdCAM
antibodies may be glycosylated or non-glycosylated. When the
anti-MAdCAM antibodies are glycosylated they may have any possible
glycosylation pattern. Moreover, each heavy chain within one
antibody may have the same glycosylation pattern or the two heavy
chains may have differing glycosylation patterns.
Routes of Administration and Dosages:
[0177] The compositions of this invention may be in liquid
solutions (e.g., injectable and infusible solutions). The preferred
form depends on the intended mode of administration and therapeutic
application. Typical preferred compositions are in the form of
injectable or infusible solutions, such as compositions similar to
those used for passive immunization of humans. The preferred mode
of administration is parenteral (e.g., intravenous, subcutaneous,
intradermal, intraperitoneal, intramuscular, and intrasternally) or
by infusion techniques, in the form of sterile injectable liquid or
olagenous suspensions. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. In a preferred embodiment, the
antibody is administered by intravenous infusion or injection. In
another preferred embodiment, the antibody is administered by
intramuscular, subcutaneous or intradermal injection.
[0178] Therapeutic compositions typically are sterile and stable
under the conditions of manufacture and storage.
[0179] The composition can be formulated as a solution,
microemulsion, dispersion, or liposome. Sterile injectable
solutions can be prepared by incorporating the anti-MAdCAM antibody
in the required amount in an appropriate diluent with one or a
combination of ingredients enumerated above, as required, followed
by sterilization (e.g., filter sterilization). Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. Such
suspensions may be formulated according to the known art using
those suitable dispersing of wetting agents and suspending agents
or other acceptable agents. The sterile injectable preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed, including
synthetic mono- or diglycerides. In addition, n-3 polyunsaturated
fatty acids may find use in the preparation of injectables.
[0180] In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying that yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. The proper fluidity
of a solution can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants.
[0181] Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin or by
formulating the composition into prolonged absorption forms such
as, depots, liposomes, polymeric microspheres, polymeric gels, and
implants.
[0182] Other methods for administration of the antibodies described
herein include dermal patches that release the medications directly
into a subject's skin. Such patches can contain the antibodies of
the present invention in an optionally buffered, liquid solution,
dissolved and/or dispersed in an adhesive, or dispersed in a
polymer.
[0183] Still other methods for administration of the antibodies
described herein include liquid opthalmological drops for the
eyes.
[0184] The antibody may be administered once, but more preferably
is administered multiple times. For example, the antibody may be
administered from once daily to once every six months or longer.
The administering may be on a schedule such as three times daily,
twice daily, once daily, once every two days, once every three
days, once weekly, once every two weeks, once every month, once
every two months, once every three months and once every six
months.
[0185] The antibody may also be administered continuously via a
minipump. The antibody may be administered once, at least twice or
for at least the period of time until the disease is treated,
palliated or cured. The antibody typically would be administered as
part of a pharmaceutical composition as described supra.
[0186] The compositions of the invention may include a
therapeutically effective amount or a prophylactically effective
amount of an antibody or antigen-binding portion of the invention.
In preparing the composition, the therapeutically effective amount
of the anti-MAdCAM antibody present in the composition can be
determined, for example, by taking into account the desired dose
volumes and mode(s) of administration, the nature and severity of
the condition to be treated, and the age and size of the
subject.
[0187] Exemplary, non-limiting dose ranges for administration of
the pharmaceutical compositions of the present invention to a
subject are from about 0.01 mg/kg to about 200 mg/kg (expressed in
terms of milligrams (mg) of anti-MAdCAM antibody administered per
kilogram (kg) of subject weight), from about 0.1 mg/kg to about 100
mg/kg, from about 1.0 mg/kg to about 50 mg/kg, from about 5.0 mg/kg
to about 20 mg/kg, or about 15 mg/kg. For purposes of the present
invention, an average human subject weighs about 70 kg.
[0188] Ranges intermediate to any of the concentrations cited
herein, e.g., about 6-94 mg/kg, are also intended to be part of
this invention. For example, ranges of values using a combination
of any of the recited values as upper and/or lower limits are
intended to be included.
[0189] Dosage regimens can also be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response) by
administering several divided doses to a subject over time or the
dose can be proportionally reduced or increased as indicated by the
exigencies of the therapeutic situation. It is especially
advantageous to formulate parenteral compositions in dosage unit
form for ease of administration and uniformity of dosage.
[0190] Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the mammalian subjects
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the anti-MAdCAM antibody or portion and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
antibody for the treatment of sensitivity in individuals.
[0191] The liquid compositions of the present invention can be
prepared as unit dosage forms. For example, a unit dosage per vial
may contain from 1 to 1000 milliliters (mls) of different
concentrations of an anti-MAdCAM antibody. In other embodiments, a
unit dosage per vial may contain about 1 ml, 2 ml, 3 ml, 4 ml, 5
ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50
ml or 100 ml of different concentrations of an anti-MAdCAM
antibody. If necessary, these preparations can be adjusted to a
desired concentration by adding a sterile diluent to each vial.
Stability Assessment:
[0192] The present invention comprises stable liquid pharmaceutical
compositions comprising an anti-MAdCAM antibody as described herein
and a pharmaceutically acceptable chelating agent. A stable
composition is desirable to maintain or resist changes in, for
example, product appearance and integrity (including physical or
chemical degradation potentially leading to a reduction in
biological activity). Various analytical techniques and indicators
for measuring protein stability are reported in the literature and
a number of these techniques and indicators are reviewed in Peptide
and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker,
Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery
Rev. 10: 29-90 (1993). In general, the liquid pharmaceutical
compositions of the present invention exhibit improved stability
when subjected to low storage temperatures over a period of time,
and/or when subjected to one or more freeze/thaw cycles.
[0193] In one embodiment, the composition when stored at a
temperature from about 2.degree. C. to about 8.degree. C. for at
least about 12 months, preferably at least about 18 months and more
preferably at least about 24 months, is more stable than an
otherwise identical composition lacking the chelating agent that is
stored under the same conditions for the same time.
[0194] In another embodiment, the composition when stored at a
temperature from about 25.degree. C. to about 30.degree. C. for at
least about 3 months, preferably at least 6 months, and more
preferably at least about 12 months, is more stable than an
otherwise identical composition lacking the chelating agent that is
stored under the same conditions for the same time.
[0195] In another embodiment, the composition when stored at a
temperature of about 40.degree. C. for at least about 1 month,
preferably at least about 2 months, and more preferably at least
about 3 months is more stable than an otherwise identical
composition lacking the chelating agent that is stored under the
same conditions for the same time.
[0196] As used herein, the term "a freeze/thaw cycle" refers to
techniques for using a liquid antibody sample after frozen storage,
wherein the temperature of the sample is lowered to a temperature
of 0.degree. C. or lower in order to freeze the liquid sample, and
then subjecting the sample to a temperature which will restore its
liquid state for a sufficient period of time to permit use of the
sample, followed by and return to frozen storage, preferably at a
temperature of 0.degree. C. or lower. As used herein, the term
"frozen storage" refers to freezing and maintaining a previously
liquid antibody sample at a temperature of 0.degree. C. or below,
and preferably -20.degree. C. or lower.
[0197] In one embodiment, the composition when subjected to at
least 1 freeze/thaw cycle, preferably at least 2 freeze/thaw
cycles, more preferably at least 3 freeze/thaw cycles, still more
preferably at least 4 freeze/thaw cycles, still more preferably at
least 5 freeze/thaw cycles, and still more preferably at least 6
freeze/thaw cycles, is more stable than an otherwise identical
composition lacking the chelating agent that is subjected to the
same freeze/thaw conditions.
[0198] In another embodiment, the composition satisfies two or more
of the following conditions:
[0199] (a) the composition when stored at a temperature from about
2.degree. C. to about 8.degree. C. for at least about 12 months,
preferably at least about 18 months and more preferably at least
about 24 months, is more stable than an otherwise identical
composition lacking the chelating agent that is stored under the
same conditions for the same time;
[0200] (b) the composition when stored at a temperature from about
25.degree. C. to about 30.degree. C. for at least about 3 months,
preferably at least 6 months, and more preferably at least about 12
months, is more stable than an otherwise identical composition
lacking the chelating agent that is stored under the same
conditions for the same time;
[0201] (c) the composition when stored at a temperature of about
40.degree. C. for at least about 1 month, preferably at least about
2 months, and more preferably at least about 3 months is more
stable than an otherwise identical composition lacking the
chelating agent that is stored under the same conditions for the
same time; or
[0202] (d) the composition when subjected to at least 1 freeze/thaw
cycle, preferably at least 2 freeze/thaw cycles, more preferably at
least 3 freeze/thaw cycles, still more preferably at least 4
freeze/thaw cycles, still more preferably at least 5 freeze/thaw
cycles, and still more preferably at least 6 freeze/thaw cycles, is
more stable than an otherwise identical composition lacking the
chelating agent that is subjected to the same freeze/thaw
conditions.
[0203] In another embodiment, the composition satisfies three or
more of the conditions discussed immediately above.
[0204] For purposes of the present application, antibody
aggregation, antibody fragmentation, and/or composition
discoloration, for example, can be used as indicators of the
stability of the composition. In general, the liquid pharmaceutical
compositions of the present invention exhibit a lower level of at
least one of antibody aggregation, antibody fragmentation and
composition discoloration when subjected to one or more of the
above-described storage or freeze/thaw conditions relative to
otherwise identical compositions lacking the chelating agent that
are subjected to the same conditions.
