U.S. patent application number 15/353116 was filed with the patent office on 2017-05-04 for modulated lysine variant species compositions and methods for producing and using the same.
This patent application is currently assigned to AbbVie, Inc.. The applicant listed for this patent is AbbVie, Inc.. Invention is credited to Christopher M. CHUMSAE, Ying JING, Zehra KAYMAKCALAN, Kartik SUBRAMANIAN, Mayda Perez THIELE, Chee Furng WONG, Xiaobei ZENG.
Application Number | 20170121403 15/353116 |
Document ID | / |
Family ID | 52826376 |
Filed Date | 2017-05-04 |
United States Patent
Application |
20170121403 |
Kind Code |
A1 |
SUBRAMANIAN; Kartik ; et
al. |
May 4, 2017 |
MODULATED LYSINE VARIANT SPECIES COMPOSITIONS AND METHODS FOR
PRODUCING AND USING THE SAME
Abstract
The instant invention relates to modulated lysine variant
species compositions comprising a protein, e.g., an antibody, or
antigen-binding portion thereof, and methods, e.g., cell culture
and/or protein purification methods, for producing such modulated
lysine variant species compositions. Methods for using such
compositions to treat a disorder, e.g., a disorder in which
TNF.alpha. is detrimental, are also provided.
Inventors: |
SUBRAMANIAN; Kartik;
(Northborough, MA) ; THIELE; Mayda Perez; (Vega
Alta, PR) ; ZENG; Xiaobei; (Carolina, PR) ;
WONG; Chee Furng; (Singapore, SG) ; KAYMAKCALAN;
Zehra; (Westborough, MA) ; JING; Ying;
(Wellesley, MA) ; CHUMSAE; Christopher M.; (North
Andover, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie, Inc. |
North Chicago |
IL |
US |
|
|
Assignee: |
AbbVie, Inc.
North Chicago
IL
|
Family ID: |
52826376 |
Appl. No.: |
15/353116 |
Filed: |
November 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14923966 |
Oct 27, 2015 |
9499616 |
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15353116 |
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14077988 |
Nov 12, 2013 |
9181337 |
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14923966 |
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61893088 |
Oct 18, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/21 20130101;
C07K 2317/565 20130101; C12P 21/005 20130101; C07K 2317/14
20130101; A61K 2039/505 20130101; C07K 2317/56 20130101; C07K
16/241 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; C12P 21/00 20060101 C12P021/00 |
Claims
1. A method for treating a subject having a disorder in which
TNF.alpha. activity is detrimental, the method comprising
administering a therapeutically effective amount of a composition
comprising a human anti-TNF.alpha. antibody, wherein less than 65%
of the lysine variant species in said composition have zero
C-terminal lysines (Lys 0), wherein the lysine variant species
include the main peak and peaks that elute at a relative residence
time later than the main peak, as detected using weak
cation-exchange chromatography, and wherein the human
anti-TNF.alpha. antibody comprises a light chain variable region
(LCVR) having a CDR1 domain comprising the amino acid sequence of
SEQ ID NO:7, a CDR2 domain comprising the amino acid sequence of
SEQ ID NO:5, and a CDR3 domain comprising the amino acid sequence
of SEQ ID NO:3; and a heavy chain variable region (HCVR) having a
CDR1 domain comprising the amino acid sequence of SEQ ID NO:8, a
CDR2 domain comprising the amino acid sequence of SEQ ID NO:6, and
a CDR3 domain comprising the amino acid sequence of SEQ ID NO:4;
thereby treating the TNF.alpha.-associated disease or disorder.
2. The method of claim 1, wherein the disorder in which TNF.alpha.
activity is detrimental is selected from the group consisting of
rheumatoid arthritis (RA), juvenile idiopathic arthritic, psoriatic
arthritis, ankylosing spondylitis, Crohn's Disease, ulcerative
colitis, plaque psoriasis, hidradenitis suppurativa (HS), uveitis,
active axial spondyloarthritis (active axSpA) and non-radiographic
axial spondyloarthritis (nr-axSpA).
3. The method of claim 1, wherein said human anti-TNF.alpha.
antibody is adalimumab.
4. The method of claim 3, wherein less than 60% of the lysine
variant species in said composition have zero C-terminal lysines
(Lys 0).
5. The method of claim 3, wherein 50-60% of the lysine variant
species in said composition have zero C-terminal lysines (Lys
0).
6. The method of claim 3, wherein less than 55% of the lysine
variant species in said composition have zero C-terminal lysines
(Lys 0).
7. The method of claim 1, wherein said composition is
lyophilized.
8. The method of claim 3, wherein said adalimumab is produced in a
mammalian host cell grown in cell culture.
9. The method of claim 8, wherein the mammalian host cell is
selected from the group consisting of a CHO cell, an NSO cell, a
COS cell, and an SP2 cell.
10. The method of claim 1, wherein the composition is a
pharmaceutical composition further comprising pharmaceutically
acceptable carrier.
11. The method of claim 10, wherein adalimumab is present in said
pharmaceutical composition at a concentration of 25-100 mg/ml.
12. The method of claim 10, wherein said pharmaceutical composition
comprises one or more excipient selected from the group consisting
of a buffering agent, a surfactant and a polyol, or a combination
thereof.
13. A method for treating a subject having a disorder in which
TNF.alpha. activity is detrimental, the method comprising
administering a therapeutically effective amount of a composition
comprising a human anti-TNF.alpha. antibody, wherein the sum of the
lysine variant species having one C-terminal lysine (Lys 1) and the
lysine variant species having two C-terminal lysines (Lys 2) in
said composition is greater than 35%, wherein the lysine variant
species include the main peak and peaks that elute at a relative
residence time later than the main peak, as detected using weak
cation-exchange chromatography, and wherein the human
anti-TNF.alpha. antibody comprises a light chain variable region
(LCVR) having a CDR1 domain comprising the amino acid sequence of
SEQ ID NO:7, a CDR2 domain comprising the amino acid sequence of
SEQ ID NO:5, and a CDR3 domain comprising the amino acid sequence
of SEQ ID NO:3; and a heavy chain variable region (HCVR) having a
CDR1 domain comprising the amino acid sequence of SEQ ID NO:8, a
CDR2 domain comprising the amino acid sequence of SEQ ID NO:6, and
a CDR3 domain comprising the amino acid sequence of SEQ ID NO:4;
thereby treating the TNF.alpha.-associated disease or disorder.
14. The method of claim 13, wherein the disorder in which
TNF.alpha. activity is detrimental is selected from the group
consisting of rheumatoid arthritis (RA), juvenile idiopathic
arthritic, psoriatic arthritis, ankylosing spondylitis, Crohn's
Disease, ulcerative colitis, plaque psoriasis, hidradenitis
suppurativa (HS), uveitis, active axial spondyloarthritis (active
axSpA) and non-radiographic axial spondyloarthritis (nr-axSpA).