[0205] Protein aggregation in a liquid pharmaceutical composition
can be measured by various methods known in the art. Such methods
include gel filtration chromatography to separate proteins on the
basis of their molecular weight. A "gel" is a matrix of water and a
polymer, such as agarose or polymerized acrylamide. The present
invention also encompasses the use of gel filtration HPLC (high
performance liquid chromatography). Other recognized methods of
measuring aggregation include cation exchange chromatography, which
is the general liquid chromatographic technique of ion-exchange
chromatography utilizing anion columns. The cations exchanged in
the present invention are from the protein molecules. Since
multivalent protein aggregates may have some multiple of the net
charge of the monomer of the antigen-binding protein, the
aggregates can be retained more strongly, and may be separated from
the monomer molecules. A preferred cationic exchanger is a
polyaspartic acid column. Thus, a monomeric protein can be readily
distinguished from an aggregate. However, those of ordinary skill
in the art will realize that aggregation assays of the invention
are not limited to any particular type of chromatography column, so
long as it is capable of separating the two forms of protein
molecules.
[0206] Protein fragmentation in a liquid pharmaceutical composition
can be measured by various methods known in the art. Such methods
include, for example, size exclusion chromatography, ultraviolet
detection (e.g., at 214 nanometers), SDS-PAGE and/or
matrix-assisted laser desorption ionization/time-of-flight mass
spectrometry (MALDI/TOF MS). Protein fragmentation resulting in a
charge alteration (e.g., occurring as a result of deamidation) can
be evaluated, for example, by ion-exchange chromatography or
isoelectric focusing (IEF).
[0207] Composition discoloration generally can be measured by
visual observation of the composition itself. The present liquid
pharmaceutical compositions comprising a chelating agent generally
reduce composition discoloration (e.g., pink or yellow) and/or
maintain composition clarity (e.g., turbidity, cloudiness and/or
particulate formation) relative to otherwise identical compositions
that do not contain the chelating agent. For purposes of the
present invention, the term "discoloration" refers to both changes
in color (e.g., from clear and colorless to pink or yellow) and to
changes in clarity (e.g., from clear and colorless to turbid,
cloudy and/or having particulates). Composition discoloration
generally can be measured using additional techniques such as by
ultraviolet detection at 214 nanometers, detection at other
wavelengths such as in the visible/near-UV range and/or by visual
comparison against a standard color scale of the compositions with
and without the chelating agent. See PhEur 5.0, 2005 Monograph
2.2.2.
[0208] In one embodiment, antibody aggregation is determined after
the composition is subjected to at least one of the following
conditions:
[0209] (a) the composition is stored at a temperature from about
2.degree. C. to about 8.degree. C. for at least about 12 months,
preferably at least about 18 months and more preferably at least
about 24 months;
[0210] (b) the composition is stored at a temperature from about
25.degree. C. to about 30.degree. C. for at least about 3 months,
preferably at least 6 months, and more preferably at least about 12
months;
[0211] (c) the composition is stored at a temperature of about
40.degree. C. for at least about 1 month, preferably at least about
2 months, and more preferably at least about 3 months; or
[0212] (d) the composition is subjected to at least 1 freeze/thaw
cycle, preferably at least 2 freeze/thaw cycles, more preferably at
least 3 freeze/thaw cycles, still more preferably at least 4
freeze/thaw cycles, still more preferably at least 5 freeze/thaw
cycles, and still more preferably at least 6 freeze/thaw cycles.
Antibody aggregates are then chromatographically separated from the
monomers (e.g., using HPLC) and the extent of aggregation
determined from the resulting chromatogram. The stable liquid
pharmaceutical compositions of the present invention typically have
an aggregate peak area on the chromatogram that is less than about
6%, less than about 5%, less than about 4%, less than about 3%,
less than about 2%, or less than about 1.5% of the total peak area
on the chromatogram. In one specific example of this technique for
measuring aggregation, the composition is stored for 24 weeks at
40.degree. C. and chromatographic separation is then conducted
using SE-HPLC with ultraviolet detection at 214 nanometers.
[0213] In general, the difference between the aggregate
chromatogram peak area for a stable liquid pharmaceutical
composition of the present invention and the aggregate chromatogram
peak area for an otherwise identical composition lacking the
chelating agent that is subjected to the same conditions is at
least about 2%, at least about 3%, at least about 4%, or at least
about 4.5%.
[0214] In another embodiment, antibody fragmentation is determined
after the composition is subjected to at least one of the following
conditions:
[0215] (a) the composition is stored at a temperature from about
2.degree. C. to about 8.degree. C. for at least about 12 months,
preferably at least about 18 months and more preferably at least
about 24 months;
[0216] (b) the composition is stored at a temperature from about
25.degree. C. to about 30.degree. C. for at least about 3 months,
preferably at least 6 months, and more preferably at least about 12
months;
[0217] (c) the composition is stored at a temperature of about
40.degree. C. for at least about 1 month, preferably at least about
2 months, and more preferably at least about 3 months; or
[0218] (d) the composition is subjected to at least 1 freeze/thaw
cycle, preferably at least 2 freeze/thaw cycles, more preferably at
least 3 freeze/thaw cycles, still more preferably at least 4
freeze/thaw cycles, still more preferably at least 5 freeze/thaw
cycles, and still more preferably at least 6 freeze/thaw cycles.
Antibody fragments are then electrophoretically separated from the
composition (e.g., SDS-PAGE) and the extent of fragmentation
determined from the resulting electrophorogram or gel images. The
stable liquid pharmaceutical compositions of the present invention
typically have a fragment band volume on the SDS-PAGE gel that is
less than about 9%, less than about 8%, less than about 7%, less
than about 6%, less than about 5%, or less than about 4.5% of the
total band volume on the gel. In one specific example of this
technique for measuring fragmentation, the composition is stored
for 24 weeks at 40.degree. C. and then analyzed using reduced
SDS-PAGE (rSDS-PAGE) with band volumes determined by scanning with
either a Molecular Dynamics Personal Densitometer PDQC-90 or a
Bio-Rad GS800 Imaging Densitometer.
[0219] In general, the difference between the fragment band volume
for a stable liquid pharmaceutical composition of the present
invention and the fragment band volume for an otherwise identical
composition lacking the chelating agent that is subjected to the
same conditions is at least about 2%, at least about 3%, at least
about 4%, or at least about 5%.
Methods of Prevention/Treatment:
[0220] Any of the types of antibodies described herein may be used
therapeutically. In a preferred embodiment, the anti-MAdCAM
antibody is a human antibody. In another preferred embodiment, the
MAdCAM is human and the subject is a human subject. In yet another
preferred embodiment, the anti-MAdCAM antibody is a human IgG2
antibody. Alternatively, the subject may be a mammal that expresses
a MAdCAM protein that the anti-MAdCAM antibody cross-reacts with.
The antibody may be administered to a non-human mammal expressing
MAdCAM with which the antibody cross-reacts (i.e., a primate) for
veterinary purposes or as an animal model of human disease. Such
animal models may be useful for evaluating the therapeutic efficacy
of antibodies of this invention.
[0221] The present invention provides a method for the treatment of
an inflammatory disease in a subject, comprising administering to
the subject a liquid pharmaceutical composition comprising an
anti-MAdCAM antibody; and a chelating agent alone or in combination
with other excipients chosen from a buffer, a tonicity agent, or a
surfactant, and mixtures thereof. In further embodiments, the
aforementioned subject is one that is in need of the prevention or
treatment of an inflammatory disease.
[0222] In another embodiment, the present invention provides a
method for the treatment of an inflammatory disease condition in a
subject, comprising administering to the subject a liquid
pharmaceutical composition comprising anti-MAdCAM antibody 7.16.6;
and pharmaceutically acceptable excipient comprising a chelating
agent alone or in combination with other excipients chosen from a
buffer, a tonicity agent, or a surfactant, and mixtures
thereof.
[0223] In various embodiments of this invention, the inflammatory
disease may be, but is not limited to inflammatory diseases of the
gastrointestinal tract including Crohn's disease, ulcerative
colitis, diverticula disease, gastritis, liver disease, primary
biliary sclerosis, sclerosing cholangitis. Inflammatory diseases
also include but are not limited to abdominal disease (including
peritonitis, appendicitis, biliary tract disease), acute transverse
myelitis, allergic dermatitis (including allergic skin, allergic
eczema, skin atopy, atopic eczema, atopic dermatitis, cutaneous
inflammation, inflammatory eczema, inflammatory dermatitis, flea
skin, military dermatitis, military eczema, house dust mite skin),
ankylosing spondylitis (Reiters syndrome), asthma, airway
inflammation, atherosclerosis, arteriosclerosis, biliary atresia,
bladder inflammation, breast cancer, cardiovascular inflammation
(including vasculitis, rheumatoid nail-fold infarcts, leg ulcers,
polymyositis, chronic vascular inflammation, pericarditis, chronic
obstructive pulmonary disease), chronic pancreatitis, perineural
inflammation, colitis (including amoebic colitis, infective
colitis, bacterial colitis, Crohn's colitis, ischemic colitis,
ulcerative colitis, idiopathic proctocolitis, inflammatory bowel
disease, psuodomembranouscolitis), collagen vascular disorders
(rheumatoid arthritis, SLE, progressive systemic sclerosis, mixed
connective tissue disease, diabetes mellitus), Crohn's disease
(regional enteritis, granulomatous ileitis, ileocolitis, digestive
system inflammation), demyelinating disease (including myelitis,
multiple sclerosis, disseminated sclerosis, acute disseminated
encephalomyelitis, perivenous demyelination, vitamin B12
deficiency, Guilain-Barre syndrome, MS-associated retrovirus),
dermatomyositis, diverticulitis, exudative diarrheas, gastritis,
granulomatous hepatitis, grenulomatous inflammation, cholecystitis,
insulin-dependent diabetes mellitus, liverinflammatory diseases
(liver fibrosis primary biliary cirrhosis, hepatitis, sclerosing
cholangitis), lung inflammation (idiopathic pulmonary fibrosis,
eosinophilic granuloma of the lung, pulmonary histiocytosis X,
peribronchiolar inflammation, acute bronchitis), lymphogranuloma
venereum, malignant melanoma, mouth/tooth disease (including
gingivitis, periodontal disease), mucositis, musculoskeletal system
inflammation (myositis), nonalcoholic steatohepatitis (nonalcoholic
fatty liver disease), ocular & orbital inflammation (including
uveitis, optic neuritis, peripheral rheumatoid ulceration,
peripheral corneal inflammation), osteoarthritis, osteomyelitis,
pharyngeal inflammation, polyarthritis, proctitis, psoriasis,
radiation injury, sarcoidosis, sickle cell neuropathy, superficial
thrombophlevitits, systemic inflammatory response syndrome,
thuroiditis, systemic lupus erythematosus, graft versus host
disease, acute burn injury, Behcet's syndrome, Sjogren's
syndrome.
[0224] In a more preferred embodiment, the anti-MAdCAM antibody is
administered to a subject with colitis.
Articles of Manufacture:
[0225] In another embodiment of the invention, an article of
manufacture is provided comprising a container, which holds the
liquid pharmaceutical composition comprising at least one of the
monoclonal anti-MAdCAM antibodies of the present invention in a
composition comprising a chelating agent alone or in combination
with other pharmaceutically acceptable excipients, and optionally
provides instructions for its use. Suitable containers include, for
example, bottles, vials, bags and syringes. The container may be
formed from a variety of materials such as glass or plastic. An
exemplary container is a 3-20 cc single use glass vial.
Alternatively, for a multidose composition, the container may be a
3-100 cc glass vial. The container holds the composition and the
label on, or associated with, the container may indicate directions
for use. The article of manufacture may further include other
materials desirable from a commercial and user standpoint,
including other buffers, diluents, filters, needles, syringes, and
package inserts with instructions for use, contraindications,
and/or lists of potential side-effects.
[0226] The present invention also provides a kit for preparing a
liquid composition of a stabilized antibody comprising a first
container, comprising monoclonal anti-MAdCAM antibody 7.16.6 in
solution, and a second container comprising a sufficient amount of
a chelating agent alone or in combination with other excipients in
solution to stabilize the antibody.
[0227] The following examples describe embodiments of the
invention. Other embodiments within the scope of the claims herein
will be apparent to one skilled in the art from consideration of
the specification or practice of the invention as disclosed herein.
It is intended that the specification, together with the examples,
be considered exemplary only, with the scope and spirit of the
invention being indicated by the claims, which follow the examples.
In the examples, all percentages are given on a weight basis unless
otherwise indicated. The skilled artisan will appreciate that the
weight quantities and/or weight-to-volume ratios recited in the
examples can be converted to moles and/or molarities using the
art-recognized molecular weights of the recited ingredients. Weight
quantities exemplified herein (e.g., grams) are for the volumes
(e.g., of buffer solutions, antibody composition, etc.) recited.
The skilled artisan will appreciate that the weight quantities can
be proportionally adjusted when different composition volumes are
desired.
Example 1
Generation of Anti-MAdCAM Producing Hybridomas
[0228] Antibodies of the invention were prepared in accordance with
the present Example.
[0229] Refer to PCT/US2005/000370.
Primary Immunogen Preparation:
[0230] Two immunogens were prepared for immunisation of the
XenoMouse.TM. mice: (i) a MAdCAM-IgG.sub.1 Fc fusion protein and
(ii) cell membranes prepared from cells stably transfected with
MAdCAM.
[0231] (i) MAdCAM-IgG.sub.1 Fc Fusion Protein
Expression Vector Construction:
[0232] An EcoRI/BgIII cDNA fragment encoding the mature
extracellular, immunoglobulin-like domain of MAdCAM was excised
from a pINCY Incyte clone (3279276) and cloned into EcoRI/BamHI
sites of the pIG1 vector (Simmons, D. L. (1993) in Cellular
Interactions in Development: A Practical Approach, ed. Hartley, D.
A. (Oxford Univ. Press, Oxford), pp. 93-127.)) to generate an in
frame IgG.sub.1 Fc fusion. The resulting insert was excised with
EcoRI/NotI and cloned into pcDNA3.1+ (Invitrogen). The
MAdCAM-IgG.sub.1 Fc cDNA in the vector was sequence confirmed. The
amino acid sequence of the MAdCAM-IgG.sub.1 Fc fusion protein is
shown below:
MAdCAM-IgG.sub.1 Fc Fusion Protein:
TABLE-US-00002 [0233] (SEQ ID NO: 5)
MDFGLALLLAGLLGLLLGQSLQVKPLQVEPPEPVVAVALGASRQLTCRLA
CADRGASVQWRGLDTSLGAVQSDTGRSVLTVRNASLSAAGTRVCVGSCGG
RTFQHTVQLLVYAFPDQLTVSPAALVPGDPEVACTAHKVTPVDPNALSFS
LLVGGQELEGAQALGPEVQEEEEEPQGDEDVLFRVTERWRLPPLGTPVPP
ALYCQATMRLPGLELSHRQAIPVLHSPTSPEPPDTTSPESPDTTSPESPD
TTSQEPPDTTSQEPPDTTSQEPPDTTSPEPPDKTSPEPAPQQGSTHTPRS
PGSTRTRRPEIQPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQRREPQVYTLPPS
RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Underlined: signal peptide Bold: MAdCAM extracellular domain
Recombinant Protein Expression/Purification:
[0234] CHO-DHFR cells were transfected with pcDNA3.1+ vector
containing MAdCAM-IgG.sub.1 Fc fusion protein cDNA and stable
clones expressing MAdCAM-IgG.sub.1 Fc fusion protein selected in
Iscove's media containing 600 .mu.g/mL G418 and 100 ng/mL
methotrexate. For protein expression, a hollow fibre bioreactor was
seeded with stably expressing MAdCAM-IgG.sub.1 Fc CHO cells in
Iscove's media containing 10% low IgG fetal bovine serum (Gibco),
non essential amino acids (Gibco), 2 mM glutamine (Gibco), sodium
pyruvate (Gibco), 100 .mu.g/mL G418 and 100 ng/mL methotrexate, and
used to generate concentrated media supernatant. The
MAdCAM-IgG.sub.1 Fc fusion protein was purified from the harvested
supernatant by affinity chromatography. Briefly, supernatant was
applied to a HiTrap Protein G Sepharose (5 mL, Pharmacia) column (2
mL/min), washed with 25 mM Tris pH 8, 150 mM NaCl (5 column
volumes) and eluted with 100 mM glycine pH 2.5 (1 mL/min),
immediately neutralising fractions to pH 7.5 with 1 M Tris pH 8.
Fractions containing MAdCAM-IgG.sub.1 Fc fusion protein were
identified by SDS-PAGE, pooled together and applied to a Sephacryl
S100 column (Pharmacia), pre-equilibrated with 35 mM BisTris pH
6.5, 150 mM NaCl. The gel filtration was performed at 0.35 mL/min,
collecting a peak of MAdCAM-IgG.sub.1 Fc fusion protein in ca.
3.times.5 mL fractions. These samples were pooled and applied to a
Resource Q (6 mL, Pharmacia) column, pre-equilibrated in 35 mM
BisTris pH6.5. The column was washed with 5 column volumes of 35 mM
Bis Tris pH 6.5, 150 mM NaCl (6 mL/min) and MAdCAM-IgG.sub.1 Fc
fusion protein eluted into a 4-6 mL fraction with 35 mM Bis Tris pH
6.5, 400 mM NaCl. At this stage the protein was 90% pure and
migrating as a single band at approximately 68 kD by SDS-PAGE. For
use as an immunogen and all subsequent assays, the material was
buffer exchanged into 25 mM HEPES pH 7.5, 1 mM EDTA, 1 mM DTT, 100
mM NaCl, 50% glycerol and stored as aliquots at -80.degree. C.