15. The method of claim 13, wherein said human anti-TNF.alpha.
antibody is adalimumab.
16. The method of claim 15, wherein the sum of the lysine variant
species having one C-terminal lysine (Lys 1) and the lysine variant
species having two C-terminal lysines (Lys 2) in said composition
is greater than 40%.
17. The method of claim 15, wherein the sum of the lysine variant
species having one C-terminal lysine (Lys 1) and the lysine variant
species having two C-terminal lysines (Lys 2) in said composition
is 40-50%.
18. The method of claim 15, wherein the sum of the lysine variant
species having one C-terminal lysine (Lys 1) and the lysine variant
species having two C-terminal lysines (Lys 2) in said composition
is greater than 50%.
19. The method of claim 15, wherein greater than 25% of the lysine
variant species in said composition have one C-terminal lysine (Lys
1).
20. The method of claim 15, wherein greater than 30% of the lysine
variant species in said composition have one C-terminal lysine (Lys
1).
21. The method of claim 13, wherein said composition is
lyophilized.
22. The method of claim 15, wherein said adalimumab is produced in
a mammalian host cell grown in cell culture.
23. The method of claim 22, wherein the mammalian host cell is
selected from the group consisting of a CHO cell, an NSO cell, a
COS cell, and an SP2 cell.
24. The method of claim 13, wherein the composition is a
pharmaceutical composition further comprising pharmaceutically
acceptable carrier.
25. The method of claim 24, wherein adalimumab is present in said
pharmaceutical composition at a concentration of 25-100 mg/ml.
26. The method of claim 24, wherein said pharmaceutical composition
comprises one or more excipient selected from the group consisting
of a buffering agent, a surfactant and a polyol, or a combination
thereof.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/923,966, filed on Oct. 27, 2015 and granted
as U.S. Pat. No. 9,499,616, which is a divisional of U.S. patent
application Ser. No. 14/077,988, filed on Nov. 12, 2013 and granted
as U.S. Pat. No. 9,181,337, which claims priority to U.S.
Provisional Patent Application Ser. No. 61/893,088, filed on Oct.
18, 2013. The entire disclosures of each of the foregoing
applications are incorporated herein by reference.
SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is sequence_listing.txt.
The size of the text file is 10,893 bytes, and the text file was
created on Jan. 10, 2017. The Sequence Listing does not extend
beyond the scope of the specification and does not contain new
matter.
BACKGROUND OF THE INVENTION
[0003] Large-scale production of antibodies for biopharmaceutical
applications involves the use of cell cultures that are known to
produce antibodies, and antigen-binding portions thereof,
exhibiting varying levels of heterogeneity, which may lead to
either decreased product efficacy and stability or just the
opposite depending upon the nature of the heterogeneity. One source
of antibody heterogeneity involves C-terminal lysine residues, such
as those found on the heavy chains of antibody molecules. For
example, C-terminal lysines can potentially be present on both the
heavy chains of an antibody (Lys 2), on either one of the heavy
chains (Lys 1), or neither of them (Lys 0). Since lysine can carry
a positive charge, antibodies lacking the basic C-terminal
lysine(s) differ in their charge state from ones that contain the
lysine, so that the distribution of lysine variants (% Lys 0, % Lys
1, % Lys 2 of the total lysine sum) can be detected using
ion-exchange chromatographic methods, for example, using a ProPac
WCX-10 Weak Cation-Exchange column for high-resolution separation
of protein isoforms (Dionex, CA), or other methods known in the
art, and subsequently quantified.
[0004] C-terminal lysine heterogeneity is commonly observed in
biopharmaceutical antibody and protein compositions. However, the
development of compositions comprising antibodies, or
antigen-binding portions thereof, with lower or higher levels of
certain lysine variants is an important, need in the
biopharmaceutical industry.
SUMMARY OF THE INVENTION
[0005] The present invention is based on the identification and
optimization of processes for the production of antibodies or
antigen-binding portions thereof (e.g., cell culture processes),
which result in the production of compositions comprising
antibodies, or antigen-binding portions thereof, that comprise
modified distributions of C-terminal lysine variants. C-terminal
lysines can be present on both the heavy chains of an antibody (Lys
2), on either one of them (Lys 1), or neither of them (Lys 0).
Since antibodies lacking basic C-terminal lysines differ in the
charge state from the ones that contain one or two lysines, the
distribution of lysine variants (% Lys 0, % Lys 1, % Lys 2 of the
total lysine sum in the composition) can be detected by
ion-exchange chromatographic methods such as, for example, weak
cation-exchange chromatography (WCX-10) and subsequently quantified
(FIG. 1).
[0006] The present invention provides methods for producing
compositions comprising modulated amounts (or percentages) of Lys
1, Lys 2, and/or Lys 0 variants as compared to the amount (or
percentage) of Lys 1, Lys 2, and/or Lys 0 variants in a starting
population of proteins (referred to herein as "modulated lysine
variant species compositions"). In one embodiment, the methods of
the invention are used to produce a protein composition comprising
an increased amount of Lys 1 and/or Lys 2 variants and a decreased
amount of Lys 0 variants, e.g., as compared to the amount (or
percentage) of Lys 1, Lys 2, and/or Lys 0 variants in a starting
population of proteins, e.g., a control composition. In another
embodiment, the methods of the invention are used to produce a
protein composition comprising a decreased amount of Lys 1 and/or
Lys 2 variants and an increased amount of Lys 0 variants, e.g., as
compared to the amount (or percentage) of Lys 1, Lys 2, and/or Lys
0 variants in a starting population of proteins, e.g., a control
composition.
[0007] For example, a modulated lysine variant species composition
of the invention may comprise more than about 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% Lys 1 and/or Lys 2 antibodies, or
antigen-binding portions thereof relative to the lysine sum (i.e.,
the sum of Lys 0, Lys 1 and Lys 2 in the composition). In one
embodiment, a modulated lysine variant species composition of the
invention may comprise more than about 20% to about 30%, about 30%
to about 40%, about 40% to about 50%, about 60% to about 70%, or
about 70% to about 80%, about 80% to about 90%, or about 90% to
about 100% Lys 1 and/or Lys 2 antibodies, or antigen-binding
portions thereof relative to the lysine sum. Ranges within one or
more of any of the preceding percentages are also included.
[0008] A modulated lysine variant species composition of the
invention may also comprise less than about 80%, 75%, 70%, 65%,
60%, 55%, 50%, 45%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%,
1%, or zero antibodies, or antigen-binding portions thereof that
are Lys 0, relative to the lysine sum. In one embodiment, a
modulated lysine variant species composition of the invention may
also comprise less than about 0% to about 10%, about 10% to about
20%, about 20% to about 30%, about 30% to about 40%, about 50% to
about 60%, about 60% to about 70%, or about 70% to about 80%,
antibodies or antigen-binding portions thereof that are Lys 0,
relative to the lysine sum. Ranges within one or more of any of the
preceding percentages are also included.