[0235] (ii) Cell Membranes Stably Expressing MAdCAM
[0236] A SacI/NotI fragment comprising nucleotides 645-1222 of the
published MAdCAM sequence (Shyjan A M, et al., J. Immunol., 156,
2851-7 (1996)) was PCR amplified from a colon cDNA library and
cloned into SacI/NotI sites of pIND-Hygro vector (Invitrogen). A
SacI fragment, comprising the additional 5' coding sequence was
sub-cloned into this construct from pcDNA3.1 MAdCAM-IgG.sub.1 Fc,
to generate the full length MAdCAM cDNA. A KpnI/NotI fragment
containing the MAdCAM cDNA was then cloned into corresponding sites
in a pEF5FRTV5GWCAT vector (Invitrogen) and replacing the CAT
coding sequence. The cDNA insert was sequence verified and used in
transfections to generate single stably expressing clones in FlpIn
NIH 3T3 cells (Invitrogen) by Flp recombinase technology, according
to the manufacturer's instructions. Stably expressing clones were
selected by their ability to support the binding of a
.alpha..sub.4.beta..sub.7.sup.+ JY human B lymphoblastoid cell line
(Chan B M, et al, J. Biol. Chem., 267:8366-70 (1992)), outlined
below. Stable clones of CHO cells expressing MAdCAM were prepared
in the same way, using FlpIn CHO cells (Invitrogen).
[0237] MAdCAM-expressing FlpIn NIH-3T3 cells were grown in
Dulbecco's modified Eagles Medium (Gibco), containing 2 mM
L-glutamine, 10% Donor calf serum (Gibco) and 200 .mu.g/mL
Hygromycin B (Invitrogen) and expanded in roller bottles.
MAdCAM-expressing FlpIn CHO cells were grown in Ham's
F12/Dulbecco's modified Eagles Medium (Gibco), containing 2 mM
L-glutamine, 10% Donor calf serum (Gibco) and 350 .mu.g/mL
Hygromycin B (Invitrogen) and expanded in roller bottles. Cells
were harvested by use of a non-enzymatic cell dissociation solution
(Sigma) and scraping, washing in phosphate buffered saline by
centrifugation. Cell membranes were prepared from the cell pellet
by two rounds of polytron homogenization in 25 mM Bis Tris pH 8, 10
mM MgCl.sub.2, 0.015% (w/v) aprotinin, 100 U/mL bacitracin and
centrifugation. The final pellet was resuspended in the same
buffer, and 50.times.10.sup.6 cell equivalents aliquoted into
thick-walled eppendorfs and spun at >100,000 g to generate cell
membrane pellets for XenoMouse mice immunisations. Supernatant was
decanted and membranes were stored in eppendorfs at -80.degree. C.
until required. Confirmation of protein expression in the cell
membranes was determined by SDS-PAGE and Western blotting with a
rabbit anti-peptide antibody raised against the N-terminal residues
of MAdCAM ([C]-KPLQVEPPEP).
Immunization and Hybridoma Generation:
[0238] Eight to ten week old XENOMOUSE.TM. mice were immunized
intraperitoneally or in their hind footpads with either the
purified recombinant MAdCAM-IgG.sub.1 Fc fusion protein (10
.mu.g/dose/mouse), or cell membranes prepared from either stably
expressing MAdCAM-CHO or NIH 3T3 cells (10.times.10.sup.6
cells/dose/mouse). This dose was repeated five to seven times over
a three to eight week period. Four days before fusion, the mice
received a final injection of the extracellular domain of human
MAdCAM in PBS. Spleen and lymph node lymphocytes from immunized
mice were fused with the non-secretory myeloma P3-X63-Ag8.653 cell
line and were subjected to HAT selection as previously described
(Galfre and Milstein, Methods Enzymol. 73:3-46 (1981)). A panel of
hybridomas secreting MAdCAM specific human IgG.sub.2K was recovered
and sub-cloned.
[0239] The following hybridoma producing anti-MAdCAM antibodies
designated as follows was deposited at the European Collection of
cell Cultures (ECACC), H.P.A. at CAMR, Porton Down, Salisbury,
Wiltshire SP4 0JG on 9 Sep. 2003: Hybridoma 7.16.6, Deposit No.
03090909.
Example 2
Antibody Compositions
[0240] The following compositions are referred to in the Examples
that follow:
TABLE-US-00003 TABLE 2 mAb Chelating Composition 7.16.6 Buffer
Tonicifier Surfactant agent Other ID mg/ml mM; pH mg/ml mg/ml Mg/ml
Excipients 1 10 .+-. 1 Acetate, 20 mM, pH 5.5 2 10 .+-. 1 Acetate,
7 mM Citrate, 7 mM Phosphate, 7 mM, pH 5.5 3 10 .+-. 1 EDTA, 20 mM
pH 5.5 4 8 + 1 Sodium NaCl, acetate, 140 mM 20 mM, pH 5.5 5 8 .+-.
1 Sodium Mannitol, acetate, 20 mM, 45 mg/ml pH 5.5 6 8 .+-. 1
Sodium acetate, 10 mM, pH 5.5 7 8 .+-. 1 Sodium EDTA, 10 mM, pH 5.5
8 8 .+-. 1 Sodium NaCl, EDTA, 10 mM, 140 mM pH 5.5 9 8 .+-. 1
Sodium Mannitol, EDTA, 10 mM, 45 mg/ml pH 5.5 10 8 .+-. 2 Sodium
Mannitol, PS80, 0.2 mg/ml acetate, 20 mM, 45 mg/ml pH 5.5 11 8 .+-.
2 Sodium Mannitol, PS80, 0.2 mg/ml Na.sub.2EDTA.cndot.2H.sub.20,
acetate 20 mM, 45 mg/ml 0.02 mg/ml pH 5.5 12 8 .+-. 2 Sodium NaCl,
8.2 mg/ml PS80, 0.2 mg/ml acetate 20 mM, pH 5.5 13 8 .+-. 2 Sodium
Mannitol, PS80, 0.2 mg/ml CaCl.sub.2.cndot.2H.sub.2O, acetate 20
mM, 45 mg/ml 0.3 mg/ml pH 5.5 14 30 .+-. 6 Sodium Mannitol, PS80,
0.4 mg/ml acetate 20 mM, 45 mg/ml pH 5.5 15 30 .+-. 6 Sodium
Mannitol, PS80, 0.4 mg/ml Na.sub.2EDTA.cndot.2H.sub.20, acetate 20
mM, 45 mg/ml 0.02 mg/ml pH 5.5 16 30 .+-. 6 Sodium NaCl, 8.2 mg/ml
PS80, 0.4 mg/ml acetate 20 mM, pH 5.5 17 30 .+-. 6 Sodium Mannitol,
PS80, 0.4 mg/ml CaCl.sub.2.cndot.2H.sub.2O, acetate 20 mM, 45 mg/ml
0.3 mg/ml pH 5.5 18 50 .+-. 6 Histidine, Mannitol, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 45 mg/ml 0.02 mg/ml 5.5 19
10 .+-. 2 Sodium Mannitol, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, acetate 20 mM, 45 mg/ml 0.02 mg/ml pH
5.5 20 10 .+-. 2 Histidine, Mannitol, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 45 mg/ml 0.02 mg/ml 5.5 21
10 .+-. 2 Histidine, Mannitol, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 45 mg/ml 0.02 mg/ml 5.5 22
50 .+-. 6 Sodium Mannitol, PS80, 0.4 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, acetate 20 mM, 45 mg/ml 0.02 mg/ml pH
5.5 23 50 .+-. 6 Histidine, Mannitol, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 45 mg/ml 0.02 mg/ml 6.0 24
150 .+-. 6 Histidine, Mannitol, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 45 mg/ml 0.02 mg/ml 5.5 25
50 .+-. 5 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 26
75 .+-. 5 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 27
100 .+-. 7 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 28
150 .+-. 10 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 29
190 .+-. 2 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 30
80 .+-. 10 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.02 mg/ml 5.5 31
80 .+-. 10 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.10 mg/ml 5.5 32
80 .+-. 10 Histidine, Trehalose, PS80, 0.4 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 33
80 .+-. 10 Histidine, Trehalose, PS80, 1.0 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 34
85 .+-. 15 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.10 mg/ml 5.5 35
85 .+-. 15 Histidine, Trehalose, PS80, 0.4 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.10 mg/ml 5.5 36
85 .+-. 15 Histidine, Trehalose, PS80, 1.0 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.10 mg/ml 5.5 37
80 .+-. 10 Citrate, 5 mM, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, pH 5.5 90 mg/ml 0.05 mg/ml 38 80 .+-.
10 Succinate, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 5 mM pH 90 mg/ml 0.05 mg/ml 5.5 39 80
.+-. 10 Histidine, Sucralose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 85 mg/ml 0.05 mg/ml 5.5 40
80 .+-. 10 Histidine, Sorbitol PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 45 mg/ml 0.05 mg/ml 5.5 41
80 .+-. 10 Histidine, Xylitol PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 35 mg/ml 0.05 mg/ml 5.5 42
80 .+-. 10 Histidine, Trehalose, PEG.sub.3350,
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 10 mg/ml 0.05
mg/ml 5.5 43 80 .+-. 10 Histidine, Trehalose, NOF PS80
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.2 mg/ml 0.05
mg/ml 5.5 44 80 .+-. 10 Histidine, Trehalose, Poloxamer
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 407, 0.05 mg/ml
5.5 1.0 mg/ml 45 80 .+-. 10 Histidine, Trehalose, Poloxamer
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 188 mg/ml 0.05
mg/ml 5.5 1.0 mg/ml 46 50 .+-. 5 Histidine, Trehalose, PS80, 0.2
mg/ml Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml
5.5 47 50 .+-. 5 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 48
75 .+-. 15 Histidine, Trehalose, PS80, 0.4 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 49
75 .+-. 15 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 50
75 .+-. 15 Histidine, Sucrose, PS80, 0.4 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 85 mg/ml 0.05 mg/ml 5.5 51
75 .+-. 5 Histidine, Trehalose, PS80, 0.2 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.05 mg/ml 5.5 52
75 .+-. 5 Histidine, Trehalose, PS80, 0.4 mg/ml
Na.sub.2EDTA.cndot.2H.sub.20, 10 mM, pH 90 mg/ml 0.10 mg/ml 5.5
Example 3
[0241] A study was conducted to evaluate the effect of several
different buffers on anti-MAdCAM antibody 7.16.6 aggregation.