[0009] A modulated lysine variant species composition also includes
a composition comprising an antibody, or antigen-binding portion
thereof, that comprises more than about 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% antibodies, or antigen-binding portions thereof
that have one C-terminal lysine (Lys 1) relative to the lysine sum.
Ranges within one or more of any of the preceding percentages are
also included. In one embodiment, a modulated lysine variant
species composition of the invention may comprise more than about
20% to about 30%, about 30% to about 40%, about 40% to about 50%,
about 60% to about 70%, or about 70% to about 80%, about 80% to
about 90%, or about 90% to about 100% Lys 1 antibodies, or
antigen-binding portions thereof relative to the lysine sum. Ranges
within one or more of any of the preceding percentages are also
included.
[0010] A modulated lysine variant species composition also includes
a composition comprising an antibody, or antigen-binding portion
thereof, that comprises more than about 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% antibodies, or antigen-binding portions thereof
that have two C-terminal lysines (Lys 2) relative to the lysine
sum. Ranges within one or more of any of the preceding percentages
are also included. In one embodiment, a modulated lysine variant
species composition of the invention may comprise more than about
20% to about 30%, about 30% to about 40%, about 40% to about 50%,
about 60% to about 70%, or about 70% to about 80%, about 80% to
about 90%, or about 90% to about 100% Lys 2 antibodies, or
antigen-binding portions thereof relative to the lysine sum. Ranges
within one or more of any of the preceding percentages are also
included.
[0011] In one embodiment, the methods of the invention, alone or in
combination, may reduce the amount or percentage of one or more
C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a protein or
antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,
3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges
within one or more of the preceding, as compared to a non-modulated
lysine variant species composition. In one aspect of this
embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants are
reduced by about 1% to about 25%, about 5% to about 20%, about 5%
to about 15%, or about 5% to about 10%, and ranges within one or
more of the preceding, as compared to a non-modulated lysine
variant species composition.
[0012] In one embodiment, the methods of the invention, alone or in
combination, may increase the amount or percentage of one or more
C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a protein or
antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,
3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges
within one or more of the preceding, as compared to a non-modulated
lysine variant species composition. In one aspect of this
embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants are
increased by about 1% to about 25%, about 5% to about 20%, about 5%
to about 15%, or about 5% to about 10%, and ranges within one or
more of the preceding, as compared to a non-modulated lysine
variant species composition,
[0013] As demonstrated herein, modulated lysine variant species
compositions of the invention comprising adalimumab (e.g.,
modulated lysine variant species compositions that comprise only
Lys 1 and Lys 2 variants, and not Lys 0 variants) have increased
therapeutic efficacy and improved biological properties, such as,
for example, increased tissue penetration (such as cartilage),
reduced tissue destruction (such as cartilage), reduced synovial
proliferation, reduced bone erosion, increased protection against
the development of arthritic scores and/or histopathology scores,
reduced cell infiltration, reduced proteoglycan loss, reduced
chondrocyte death, and/or increased TNF.alpha. affinity, e.g., as
compared to a control composition wherein the Lys distribution has
not been modified by the methods described herein (see Example
5).
[0014] In one embodiment, a modulated lysine variant species
composition of the invention comprises about 0.0% to about 10%
acidic species (AR), about 0.0% to about 5% AR, about 0.0% to about
4% AR, about 0.0% to about 3% AR, about 0.0% to about 2% AR, about
3% to about 5% AR, about 5% to about 8% AR, or about 8% to about
10% AR, or about 10% to about 15% AR, and ranges within one or more
of the preceding. In another embodiment, a modulated lysine variant
species composition of the invention comprises, e.g., about 15% or
less AR. In one aspect of this embodiment, a modulated lysine
variant species composition comprises about 14% or less AR, 13% or
less AR, 12% or less AR, 11% or less AR, 10% or less AR, 9% or less
AR, 8% or less AR, 7% or less AR, 6% or less AR, 5% or less AR,
4.5% or less AR, 4% or less AR, 3.5% or less AR, 3% or less AR,
2.5% or less AR, 2% or less AR, 1.9% or less AR, 1.8% or less AR,
1.7% or less AR, 1.6% or less AR, 1.5% or less AR, 1.4% or less AR,
1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1% or less AR,
0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% or less AR,
0.5% or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or less AR,
0.1% or less AR, or 0.0% AR, and ranges within one or more of the
preceding.
[0015] In one embodiment, the acidic species comprise acidic region
1 (AR1) and acidic region 2 (AR2). In one aspect of this
embodiment, a modulated lysine variant species composition
comprises about 0.1% or less AR1 and about 3% or less AR2. In
another aspect of this embodiment, the composition comprises about
0.0% AR1 and about 1.4% AR2. In another aspect of this embodiment,
the composition comprises about 0% to about 3% AR, e.g., about 3%,
2.5%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,
0.4%, 0.3%, 0.2%, 0.1%, or less AR1. In another aspect of this
embodiment, the composition comprises about 0% to about 3% AR,
e.g., about 3%, 2.5%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%,
0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less AR2.
[0016] In one embodiment, the distribution or amount of C-terminal
lysine variants produced by cell culture is modulated by employing
certain media components during production of a protein, for
example, an antibody, of interest. In some embodiments, the
modulated lysine variant species compositions of the invention are
produced by supplementing the media of cells expressing the protein
of interest with one or more amino acids during cell culture. In
certain embodiments, one or more of the amino acids belong to a
group of basic amino acids. In certain embodiments, the one or more
amino acids is arginine, lysine, histidine, or combinations
thereof, including combinations of arginine and/or lysine with
ornithine. In certain embodiments, supplementing the media of cells
expressing the protein of interest with one or more amino acids
reduces the relative amount of a Lys 0 lysine variant, and
increases the relative amount of a Lys 1 and/or Lys 2 lysine
variant in the cell culture or a composition comprising the protein
or antibody purified from the cell culture. In certain embodiments,
these amino acids may be supplemented as dipeptides or tri-peptides
of different combinations for lysine variant modulation
[0017] In some embodiments, the modulated lysine variant species
compositions of the invention are produced by supplementing the
media of cells expressing the protein of interest with zinc during
cell culture. In certain embodiments, supplementing the media of
cells expressing the protein of interest with zinc to an overall
concentration of less than about 10 .mu.M reduces the relative
amount of a Lys 0 lysine variant, and increases the relative amount
of a Lys 1 and/or Lys 2 lysine variant in the cell culture or a
composition comprising the protein or antibody purified from the
cell culture. In certain embodiments, modulation of concentration
of zinc in combination with that of the basic amino acids is used
to modulate the lysine variant distribution.