Preparation of Buffer Solutions:
[0242] The buffer solutions were prepared by first dissolving an
amount of the buffer species in water (approximately 90% of
target). The pH of each buffer solutions was then adjusted to 5.5
by addition of a sufficient amount of an acid or base solution.
After adjustment of pH, an additional amount of water was added to
provide a final buffer concentration of 20 mM. The buffer
concentration of 20 mM was selected to ensure reasonable pH
stability at the selected pH of 5.5. The buffer solution was then
filtered through a sterilization filter (0.22 micron pore size)
into a sterilized receptacle for subsequent use.
Preparation of Antibody Compositions:
[0243] The antibody compositions were prepared as follows. An
antibody bulk solution was obtained as 10.5 mg/ml in 20 mM sodium
acetate buffer pH 5.5+140 mM sodium chloride. Buffer exchanges of
this bulk solution into the composition solutions were carried out
by centrifugation at 4500.times.g using a molecular weight cut-off
membrane (e.g. 30 kD). Approximately 8 volume exchanges were made
and the final antibody solution was prepared at about 10 mg/ml
concentration. Antibody concentrations were determined by
Ultraviolet-Visible spectrometry (UV-Vis) method using an
extinction coefficient of 1.56 (mg/ml).sup.-1 cm.sup.-1 at 280 nm.
The compositions with all ingredients were then sterilized by
filtration through sterile 0.22 micron membrane filter. The
filtered compositions were then filled into washed and autoclaved
vials, which were closed with Dalkyo777-1 Fluorotec.RTM. coated
stoppers, crimp sealed and placed in stability chambers.
[0244] Specifically, three liquid compositions comprising
anti-MAdCAM antibody 7.16.6 and buffered with acetate, EDTA, or a
combination of acetate, citrate and phosphate were prepared. The
compositions were then stored for 6 weeks at 40.degree. C. and
aggregation measurements were taken.
TABLE-US-00004 TABLE 3 Relative Compo- increase sition 7.16.6 mAb %
in % ID mg/mL Description Aggregation Aggregation* 1 10 .+-. 1 20
mM Acetate 0.8 70 pH 5.5 2 10 .+-. 1 Combination, pH 0.7 60 5.5
(acetate 7 mM, citrate 7 mM, phosphate 6 mM) 3 10 .+-. 1 20 mM
EDTA, 0.5 40 pH 5.5 *Relative increase in % Aggregation is
calculated by: [{(% aggregation at 6 wk/40 C.) - (% aggregation at
initial)} * 100]/[% aggregation at initial]
Aggregation Analysis:
[0245] The antibody compositions were stored at 40.degree. C. At
six weeks, each composition was analyzed for aggregation using size
exclusion chromatography (SEC). The size exclusion chromatography
was carried out using a TSK gel G3000SWXL-G2000SWXL column, 0.2M
sodium phosphate, pH7 mobile phase, a flow rate of 0.7 mL/min, and
UV detection at 214 nm. Aggregation levels were calculated by
integrating the areas under the chromatogram peaks for each
composition and reporting the integrated areas under the high
molecular weight species peaks as a percentage of total peak area.
As is shown in Table 3, the EDTA buffered composition showed the
lowest level of aggregation and lowest relative increase in
aggregation.
Example 4
[0246] A study was conducted to evaluate the effect of buffer
concentration and presence/absence of other excipients on
anti-MAdCAM antibody 7.16.6 fragmentation.
[0247] The compositions shown in Table 4 were prepared by the
methods described in Example 2 and evaluated by the methods in
Example 3.
TABLE-US-00005 TABLE 4 Compo- 7.16.6 sition mAb % ID mg/mL Buffer
Excipient fragmentation 4 8 .+-. 1 20 mM Sodium NaCl, 140 mM 0.9
acetate, pH 5.5 5 8 .+-. 1 20 mM Sodium Mannitol, 45 mg/ml 0.6
acetate, pH 5.5 6 8 .+-. 1 10 mM Sodium -- 0.5 acetate, pH 5.5 7 8
.+-. 1 10 mM Sodium -- 0 EDTA, pH 5.5 8 8 .+-. 1 10 mM Sodium NaCl,
140 mM 0 EDTA, pH 5.5 9 8 .+-. 1 10 mM Sodium Mannitol, 45 mg/ml 0
EDTA, pH 5.5
[0248] As shown in Table 4, the presence of EDTA in the liquid
compositions results in less LMM formation than in compositions
containing no EDTA.
Example 5
[0249] A study was conducted to assess the effect of EDTA, in
liquid anti-MAdCAM antibody compositions, on aggregation and
fragmentation.
[0250] The buffer solutions were prepared by the methods described
in Example 3. The antibody compositions were prepared as follows.
An antibody bulk solution was obtained as 9.6 mg/ml in 20 mM sodium
acetate buffer pH 5.5. Buffer exchanges of this bulk solution into
the composition solutions were carried out by centrifugation at
5000.times.g using a molecular weight cut-off membrane (e.g. 30
kD). Approximately 8 volume exchanges were made and the final
antibody solution was prepared at about 8 mg/ml or about 30 mg/ml
protein concentration. Antibody concentrations were determined by
Ultraviolet-Visible spectrometry (UV-Vis) method using an
extinction coefficient of 1.56 (mg/ml).sup.-1 cm.sup.-1 at 280 nm.
A concentrate solution of polysorbate 80 (PS80) (typically 20
mg/ml) was prepared by dilution and dissolution of PS80 by the
appropriate buffer. The PS80 concentrate was then added to the
antibody solutions to obtain the final compositions described. The
compositions with all ingredients were then sterilized by
filtration through sterile 0.22 micron membrane filter. The
filtered compositions were then filled into washed, autoclaved
vials. The vials were closed with Daikyo 777-1 Fluorotec.RTM.
coated stoppers, crimp sealed, and placed in stability
chambers.
[0251] The compositions in Table 5 were stored at 40.degree. C. for
26 weeks, and evaluated by the SEC method described in Example
3.
TABLE-US-00006 TABLE 5 7.16.6 Buffer PS- Composition mAb (pH 80,
Other % % ID mg/mL 5.5) Excipient mg/mL excipients Aggregate
fragmentation 10 8 .+-. 2 Sodium Mannitol, 0.2 -- 6.9 0.6 acetate,
45 mg/mL 20 mM, pH 5.5 11 8 .+-. 2 Sodium Mannitol, 0.2
Na.sub.2EDTA.cndot.2H.sub.2O, 3.7 0 acetate, 45 mg/mL 0.02 mg/mL 20
mM, pH 5.5 12 8 .+-. 2 Sodium NaCl, 0.2 -- 6.0 0.5 acetate, 8.2
mg/mL 20 mM, pH 5.5 13 8 .+-. 2 Sodium Mannitol, 0.2
CaCl.sub.2.cndot.2H.sub.2O, 6.8 0.8 acetate, 45 mg/mL 0.3 mg/mL 20
mM, pH 5.5 14 30 .+-. 6 Sodium Mannitol, 0.4 -- 7.2 0.2 acetate, 45
mg/mL 20 mM, pH 5.5 15 30 .+-. 6 Sodium Mannitol, 0.4
Na.sub.2EDTA.cndot.2H.sub.2O, 3.7 0 acetate, 45 mg/mL 0.02 mg/mL 20
mM, pH 5.5 16 30 .+-. 6 Sodium NaCl, 0.4 -- 7.7 0.4 acetate, 8.2
mg/mL 20 mM, pH 5.5 17 30 .+-. 6 Sodium Mannitol, 0.4
CaCl.sub.2.cndot.2H.sub.2O, 9.8 0.7 acetate, 45 mg/mL 0.3 mg/mL 20
mM, pH 5.5
[0252] The compositions in Table 6 were stored at 25.degree. C. for
26 weeks and evaluated by the SEC method described in Example
3.
TABLE-US-00007 TABLE 6 7.16.6 Composition mAb, PS-80, Other % ID
mg/mL Buffer Excipient mg/mL excipients Aggregate 14 30 .+-. 6
Sodium Mannitol, 0.4 -- 1.6 acetate, 45 mg/mL 20 mM, pH 5.5 15 30
.+-. 6 Sodium Mannitol, 0.4 Na.sub.2EDTA.cndot.2H.sub.2O, 0.7
acetate, 45 mg/mL 0.02 mg/mL 20 mM, pH 5.5 18 50 .+-. 6 Histidine
Mannitol, 0.2 Na.sub.2EDTA.cndot.2H.sub.2O, 0.5 10 mM, 45 mg/mL
0.02 mg/mL pH 5.5
[0253] This example shows that the presence of EDTA in liquid
composition results in less aggregation and less LMM species
formation.