[0018] In another embodiment, the modulated lysine variant species
compositions of the invention are produced by adjusting the pH,
and/or temperature of a cell culture expressing a protein or
antibody of interest. In certain embodiments, increasing the pH of
the cell culture expressing the protein or antibody of interest
reduces the amount of a Lys 0 lysine variant, and increases the
amount of a Lys 1 and/or Lys 2 lysine variant in the cell culture,
or a composition comprising the protein or antibody purified from
the cell culture, compared to a control cell culture at a lower pH.
In certain embodiments, the final pH of the cell culture is
adjusted to a pH of about 6.7 to about 7.1. In certain embodiments,
the temperature of the cell culture is adjusted to a temperature of
about 31.degree. C. to about 37.degree. C. In certain embodiments,
decreasing the temperature of the cell culture expressing the
protein or antibody of interest reduces the amount of a Lys 0
lysine variant, and increases the amount of a Lys 1 and/or Lys 2
lysine variant in the cell culture, or a composition comprising the
protein or antibody purified from the cell culture, compared to a
control cell culture at a higher temperature.
[0019] In certain embodiments, the methods of the invention
modulate the distribution, reduce the amount, or increase the
amount of lysine variants present in the resulting composition. In
certain embodiments, the resulting composition has a reduced amount
of Lys 0 lysine variant, and an increased amount of Lys 1 and Lys 2
lysine variants compare to a composition that was not prepared
according to the methods of the present application. In one aspect,
the sample comprises a cell harvest wherein the cell line is
employed to produce specific proteins of the present invention. In
a particular aspect, the sample is prepared from a cell line used
to produce anti-TNF-.alpha. antibodies.
[0020] In certain embodiments, the methods of the invention for
modulating the C-terminal lysine variants in a protein composition
described herein is exerted by employing one or more of the
foregoing methods during the production and purification of the
desired protein, such as antibodies or antigen-binding portions
thereof.
[0021] The purity/heterogeneity of the proteins of interest in the
resultant sample product can be analyzed using methods well known
to those skilled in the art, e.g., weak cation exchange
chromatography (WCX), capillary isoelectric focusing (cIEF),
size-exclusion chromatography, Poros.TM. A HPLC Assay, Host Cell
Protein ELISA, Protein A ELISA, and western blot analysis.
[0022] In one embodiment, the antibody, or antigen-binding portion
thereof, of the compositions disclosed herein is an anti-TNF.alpha.
antibody, or antigen-binding portion thereof. For example, in one
aspect of this embodiment, the anti-TNF.alpha. antibody, or
antigen-binding portion thereof, dissociates from human TNF.alpha.
with a K.sub.d of about 1.times.10.sup.-8 M or less and a K.sub.off
rate constant of 1.times.10.sup.-3 S.sup.-1 or less. In another
aspect of this embodiment, the anti-TNF.alpha. antibody, or
antigen-binding portion thereof, comprises a light chain variable
region (LCVR) having a CDR1 domain comprising the amino acid
sequence of SEQ ID NO: 7, a CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 5, and a CDR3 domain comprising the amino
acid sequence of SEQ ID NO: 3; and a heavy chain variable region
(HCVR) having a CDR1 domain comprising the amino acid sequence of
SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of
SEQ ID NO: 6, and a CDR3 domain comprising the amino acid sequence
of SEQ ID NO: 4. In still another aspect of this embodiment, the
anti-TNF.alpha. antibody, or antigen-binding portion thereof,
comprises a light chain variable region comprising the amino acid
sequence set forth in SEQ ID NO: 1 and a heavy chain variable
region comprising the amino acid sequence set forth in SEQ ID NO:
2. In yet another aspect of this embodiment, the anti-TNF.alpha.
antibody, or antigen-binding portion thereof, is adalimumab, or an
antigen binding-portion thereof.
[0023] In one embodiment, a modulated lysine variant species
composition of the invention comprises adalimumab, and has a
percentage of Lys 1/Lys 2 variants that is not the same as the
percentage of Lys 1/Lys 2 variants present in adalimumab formulated
as HUMIRA.RTM. as currently approved and described in the
"Highlights of Prescribing Information" for HUMIRA.RTM.
(adalimumab) Injection (Revised January 2008), the contents of
which are hereby incorporated herein by reference.
[0024] In another embodiment, a modulated lysine variant species
composition of the invention comprises adalimumab, and has a
percentage of Lys 1/Lys 2 variants that is higher than the
percentage of Lys 1/Lys 2 variants present in adalimumab formulated
as HUMIRA.RTM. as currently approved and described in the
"Highlights of Prescribing Information" for HUMIRA.RTM.
(adalimumab) Injection (Revised January 2008).
[0025] As demonstrated herein, a modulated lysine variant species
composition of the present invention exhibits increased efficacy in
treating or preventing diseases in a subject compared to a
corresponding non-modulated lysine variant species composition. In
particular, modulated lysine variant species compositions
comprising anti-TNF.alpha. antibodies, or antigen binding portions
thereof, e.g., adalimumab, exhibit increased cartilage tissue
penetration, reduced cartilage destruction, reduced synovial
proliferation, and/or reduced bone erosion as compared to a
non-modulated lysine variant species composition. In another aspect
of this embodiment, modulated lysine variant species compositions
comprising anti-TNF.alpha. antibodies, or antigen binding portions
thereof, e.g., adalimumab, exhibit increased protection against the
development of arthritic scores and/or increased protection against
the development of histopathology scores as compared to a
non-modulated lysine variant species composition, when administered
to an animal model of arthritis.
[0026] In still another embodiment, a modulated lysine variant
species composition comprising an anti-TNF.alpha. antibody, or
antigen binding portion thereof, e.g., adalimumab, exhibits reduced
cell infiltration, reduced proteoglycan loss, and/or reduced
chondrocyte death as compared to a non-modulated lysine variant
species composition.
[0027] In another embodiment, a modulated lysine variant species
composition of the invention comprises anti-TNF.alpha. antibodies,
or antigen-binding portions thereof, e.g., adalimumab, exhibit
increased TNF.alpha. affinity as compared to a non-modulated lysine
variant species composition.
[0028] Another aspect of the invention provides methods for
treating a subject having a disorder in which TNF.alpha. activity
is detrimental, e.g., rheumatoid arthritis, juvenile idiopathic
arthritic, psoriatic arthritis, ankylosing spondylitis, Crohn's
Disease, plaque psoriasis, active axial spondyloarthritis (active
axSpA) and non-radiographic axial spondyloarthritis (nr-axSpA),
comprising administering a therapeutically effective amount of the
composition of a modulated lysine variant species composition to
the subject, thereby treating the TNF.alpha.-associated disease or
disorder.
[0029] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a representation of the lysine variants in a
sample WCX-10 chromatogram and quantification scheme of each of the
variants.
[0031] FIG. 2 depicts the effect of zinc modulation in adalimumab
producing cell line 1, media 1 on viable cell density (n=2).
[0032] FIG. 3 depicts the effect of zinc modulation in adalimumab
producing cell line 1, media 1 on viability (n=2).