Example 6
[0254] A study was conducted to compare the effect of acetate and
histidine buffers on aggregation and fragmentation of anti-MAdCAM
antibody 7.16.6. The compositions shown in Tables 7 and 8 were
prepared by the methods described in Example 5. The compositions in
Table 7 were stored at 40.degree. C. for 26 weeks and analyzed by
the SEC methods described in Example 3.
TABLE-US-00008 TABLE 7 7.16.6 Composition mAb PS-80,
Na.sub.2EDTA.cndot.2H.sub.2O, % % ID mg/mL Buffer pH Excipient
mg/mL mg/mL Aggregate fragmentation 19 10 .+-. 2 Sodium 5.5
Mannitol, 0.2 0.02 3.8 0 acetate, 45 mg/mL 20 mM 20 10 .+-. 2
Histidine 6.0 Mannitol, 0.2 0.02 1.2 0 10 mM 45 mg/mL 21 10 .+-. 2
Histidine 5.5 Mannitol, 0.2 0.02 1.5 0 10 mM 45 mg/mL 22 50 .+-. 6
Sodium 5.5 Mannitol, 0.4 0.02 5.2 0 acetate, 45 mg/mL 20 mM 23 50
.+-. 6 Histidine 6.0 Mannitol, 0.2 0.02 2.3 0 10 mM 45 mg/mL 18 50
.+-. 6 Histidine 5.5 Mannitol, 0.2 0.02 2.8 0.1 10 mM 45 mg/mL
[0255] The compositions in Table 8 were stored at 25.degree. C. for
52 weeks, and analysed by the SEC methods described in Example
3.
TABLE-US-00009 TABLE 8 7.16.6 PS- Composition mAb 80,
Na.sub.2EDTA.cndot.2H.sub.2O, % % ID mg/mL Buffer pH Excipient
mg/mL mg/mL Aggregate fragmentation 19 10 .+-. 2 Sodium 5.5
Mannitol, 0.2 0.02 0.9 0 acetate, 45 mg/mL 20 mM 20 10 .+-. 2
Histidine 6.0 Mannitol, 0.2 0.02 0.4 0 10 mM 45 mg/mL 21 10 .+-. 2
Histidine 5.5 Mannitol, 0.2 0.02 0.3 0 10 mM 45 mg/mL 22 50 .+-. 6
Sodium 5.5 Mannitol, 0.4 0.02 1.3 0 acetate, 45 mg/mL 20 mM 23 50
.+-. 6 Histidine 6.0 Mannitol, 0.2 0.02 0.8 0 10 mM 45 mg/mL 18 50
.+-. 6 Histidine 5.5 Mannitol, 0.2 0.02 0.9 0 10 mM 45 mg/mL
[0256] This example shows that the compositions using histidine as
a buffer have less aggregate formation than the compositions using
acetate at the same pH.
Example 7
[0257] A study was conducted to evaluate the aggregation propensity
of anti-MAdCAM antibody 7.16.6 in compositions at various antibody
concentrations. In this study the compositions in Table 9 were
prepared by the methods described in Example 5. The compositions in
Table 9 were stored at 5.degree. C., 25.degree. C. or 40.degree. C.
for 26 weeks and then analyzed by the SEC methods described in
Example 3.
TABLE-US-00010 TABLE 9 % % % mAb Aggregation Aggregation
Aggregation Composition Buffer, 7.16.6, Trehalose.cndot.2H.sub.2O,
Na.sub.2EDTA.cndot.2H.sub.2O, PS80, at at at ID mM pH mg/mL mg/mL
mg/mL mg/mL 5 C. 25 C. 40 C. 25 His, 5.5 47 90 0.05 0.2 0.3 0.6 2.7
10 26 His, 5.5 75 90 0.05 0.2 0.4 0.7 3.6 10 27 His, 5.5 99 90 0.05
0.2 0.5 0.8 4.4 10 28 His, 5.5 145 90 0.05 0.2 0.7 1.2 6.0 10 29
His, 5.5 183 90 0.05 0.2 0.6 1.3 7.5 10
[0258] As shown in Table 9, the propensity for aggregation
increases with increasing concentrations of antibody. However,
after storage for 26 weeks, the stabilizing effect of the
compositions shown in Table 9 is demonstrated by the accelerated
condition data (storage at 25.degree. C. and 40.degree. C.) that
shows relatively low levels of aggregation with high concentration
compositions.
Example 8
[0259] A study was conducted to evaluate the aggregation propensity
of anti-MAdCAM antibody 7.16.6 in compositions with various levels
of EDTA. The compositions were prepared as described above in
example 5, except that the final antibody concentration was
adjusted to 80.+-.10 mg/ml. The compositions in Table 10 were
stored for 26 weeks at 5.degree. C. or 26.degree. C. and then
analyzed by SEC as described above.
TABLE-US-00011 TABLE 10 % % Composition Buffer,
Trehalose.cndot.2H.sub.2O, Na.sub.2EDTA.cndot.2H.sub.2O, PS80,
Aggregation Aggregation ID mM pH mg/mL mg/mL mg/mL at 5 C. at 25 C.
30 His, 5.5 90 0.02 0.2 0.6 0.9 10 26 His, 5.5 90 0.05 0.2 0.4 0.7
10 31 His, 5.5 90 0.10 0.2 0.5 0.8 10
[0260] As shown, there is an improvement at 0.05 mg/ml and 0.10
mg/mL relative to 0.02 mg/mL EDTA, and in each case, aggregation in
the presence of EDTA is low after storage at 5.degree. C. for 26
weeks.
Example 9
[0261] A study was conducted to evaluate stability of compositions
of anti-MAdCAM antibody 7.16.6 with various levels of
Polysorbate-80. The compositions were prepared as described above,
but with the final antibody concentration adjusted to 80.+-.mg/ml.
The compositions in Table 11 were stored for 26 weeks at 25.degree.
C. or 40.degree. C. and then analyzed by SEC as described
above.
TABLE-US-00012 TABLE 11 % % Composition Buffer,
Trehalose.cndot.2H.sub.2O, Na.sub.2EDTA.cndot.2H.sub.2O, PS80,
Aggregation Aggregation ID mM pH mg/mL mg/mL mg/mL at 25 C. at 40
C. 26 His, 5.5 90 0.05 0.2 0.7 3.6 10 32 His, 5.5 90 0.05 0.4 0.8
4.2 10 33 His, 5.5 90 0.05 1.0 1.0 4.8 10
[0262] The compositions in Table 12 were subjected to shaking
stress applied by orbital shaking at 300 rpm for 24 hours at
ambient temperature. The compositions were prepared as described
above, but with final antibody concentration adjusted to
85.+-.mg/ml
TABLE-US-00013 TABLE 12 Composition Buffer,
Trehalose.cndot.2H.sub.2O, Na.sub.2EDTA.cndot.2H.sub.2O, PS80, ID
mM pH mg/mL mg/mL mg/mL Appearance 34 His, 5.5 90 0.10 0.2 Presence
of few 10 particulates 35 His, 5.5 90 0.10 0.4 No particulates 10
36 His, 5.5 90 0.10 1.0 No particulates 10
[0263] Although the above-described storage stability study shows a
slight increase in soluble aggregation levels with increasing
levels of polysorbate-80, the shaking stress study indicates that a
polysorbate-80 level of 0.4 mg/mL provides adequate protection from
shaking stress.
Example 10
[0264] A study was undertaken to evaluate aggregation propensity of
anti-MAdCAM antibody 7.16.6 in compositions with various buffers.
The compositions were prepared as described above, and adjusted to
a final concentration of antibody of 80.+-.10 mg/ml. The
compositions in Table 13 were stored at 25.degree. C. or 40.degree.
C. for 26 weeks.
TABLE-US-00014 TABLE 13 % % Aggregation Composition Buffer,
Trehalose.cndot.2H.sub.2O, Na.sub.2EDTA.cndot.2H.sub.2O, PS80,
Aggregation at ID mM pH mg/mL mg/mL mg/mL at 25 C. 40 C. 26 His, 10
5.5 90 0.05 0.2 0.7 3.6 37 Citrate, 5 5.5 90 0.05 0.2 1.7 6.0 38
Succinate, 5 5.5 90 0.05 0.2 1.0 4.9
[0265] The level of aggregation was lowest in the composition with
histidine buffer.
Example 11
[0266] A study was undertaken to evaluate aggregation propensity of
anti-MAdCAM antibody 7.16.6 in compositions with various sugars and
polyols. The compositions were prepared as described above, and
adjusted to a final antibody concentration of 80.+-.10 mg/ml. The
compositions in Table 14 were stored at 40.degree. C. for 26
weeks.
TABLE-US-00015 TABLE 14 Composition Buffer, Sugar/Polyol,
Na.sub.2EDTA.cndot.2H.sub.2O, PS80, % Aggregation ID mM pH mg/mL
mg/mL mg/mL at 40 C. 26 His, 10 5.5 Trehalose.cndot.2H.sub.2O, 0.05
0.2 3.6 90 39 His, 10 5.5 Sucrose, 85 0.05 0.2 4.8 40 His, 10 5.5
Sorbitol, 45 0.05 0.2 4.8 41 His, 10 5.5 Xylitol, 35 0.05 0.2
4.6
[0267] The level of aggregation was lower with the composition
containing trehalose.
Example 12
[0268] A study was undertaken to evaluate antibody aggregation
propensity of anti-MAdCAM antibody 7.16.6 in compositions with
various surfactants and PEG. The compositions were prepared as
described above, and adjusted to a final antibody concentration of
80.+-.10 mg/ml. The final concentration of surfactant of PEG in the
antibody compositions was achieved by addition of an appropriate
quantity from concentrate stock solutions of surfactant or PEG. The
compositions in Table 15 were stored at 40.degree. C. for 26
weeks.
TABLE-US-00016 TABLE 15 % Composition Buffer,
Trehalose.cndot.2H.sub.2O, Na.sub.2EDTA.cndot.2H.sub.2O, Surfactant
or Aggregation ID mM pH mg/mL mg/mL PEG, mg/mL at 40 C. 26 His, 5.5
90 0.05 PS80, 0.2 3.6 10 42 His, 5.5 90 0.05 PEG.sub.3350, 10 3.9
10 43 His, 5.5 90 0.05 NOF PS80, 3.9 10 0.2 44 His, 5.5 90 0.05
Poloxamer 3.4 10 407, 1.0 45 His, 5.5 90 0.05 Poloxamer 4.1 10 188,
1.0
[0269] Compositions containing PS80 and Poloxamer 407 performed
marginally better than the other surfactants and amphiphiles.
Example 13
[0270] A study was undertaken to evaluate Met256 oxidation in
compositions with either trehalose or sucrose. The compositions
were prepared as described above, and adjusted to a final antibody
concentration of 80.+-.10 mg/ml. The compositions in Table 16 were
stored at 5.degree. C. or 40.degree. C. for 26 weeks. Methionine
oxidation was measured by enzymatically digesting the protein using
Lysyl endoproteinase and the resulting peptide fragments were
separated by reversed-phase HPLC with 214 nm absorbance detection.
The peptide fragment containing methionine or its oxidized form was
monitored. Percentage oxidation was calculated by peak area of
oxidized methionine relative to that of parent methionine.
TABLE-US-00017 TABLE 16 % % Met256 Met256 Composition Buffer,
Sugar, Na.sub.2EDTA.cndot.2H.sub.2O, PS80, oxidation oxidation ID
mM pH mg/mL mg/mL mg/mL at 5 C. at 40 C. 26 His, 5.5
Trehalose.cndot.2H.sub.2O, 0.05 0.2 3.3 7.2 10 90 32 His, 5.5
Trehalose.cndot.2H.sub.2O, 0.05 0.4 3.2 7.3 10 90 39 His, 5.5
Sucrose, 85 0.05 0.2 3.2 9.0 10
[0271] The compositions containing trehalose show lower propensity
for methionine oxidation relative to those containing sucrose.
Example 14
[0272] A study was undertaken to evaluate the chemical stability
performance of high antibody concentration compositions. The
compositions in Tables 17 and 18 were prepared as described above,
and adjusted to a final antibody concentration of 80.+-.10 mg/ml.
The compositions in Table 17 were stored at 5.degree. C. for 26
weeks. Chemical stability was assessed by iCE. Measurements were
conducted by preparing the protein mix with pl markers,
methylcellulose, and pharmalytes to a final protein concentration
of approximately 0.22 .mu.g/.mu.L. The electrophoresis run was
performed with focusing time of 6 min. at 3000 volts and absorbance
probe at 280 nm. Relative percentage of various charged species was
determined by their respective area under the peak.
TABLE-US-00018 TABLE 17 % Major % Major band band by Composition
Buffer, Trehalose.cndot.2H.sub.2O, Na.sub.2EDTA.cndot.2H.sub.2O,
PS80, by iCE iCE (26 ID mM pH mg/mL mg/mL mg/mL (initial) weeks) 26
His, 5.5 90 0.05 0.2 66.5 67.9 10 32 His, 5.5 90 0.05 0.4 66.6 67.3
10 31 His, 5.5 90 0.10 0.2 65.7 65.5 10
[0273] The compositions in Table 17 show good chemical stability,
as no significant change in the major species as well as in total
acidic species or total basic species.
[0274] The compositions in Table 18 were stored at 5.degree. C. for
26 weeks, and assayed by reduced SDS-PAGE to determine purity. The
SDS-PAGE gels were run using NuPAGE 4-12% Bis-Tris gel, and
colloidal blue (Coomassie blue) stain. For the reduced gels,
reduction was achieved by Nu-PAGE reducing agent. Percent purity in
reduced gels was estimated densitometrically by: % purity=(% heavy
chain+% light chain).
TABLE-US-00019 TABLE 18 % % Purity Purity by by reduced reduced
SDS- SDS- PAGE Composition Buffer, Trehalose.cndot.2H.sub.2O,
Na.sub.2EDTA.cndot.2H.sub.2O, PS80, PAGE (26 ID mM pH mg/mL mg/mL
mg/mL (initial) weeks) 26 His, 5.5 90 0.05 0.2 99.7 98.8 10 32 His,
5.5 90 0.05 0.4 99.7 98.8 10 31 His, 5.5 90 0.10 0.2 99.7 98.8
10
[0275] The compositions in Table 18 show no significant change in
purity after storage for 26 weeks at 5.degree. C., indicating good
chemical stability.
Example 15
[0276] A study was undertaken to evaluate the performance of high
concentration compositions against freeze-thaw stress.
[0277] The compositions in Table 19 were prepared as described
above, and adjusted to a final antibody concentration of 50.+-.10
mg/ml. The compositions in Table 19 were subjected to three
freeze-thaw cycles at -70.degree. C./5.degree. C. or -20.degree.
C./5.degree. C. The studies were conducted in 2 ml glass vials with
1 ml fill. SE_HPLC measurements were conducted using 0.2M sodium
phosphate, pH 7 mobile phase, TSK gel G3000SWXL columns, at a flow
rate of 0.7 ml/min, probe at 214 nm. Aggregate quantity was
determined by summing antibody related peaks that eluted prior to
the antibody monomer.
TABLE-US-00020 TABLE 19 Change in % soluble Appearance aggregation
after three after three Freeze-thaw freeze-thaw freeze-thaw
Composition ID Composition Description cycle cycles cycles 46 50
mg/mL mAb 7.16.6, -70 C./5 C. Clear; No 0.0 10 mM histidine, pH
5.5, particulates 90 mg/mL trehalose dihydrate, 0.05 mg/mL disodium
EDTA dihydrate, 0.2 mg/mL polysorbate 80 47 50 mg/mL mAb 7.16.6,
-20 C./5 C. Clear; No 0.0 10 mM histidine, pH 5.5, particulates 90
mg/mL trehalose dihydrate, 0.05 mg/mL disodium EDTA dihydrate, 0.2
mg/mL polysorbate 80
[0278] The compositions in Table 19 show no increase in aggregation
after 3 freeze-thaw cycles at either -70.degree. C./5.degree. C. or
-20.degree. C./5.degree. C.
[0279] The compositions in Table 20 were prepared as described
above, and adjusted to a final antibody concentration of 75.+-.15
mg/ml. The compositions in Table 20 were subjected to four
freeze-thaw cycles at -20.degree. C./5.degree. C. These freeze-thaw
studies were conducted in 10 ml glass vials with 10 ml fill. SEC
measurements were conducted as indicated above in this example.
TABLE-US-00021 TABLE 20 Change in % soluble Freeze- Appearance
after aggregation after Composition thaw four freeze-thaw four
freeze-thaw ID Composition Description cycle cycles cycles 48 75
mg/mL mAb 7.16.6, -20 C./5 C. Clear; No 0.0 10 mM histidine, pH
5.5, particulates 90 mg/mL trehalose dihydrate, 0.05 mg/mL disodium
EDTA dihydrate, 0.4 mg/mL polysorbate 80 49 75 mg/mL mAb 7.16.6,
-20 C./5 C. Clear; No 0.0 10 mM histidine, pH 5.5, particulates 90
mg/mL trehalose dihydrate, 0.05 mg/mL disodium EDTA dihydrate, 0.2
mg/mL polysorbate 80 50 75 mg/mL mAb 7.16.6, -20 C./5 C. Clear; No
0.0 10 mM histidine, pH 5.5, particulates 85 mg/mL sucrose, 0.05
mg/mL disodium EDTA dihydrate, 0.4 mg/mL polysorbate 80
[0280] The compositions in Table 20 show no increase in aggregation
after four freeze-thaw cycles at -20.degree. C./5.degree. C.
Example 16
[0281] A study was undertaken to assess the stability of a high
concentration composition during frozen storage. The composition in
Table 21 was prepared as described above, and final antibody
concentration was adjusted to about 75 mg/ml. The composition in
Table 21 was stored at -20.degree. C. for 13 weeks, and aggregation
assessed as described in Example 15.
TABLE-US-00022 TABLE 21 Composition Buffer,
Trehalose.cndot.2H.sub.2O, Na.sub.2EDTA.cndot.2H.sub.2O, PS80, %
Increase in ID mM pH mg/mL mg/mL mg/mL aggregation at -20 C. 51
His, 10 5.5 90 0.05 0.2 0.0
[0282] The high concentration (75 mg/ml antibody) composition shows
no increase in aggregation after 13 weeks of storage at -20.degree.