[0033] FIG. 4 depicts the effect of zinc modulation in adalimumab
producing cell line 1, media 1 on harvest titer (n=2)
[0034] FIG. 5 depicts the effect of zinc modulation in adalimumab
producing cell line 1, media 1 on day 10 WCX-10 profile relative
lysine distribution (n=2).
[0035] FIG. 6 depicts the effect of zinc modulation in adalimumab
producing cell line 1, media 1 on viable cell density (n=2).
[0036] FIG. 7 depicts the effect of zinc modulation in adalimumab
producing cell line 1, media 1 on viability (n=2).
[0037] FIG. 8 depicts the effect of zinc modulation in adalimumab
producing cell line 1, media 1 on harvest titer (n=2).
[0038] FIG. 9 depicts the effect of zinc modulation in adalimumab
producing cell line 1, media 1 on day 10 WCX-10 profile relative
lysine distribution (n=2).
[0039] FIG. 10 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 2, media 1 on
viable cell density (n=2).
[0040] FIG. 11 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 2, media 1 on
viability (n=2).
[0041] FIG. 12 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 2, media 1 on
harvest titer (n=2).
[0042] FIG. 13 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 2, media 1 on day
10 on WCX-10 profile relative lysine distribution (n=2).
[0043] FIG. 14 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 2, media 1 on day
12 on WCX-10 profile relative lysine distribution (n=2).
[0044] FIG. 15 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 3, media 1 on
viable cell density (n=2).
[0045] FIG. 16 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 3, media 1 on
viability (n=2).
[0046] FIG. 17 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 3, media 1 on
harvest titer (n=2).
[0047] FIG. 18 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 3, media 1 on
WCX-10 profile relative lysine distribution (n=2).
[0048] FIG. 19 depicts the effect of varying total arginine
concentration in adalimumab producing cell line 1, media 1 on
WCX-10 profile relative lysine distribution (n=2).
[0049] FIG. 20 depicts the effect of arginine addition to
adalimumab producing cell line 1, media 2 on day 11 on WCX-10
profile relative lysine distribution (n=2).
[0050] FIG. 21 depicts the effect of arginine addition to
adalimumab producing cell line 2, media 3 on WCX-10 profile
relative lysine distribution (n=2).
[0051] FIG. 22 depicts the effect of varying total arginine
concentration in mAb1 producing cell line on WCX-10 profile
relative lysine distribution (n=1).
[0052] FIG. 23 depicts the effect of varying total arginine
concentration in mAb2 producing cell line on WCX-10 profile
relative lysine distribution (n=2).
[0053] FIG. 24 depicts the effect of varying total lysine
concentration in adalimumab producing cell line 2, media 1 on
viable cell density (n=2).
[0054] FIG. 25 depicts the effect of varying total lysine
concentration in adalimumab producing cell line 2, media 1 on
viability (n=2).
[0055] FIG. 26 depicts the effect of varying total lysine
concentration in adalimumab producing cell line 2, media 1 on
harvest titer (n=2).
[0056] FIG. 27 depicts the effect of varying total lysine
concentration in adalimumab producing cell line 2, media 1 on
WCX-10 profile relative lysine distribution (n=2).
[0057] FIG. 28 depicts the effect of varying total lysine
concentration in adalimumab producing cell line 3, media 1 on
viable cell density (n=2).
[0058] FIG. 29 depicts the effect of varying total lysine
concentration in adalimumab producing cell line 3, media 1 on
viability (n=2).
[0059] FIG. 30 depicts the effect of varying total lysine
concentration in adalimumab producing cell line 3, media 1 on
harvest titer (n=2).
[0060] FIG. 31 depicts the effect of varying total lysine
concentration in adalimumab producing cell line 3, media 1 on
WCX-10 profile relative lysine distribution (n=2).
[0061] FIG. 32 depicts the effect of varying total lysine
concentration in adalimumab producing cell line 1, media 1 on
WCX-10 profile relative lysine distribution (n=2).
[0062] FIG. 33 depicts the effect of lysine addition to adalimumab
producing cell line 1, media 2 on WCX-10 profile relative lysine
distribution (n=2).
[0063] FIG. 34 depicts the effect of lysine addition to adalimumab
producing cell line 2, media 3 on WCX-10 profile relative lysine
distribution (n=2).
[0064] FIG. 35 depicts the effect of varying total lysine
concentration in mAb1 producing cell line on WCX-10 profile
relative lysine distribution (n=1).
[0065] FIG. 36 depicts the effect of varying total lysine
concentration in mAb2 producing cell line on WCX-10 profile
relative lysine distribution (n=2).
[0066] FIG. 37 depicts the effect of varying total histidine
concentration in adalimumab producing cell line 2, media 1 on
viable cell density (n=2).
[0067] FIG. 38 depicts the effect of varying total histidine
concentration in adalimumab producing cell line 2, media 1 on
viability (n=2).
[0068] FIG. 39 depicts the effect of varying total histidine
concentration in adalimumab producing cell line 2, media 1 on
harvest titer (n=2).
[0069] FIG. 40 depicts the effect of varying total histidine
concentration in adalimumab producing cell line 2, media 1 on
WCX-10 profile relative lysine distribution (n=2).
[0070] FIG. 41 depicts the effect of varying total histidine
concentration in adalimumab producing cell line 3, media 1 on
viable cell density (n=2).
[0071] FIG. 42 depicts the effect of total histidine concentration
in adalimumab producing cell line 3, media 1 on viability
(n=2).
[0072] FIG. 43 depicts the effect of varying total histidine
concentration in adalimumab producing cell line 3, media 1 on
harvest titer (n=2).
[0073] FIG. 44 depicts the effect of varying total histidine
concentration in adalimumab producing cell line 3, media 1 on
WCX-10 profile relative lysine distribution (n=2).
[0074] FIG. 45 depicts the effect of varying total histidine
concentration in adalimumab producing cell line 1, media 1 on
WCX-10 profile relative lysine distribution (n=2).
[0075] FIG. 46 depicts the effect of histidine addition to
adalimumab producing cell line 1, media 2 on WCX-10 profile
relative lysine distribution (n=2).
[0076] FIG. 47 depicts the effect of histidine addition to
adalimumab producing cell line 2, media 3 on WCX-10 profile
relative lysine distribution (n=2).
[0077] FIG. 48 depicts the effect of varying total histidine
concentration in mAb1 producing cell line on WCX-10 profile
relative lysine distribution (n=1).
[0078] FIG. 49 depicts the effect of varying total histidine
concentration in mAb2 producing cell line on WCX-10 profile
relative lysine distribution (n=2).
[0079] FIG. 50 depicts the effect of concentration modulation of
multiple amino acids to adalimumab producing cell line 1, media 1
on viable cell density (n=2).
[0080] FIG. 51 depicts the effect of concentration modulation of
multiple amino acids to adalimumab producing cell line 1, media 1
on viability.
[0081] FIG. 52 depicts the effect of concentration modulation of
multiple amino acids to adalimumab producing cell line 1, media 1
on harvest titer.
[0082] FIG. 53 depicts the effect of concentration modulation of
multiple amino acids to adalimumab producing cell line 1, media 1
on WCX-10 profile relative lysine distribution.
[0083] FIG. 54 depicts the effect of concentration modulation of
multiple amino acids to adalimumab producing cell line 3, media 1
on viable cell density (n=2).
[0084] FIG. 55 depicts the effect of concentration modulation of
multiple amino acids to adalimumab producing cell line 3, media 1
on viability.
[0085] FIG. 56 depicts the effect of concentration modulation of
multiple amino acids to adalimumab producing cell line 3, media 1
on harvest titer.
[0086] FIG. 57 depicts the effect of concentration modulation of
multiple amino acids to adalimumab producing cell line 3, media 1
on WCX-10 profile relative lysine distribution.
[0087] FIG. 58 depicts the effect of concentration modulation of
multiple amino acids to adalimumab producing cell line 2, media 1
on WCX-10 profile relative lysine distribution (n=2).
[0088] FIGS. 59A-B depict the effect of concentration modulation of
zinc and multiple amino acids to adalimumab producing cell line 1,
media 1 on WCX-10 profile relative lysine distribution A) overall
prediction plot, B) prediction plots for each additive.
[0089] FIG. 60 depicts the effect of peptides of varying length
added to adalimumab producing cell line 2, media 1 on WCX-10
profile relative lysine distribution (n=2).
[0090] FIG. 61 depicts the effect of pH modulation on adalimumab
producing cell line 1, media 1 on viable cell density.
[0091] FIG. 62 depicts the effect of pH modulation on adalimumab
producing cell line 1, media 1 on viability.
[0092] FIG. 63 depicts the effect of pH modulation on adalimumab
producing cell line 1, media 1 on harvest titer.
[0093] FIG. 64 depicts the effect of pH modulation on adalimumab
producing cell line 1, media 1 on WCX-10 profile relative lysine
distribution.
[0094] FIG. 65 depicts the effect of pH modulation on adalimumab
producing cell line 1, media 2 on viable cell density.
[0095] FIG. 66 depicts the effect of pH modulation on adalimumab
producing cell line 1, media 2 on viability.
[0096] FIG. 67 depicts the effect of pH modulation on adalimumab
producing cell line 1, media 2 on harvest titer.
[0097] FIG. 68 depicts the effect of pH modulation on adalimumab
producing cell line 1, media 2 on WCX-10 profile relative lysine
distribution.
[0098] FIG. 69 depicts the effect of pH modulation on adalimumab
producing cell line 3, media 1 on viable cell density.
[0099] FIG. 70 depicts the effect of pH modulation on adalimumab
producing cell line 3, media 1 on viability.
[0100] FIG. 71 depicts the effect of pH modulation on adalimumab
producing cell line 3, media 1 on harvest titer.
[0101] FIG. 72 depicts the effect of pH modulation on adalimumab
producing cell line 3, media 1 on WCX-10 profile relative lysine
distribution.
[0102] FIG. 73 depicts the effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 2 at 35.degree. C. on
viable cell density.
[0103] FIG. 74 depicts the effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 2 at 35.degree. C. on
viability.
[0104] FIG. 75 depicts the effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 2 at 35.degree. C. on
harvest titer.
[0105] FIG. 76 depicts the effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 2 at 35.degree. C. on
WCX-10 profile relative lysine distribution.
[0106] FIG. 77 depicts the effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 2 at 33.degree. C. on
viable cell density.
[0107] FIG. 78 depicts the effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 2 at 33.degree. C. on
viability.
[0108] FIG. 79 depicts the effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 2 at 33.degree. C. on
harvest titer.
[0109] FIG. 80 depicts the effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 2 at 33.degree. C. on
WCX-10 profile relative lysine distribution.
[0110] FIG. 81 depicts effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 1 on viable cell
density.
[0111] FIG. 82 depicts effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 1 on viability.
[0112] FIG. 83 depicts effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 1 on harvest titer.
[0113] FIG. 84 depicts effect of dissolved oxygen modulation on
adalimumab producing cell line 1, media 1 on WCX-10 profile
relative lysine distribution.
[0114] FIG. 85 depicts the effect of temperature modulation on
adalimumab producing cell line 1, media 1 on viable cell density
(n=2).
[0115] FIG. 86 depicts the effect of temperature modulation on
adalimumab producing cell line 1, media 1 on viability (n=2).
[0116] FIG. 87 depicts the effect of temperature modulation on
adalimumab producing cell line 1, media 1 on harvest titer
(n=2).
[0117] FIG. 88 depicts the effect of temperature modulation on
adalimumab producing cell line 1, media 1 on WCX-10 profile
relative lysine distribution (n=2).
[0118] FIG. 89 depicts the effect of temperature modulation on
adalimumab producing cell line 1, media 1 on viable cell density
(n=2).
[0119] FIG. 90 depicts the effect of temperature modulation on
adalimumab producing cell line 1, media 1 on viability (n=2).
[0120] FIG. 91 depicts the effect of temperature modulation on
adalimumab producing cell line 1, media 1 on harvest titer
(n=2).
[0121] FIG. 92 depicts the effect of temperature modulation on
adalimumab producing cell line 1, media 1 on WCX-10 profile
relative lysine distribution (n=2).
[0122] FIG. 93 depicts the effect of dissolved oxygen and
temperature modulation on adalimumab producing cell line 3, media 1
on viable cell density.
[0123] FIG. 94 depicts the effect of dissolved oxygen and
temperature modulation on adalimumab producing cell line 3, media 1
on viability.
[0124] FIG. 95 depicts the effect of dissolved oxygen and
temperature modulation on adalimumab producing cell line 3, media 1
on harvest titer.
[0125] FIG. 96 depicts the effect of dissolved oxygen and
temperature modulation on adalimumab producing cell line 3, media 1
on WCX-10 profile relative lysine distribution.
[0126] FIGS. 97A-D depict the effect of varying phosphate
concentration in chemically defined media with cell line 1 on (A)
viable cell density profile; (B) viability profile; (C) harvest
titer; and (D) lysine variant distribution (n=2).
[0127] FIGS. 98A-D depict the effect of varying phosphate
concentration in chemically defined media with cell line 2 on (A)
viable cell density profile; (B) viability profile; (C) harvest
titer; and (D) lysine variant distribution (n=2).
[0128] FIGS. 99A-F depict the effects of varying lysine, arginine,
zinc and phosphate concentration in chemically defined media with
cell line 1 on (A) peak viable cell density; (B) harvest viability;
(C) harvest titer; (D) the relative fraction of Lys 0; (E) the
relative fraction of Lys 1; and (F) the relative fraction of Lys
2.