C.
Example 17
[0283] A study was conducted to assess the viscosity of a high
concentration composition. The composition in Table 22 was prepared
as described above, and final antibody concentration was adjusted
to about 75 mg/ml. Viscosity measurements were conducted by
applying an average shear rate of 300.sup.s-1 to the composition
placed on a rheometer plate.
TABLE-US-00023 mAb 7.16.6, Viscosity at 5 C., Composition 52: mg/mL
cP 10 mM histidine, pH 5.5, 75 5.7 90 mg/mL trehalose dihydrate,
0.10 mg/mL disodium EDTA dihydrate, 0.4 mg/mL polysorbate 80
[0284] The composition shows a viscosity suitable for subcutaneous
administration.
Sequence CWU 1
1
511410DNAHomo sapiens 1atggactgga cctggagcat ccttttcttg gtggcagcag
caacaggtgc ccactcccag 60gttcagctgg tgcagtctgg agctgaggtg aagaagcctg
gggcctcagt gaaggtctcc 120tgcaaggctt ctggttacac ctttaccagc
tatggtatca actgggtgcg acaggcccct 180ggacaagggc ttgagtggat
gggatggatc agcgtttaca gtggtaacac aaactatgca 240cagaaggtcc
agggcagagt caccatgacc gcagacacat ccacgagcac agcctacatg
300gacctgagga gcctgagatc tgacgacacg gccgtgtatt actgtgcgag
agagggtagc 360agctcgtccg gagactacta ttacggtatg gacgtctggg
gccaagggac cacggtcacc 420gtctcctcag cctccaccaa gggcccatcg
gtcttccccc tggcgccctg ctccaggagc 480acctccgaga gcacagcggc
cctgggctgc ctggtcaagg actacttccc cgaaccggtg 540acggtgtcgt
ggaactcagg cgctctgacc agcggcgtgc acaccttccc agctgtccta
600cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag
caacttcggc 660acccagacct acacctgcaa cgtagatcac aagcccagca
acaccaaggt ggacaagaca 720gttgagcgca aatgttgtgt cgagtgccca
ccgtgcccag caccacctgt ggcaggaccg 780tcagtcttcc tcttcccccc
aaaacccaag gacaccctca tgatctcccg gacccctgag 840gtcacgtgcg
tggtggtgga cgtgagccac gaagaccccg aggtccagtt caactggtac
900gtggacggcg tggaggtgca taatgccaag acaaagccac gggaggagca
gttcaacagc 960acgttccgtg tggtcagcgt cctcaccgtt gtgcaccagg
actggctgaa cggcaaggag 1020tacaagtgca aggtctccaa caaaggcctc
ccagccccca tcgagaaaac catctccaaa 1080accaaagggc agccccgaga
accacaggtg tacaccctgc ccccatcccg ggaggagatg 1140accaagaacc
aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc
1200gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacacc
tcccatgctg 1260gactccgacg gctccttctt cctctacagc aagctcaccg
tggacaagag caggtggcag 1320caggggaacg tcttctcatg ctccgtgatg
catgaggctc tgcacaacca ctacacgcag 1380aagagcctct ccctgtctcc
gggtaaatga 14102469PRTHomo sapiens 2Met Asp Trp Thr Trp Ser Ile Leu
Phe Leu Val Ala Ala Ala Thr Gly1 5 10 15Ala His Ser Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys20 25 30Pro Gly Ala Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe35 40 45Thr Ser Tyr Gly Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu50 55 60Glu Trp Met Gly
Trp Ile Ser Val Tyr Ser Gly Asn Thr Asn Tyr Ala65 70 75 80Gln Lys
Val Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser85 90 95Thr
Ala Tyr Met Asp Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val100 105
110Tyr Tyr Cys Ala Arg Glu Gly Ser Ser Ser Ser Gly Asp Tyr Tyr
Tyr115 120 125Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser Ala130 135 140Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser145 150 155 160Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe165 170 175Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly180 185 190Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu195 200 205Ser Ser
Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr210 215
220Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
Thr225 230 235 240Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro Pro245 250 255Val Ala Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr260 265 270Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val275 280 285Ser His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val290 295 300Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser305 310 315 320Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu325 330
335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln370 375 380Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr405 410 415Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu420 425 430Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser435 440
445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser450 455 460Leu Ser Pro Gly Lys4653723DNAHomo sapiens 3atgaggctcc
ctgctcagct cctggggctg ctaatgctct ggatacctgg atccagtgca 60gatattgtga
tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc
120atctcctgca agtctagtca gagcctcctg catactgatg gaacgaccta
tttgtattgg 180tacctgcaga agccaggcca gcctccacag ctcctgatct
atgaagtttc caaccggttc 240tctggagtgc cagataggtt cagtggcagc
gggtcaggga cagatttcac actgaaaatc 300agccgggtgg aggctgagga
tgttgggatt tattactgca tgcaaaatat acagcttccg 360tggacgttcg
gccaagggac caaggtggaa atcaaacgaa ctgtggctgc accatctgtc
420ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt
tgtgtgcctg 480ctgaataact tctatcccag agaggccaaa gtacagtgga
aggtggataa cgccctccaa 540tcgggtaact cccaggagag tgtcacagag
caggacagca aggacagcac ctacagcctc 600agcagcaccc tgacgctgag
caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 660gtcacccatc
agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgttag 720tga
7234239PRTHomo sapiens 4Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu
Met Leu Trp Ile Pro1 5 10 15Gly Ser Ser Ala Asp Ile Val Met Thr Gln
Thr Pro Leu Ser Leu Ser20 25 30Val Thr Pro Gly Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser35 40 45Leu Leu His Thr Asp Gly Thr Thr
Tyr Leu Tyr Trp Tyr Leu Gln Lys50 55 60Pro Gly Gln Pro Pro Gln Leu
Leu Ile Tyr Glu Val Ser Asn Arg Phe65 70 75 80Ser Gly Val Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe85 90 95Thr Leu Lys Ile
Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr100 105 110Cys Met
Gln Asn Ile Gln Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys115 120
125Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro130 135 140Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu145 150 155 160Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp165 170 175Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp180 185 190Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys195 200 205Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln210 215 220Gly Leu
Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230
2355543PRTHomo sapiens 5Met Asp Phe Gly Leu Ala Leu Leu Leu Ala Gly
Leu Leu Gly Leu Leu1 5 10 15Leu Gly Gln Ser Leu Gln Val Lys Pro Leu
Gln Val Glu Pro Pro Glu20 25 30Pro Val Val Ala Val Ala Leu Gly Ala
Ser Arg Gln Leu Thr Cys Arg35 40 45Leu Ala Cys Ala Asp Arg Gly Ala
Ser Val Gln Trp Arg Gly Leu Asp50 55 60Thr Ser Leu Gly Ala Val Gln
Ser Asp Thr Gly Arg Ser Val Leu Thr65 70 75 80Val Arg Asn Ala Ser
Leu Ser Ala Ala Gly Thr Arg Val Cys Val Gly85 90 95Ser Cys Gly Gly
Arg Thr Phe Gln His Thr Val Gln Leu Leu Val Tyr100 105 110Ala Phe
Pro Asp Gln Leu Thr Val Ser Pro Ala Ala Leu Val Pro Gly115 120
125Asp Pro Glu Val Ala Cys Thr Ala His Lys Val Thr Pro Val Asp
Pro130 135 140Asn Ala Leu Ser Phe Ser Leu Leu Val Gly Gly Gln Glu
Leu Glu Gly145 150 155 160Ala Gln Ala Leu Gly Pro Glu Val Gln Glu
Glu Glu Glu Glu Pro Gln165 170 175Gly Asp Glu Asp Val Leu Phe Arg
Val Thr Glu Arg Trp Arg Leu Pro180 185 190Pro Leu Gly Thr Pro Val
Pro Pro Ala Leu Tyr Cys Gln Ala Thr Met195 200 205Arg Leu Pro Gly
Leu Glu Leu Ser His Arg Gln Ala Ile Pro Val Leu210 215 220His Ser
Pro Thr Ser Pro Glu Pro Pro Asp Thr Thr Ser Pro Glu Ser225 230 235
240Pro Asp Thr Thr Ser Pro Glu Ser Pro Asp Thr Thr Ser Gln Glu
Pro245 250 255Pro Asp Thr Thr Ser Gln Glu Pro Pro Asp Thr Thr Ser
Gln Glu Pro260 265 270Pro Asp Thr Thr Ser Pro Glu Pro Pro Asp Lys
Thr Ser Pro Glu Pro275 280 285Ala Pro Gln Gln Gly Ser Thr His Thr
Pro Arg Ser Pro Gly Ser Thr290 295 300Arg Thr Arg Arg Pro Glu Ile
Gln Pro Lys Ser Cys Asp Lys Thr His305 310 315 320Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val325 330 335Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr340 345
350Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu355 360 365Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys370 375 380Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser385 390 395 400Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys405 410 415Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile420 425 430Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro435 440 445Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu450 455
460Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn465 470 475 480Gly Gln Pro Glu Asn Asn Tyr Lys Ala Thr Pro Pro
Val Leu Asp Ser485 490 495Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg500 505 510Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu515 520 525His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys530 535 540
* * * * *