[0129] FIG. 100 depicts a chromatogram wherein acidic and basic
species are identified in adalimumab and various fractions are
delineated.
[0130] FIGS. 101A-B depict the (A) average arthritic scores and (B)
growth related weight gain of mice administered low AR composition,
control AR composition, AR1 composition, modulated lysine variant
species composition (Lys-1/2), and placebo.
[0131] FIG. 102 depicts the average arthritic scores (area under
the curve) of mice administered low AR composition, control
(normal) AR composition, AR1 composition, modulated lysine variant
species composition (Lys-1/2), and placebo (PBO).
[0132] FIGS. 103A-B depict the (A) average trough serum drug levels
and (B) average trough serum ADA levels for mice administered low
AR composition, control AR composition, AR1 composition, and
modulated lysine variant species composition.
[0133] FIG. 104 depicts the average pK and ADA profiles (area under
the curve) for mice administered low AR composition, control
(normal) AR composition, AR1 composition, modulated lysine variant
species composition (Lys-1/2), and placebo (PBO).
[0134] FIG. 105 depicts complexed TNF levels (area under the curve)
and shows that the cumulative serum concentration values of
adalimumab for mice administered low AR composition, control
(normal) AR composition, AR1 composition, modulated lysine variant
species composition (Lys-1/2), and placebo (PBO) during the ten
week treatment period was highest for the modulated lysine variant
species composition, the low AR composition, and the control AR
composition, and lowest for the AR1 composition.
[0135] FIG. 106 depicts the chondrocyte death, synovial
proliferation, proteoglycan loss, cartilage destruction, and bone
erosion of mice administered low AR composition, control (normal)
AR composition, AR1 composition, modulated lysine variant species
composition (Lys-1/2), and placebo (PBO).
[0136] FIGS. 107A-D illustrate the average drug levels for various
tissues (paw, lymph node, spleen, skin, knee and serum) for mice
administered (A) low AR composition; (B) control AR composition;
(C) AR1 composition; and (D) modulated lysine variant species
composition (Lys-1/2).
[0137] FIGS. 108A-D illustrate the average ADA levels for various
tissues (paw, lymph node, spleen, skin, knee and serum) for mice
administered (A) low AR composition; (B) control AR composition;
(C) AR1 composition; and (D) modulated lysine variant species
composition (Lys-1/2).
[0138] FIGS. 109A-D show the results of a micro CT analysis of
spines and femurs obtained from TNF-Tg197 transgenic mice which
were administered placebo, low AR composition, control (normal) AR
composition, AR1 composition, and modulated lysine variant species
composition (Lys-1/2). The graphs depict the effect of the
administered compositions on (A) vertebra bone volume; (B) vertebra
trabecular number; (C) vertebra trabecular thickness; and (D)
vertebra trabecular space.
[0139] FIGS. 110A-D show the results of a micro CT analysis of
spines and femurs obtained from TNF-Tg197 transgenic mice which
were administered placebo, low AR composition, control (normal) AR
composition, AR1 composition, and modulated lysine variant species
composition (Lys-1/2). The graphs depict the effect of the
administered compositions on (A) vertebra bone loss; (B) vertebra
trabecular number; (C) vertebra trabecular thickness; and (D)
vertebra trabecular space.
[0140] FIGS. 111A-D show results of a micro CT analysis of spines
and femurs obtained from TNF-Tg197 transgenic mice which were
administered placebo, low AR composition, control (normal) AR
composition, AR1 composition, and modulated lysine variant species
composition (Lys-1/2). The graphs depict the effect of the
administered compositions on (A) trabecular bone volume/total
volume at the femoral metaphysis; (B) trabecular number at the
femoral metaphysis; (C) trabecular thickness at the femoral
metaphysis; and (D) trabecular separation at the femoral
metaphysis.
[0141] FIGS. 112A-F depict micro CT images of the spine from each
of six groups of mice administered the following compositions:
naive (A), vehicle (control) (B), low AR composition (group 5) (C),
low host cell protein (HCP) composition (group 7) (D), AR1
composition (containing only AR1 acidic variants) (group 8) (E),
and Lys-1/2 composition (containing only Lys 1 and Lys 2 variants)
(group 9) (F).
[0142] FIGS. 113A-F depict micro CT images of the femur from each
of six groups of mice administered the following compositions:
naive (A), vehicle (control) (B), low AR composition (group 5) (C),
low host cell protein (HCP) composition (group 7) (D), AR1
composition (containing only AR1 acidic variants) (group 8) (E),
and Lys-1/2 composition (containing only Lys 1 and Lys 2 variants)
(group 9) (F).
DETAILED DESCRIPTION OF THE INVENTION
[0143] The present invention is based on the identification and
optimization of processes for the production of antibodies or
antigen-binding portions thereof (e.g., cell culture processes), to
produce compositions comprising antibodies, or antigen-binding
portions thereof, that contain modified distributions of C-terminal
lysine variants in a population of proteins, e.g., antibodies or
antigen binding portions thereof. C-terminal lysines can be present
on both the heavy chains of the antibody (Lys 2), on either one of
the heavy chains of the antibody (Lys 1), or neither of the heavy
chains of the antibody (Lys 0). Antibodies lacking basic C-terminal
lysines differ in the charge state from those that contain one or
two lysines. Therefore, as shown in FIG. 1, the distribution of
lysine variants (% Lys 0, % Lys 1, % Lys 2) of the total lysine sum
in the composition can be detected by ion-exchange chromatographic
methods such as weak cation-exchange chromatography (WCX-10) and
subsequently quantified.
[0144] The present invention provides methods for producing
compositions comprising modulated amounts (or percentages) of Lys
1, Lys 2, and/or Lys 0 variants as compared to the amount (or
percentage) of Lys 1, Lys 2, and/or Lys 0 variants in a starting
population of proteins (referred to herein as "modulated lysine
variant species compositions"). In one embodiment, the methods of
the invention are used to produce a protein composition comprising
an increased amount of Lys 1 and/or Lys 2 variants and a decreased
amount of Lys 0 variants, e.g., as compared to the amount (or
percentage) of Lys 1, Lys 2, and/or Lys 0 variants in a starting
population of proteins, e.g., a control composition. In another
embodiment, the methods of the invention are used to produce a
protein composition comprising a decreased amount of Lys 1 and/or
Lys 2 variants and an increased amount of Lys 0 variants, e.g., as
compared to the amount (or percentage) of Lys 1, Lys 2, and/or Lys
0 variants in a starting population of proteins, e.g., a control
composition.
[0145] As demonstrated herein, modulated lysine variant species
compositions of the invention comprising adalimumab that have
increased Lys1/Lys2 percentages and decreased Lys 0 percentages,
e.g., compositions that comprise only Lys 1 and Lys 2 variants, and
not Lys 0 variants, have improved therapeutic efficacy and improved
biological properties, for example, increased cartilage tissue
penetration, reduced cartilage destruction, reduced synovial
proliferation, reduced bone erosion, increased protection against
the development of arthritic scores and/or histopathology scores,
reduced cell infiltration, reduced proteoglycan loss, reduced
chondrocyte death, and/or increased TNF.alpha. affinity, as
compared to a non-modulated lysine variant species composition (see
Example 5).
[0146] In one embodiment, the present invention provides methods
for culturing host cells under conditions that modulate the
distribution or amount of C-terminal lysine variants that are
expressed by the cells or are present in purified protein
compositions, e.g., compositions comprising an antibody or
antigen-binding portion thereof. In some aspects of this
embodiment, the methods described herein comprise culturing cells
in the presence of one or more amino acids. In other aspects of
this embodiment, the methods described herein comprise culturing
cells in the presence of zinc, alone or in combination with one or
more amino acids. In still other aspects of this embodiment, the
methods described herein comprise culturing cells under conditions
of increased or decreased temperatures compared to a control
temperature. In other aspects of this embodiment, the methods
described herein comprise culturing cells under conditions of
increased or decreased pH compared to a control pH. In still other
aspects of this embodiment, the methods described herein comprise
culturing cells under conditions of increased or decreased
dissolved oxygen (DO) concentration as compared to a control level
of DO. In yet a further embodiment, the methods described herein
employ culturing cells under conditions of increased or decreased
phosphate concentration compared to a control level of phosphate
concentration. Combinations of one or more of these methods are
also included within the methods of the invention.
I. DEFINITIONS
[0147] In order that the present invention may be more readily
understood, certain terms are first defined.
[0148] Ion exchange chromatography (e.g., WCX-10) analysis of
adalimumab has shown that it has three main basic charge variants
(i.e., Lys 0, Lys 1, and Lys 2). These variants, or charged
isomers, are the result of incomplete post-translational cleavage
of the C-terminal lysine residues on the heavy chains of the
antibody. In addition to the lysine variants, ion exchange
chromatography, e.g., the WCX-10 analysis, measures the presence of
acidic species (AR), e.g., AR1 and AR2.
[0149] As used herein, the term "lysine variant species" refers to
an antibody, or antigen-binding portion thereof, comprising heavy
chains with either zero, one or two C-terminal lysines. For
example, the "Lys 0" variant comprises an antibody, or
antigen-binding portion thereof, with heavy chains that do not
comprise a C-terminal lysine. The "Lys 1" variant comprises an
antibody, or antigen-binding portion thereof, with one heavy chain
that comprises a C-terminal lysine. The "Lys 2" variant comprises
an antibody, or antigen-binding portion thereof, with both heavy
chains comprising a C-terminal lysine. Lysine variants can be
detected by ion chromatography, including weak cation exchange
chromatography, for example, WCX-10, of the expression product of a
host cell expressing the antibody, or antigen-binding portion
thereof. For example, but not by way of limitation, FIG. 1 depicts
WCX analysis of adalimumab wherein the three lysine variants, as
well as two acidic species, are resolved from each other.
[0150] A composition of the invention may comprise more than one
lysine variant species of an antibody, or antigen-binding portion
thereof. For example, in one embodiment, the composition may
comprise a Lys 2 variant of an antibody, or antigen-binding portion
thereof. The composition may comprise a Lys 1 variant of an
antibody, or antigen-binding portion thereof. The composition may
comprise a Lys 0 variant of an antibody, or antigen-binding portion
thereof. In another embodiment, the composition may comprise both
Lys 1 and Lys 2, or Lys 1 and Lys 0, or Lys 2 and Lys 0 variants of
an antibody, or antigen-binding portion thereof. In another
embodiment, the composition may comprise all three lysine variant
species, i.e., Lys 0, Lys 1 and Lys 2, of an antibody, or
antigen-binding portion thereof.
[0151] As used herein, the term "modulated lysine variant species
composition," refers to a composition comprising an antibody, or
antigen-binding portion thereof, that comprises a modulated, e.g.,
increased or decreased, percentage of Lys 1, Lys 2, and/or Lys 0
variants in the composition after production using one or more of
the cell culture methods described herein, as compared to a
corresponding non-modulated lysine variant species composition
(e.g., a control composition). In one embodiment, a modulated
lysine variant species composition comprises an increased
percentage of Lys 1 and/or Lys 2 variants and a decreased
percentage of Lys 0 variants after production using one or more of
the cell culture methods described herein, as compared to a
corresponding non-modulated lysine variant species composition
(e.g., a control composition).
[0152] For example, a modulated lysine variant species composition
of the invention may comprise more than about 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% Lys 1 and/or Lys 2 antibodies, or
antigen-binding portions thereof relative to the lysine sum (i.e.,
the sum of Lys 0, Lys 1 and Lys 2 in the composition). In one
embodiment, a modulated lysine variant species composition of the
invention may comprise more than about 20% to about 30%, about 30%
to about 40%, about 40% to about 50%, about 60% to about 70%, or
about 70% to about 80%, about 80% to about 90%, or about 90% to
about 100% Lys 1 and/or Lys 2 antibodies, or antigen-binding
portions thereof relative to the lysine sum. Ranges within one or
more of any of the preceding percentages are also included.
[0153] A modulated lysine variant species composition of the
invention may also comprise less than about 80%, 75%, 70%, 65%,
60%, 55%, 50%, 45%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%,
1%, or zero antibodies, or antigen-binding portions thereof that
are Lys 0, relative to the lysine sum. In one embodiment, a
modulated lysine variant species composition of the invention may
also comprise less than about 0% to about 10%, about 10% to about
20%, about 20% to about 30%, about 30% to about 40%, about 50% to
about 60%, about 60% to about 70%, or about 70% to about 80%,
antibodies or antigen-binding portions thereof that are Lys 0,
relative to the lysine sum. Ranges within one or more of any of the
preceding percentages are also included.
[0154] A modulated lysine variant species composition also includes
a composition comprising an antibody, or antigen-binding portion
thereof, that comprises more than about 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% antibodies, or antigen-binding portions thereof
that have one C-terminal lysine (Lys 1) relative to the lysine sum.
Ranges within one or more of any of the preceding percentages are
also included. In one embodiment, a modulated lysine variant
species composition of the invention may comprise more than about
20% to about 30%, about 30% to about 40%, about 40% to about 50%,
about 60% to about 70%, or about 70% to about 80%, about 80% to
about 90%, or about 90% to about 100% Lys 1 antibodies, or
antigen-binding portions thereof relative to the lysine sum. Ranges
within one or more of any of the preceding percentages are also
included.
[0155] A modulated lysine variant species composition also includes
a composition comprising an antibody, or antigen-binding portion
thereof, that comprises more than about 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% antibodies, or antigen-binding portions thereof
that have two C-terminal lysines (Lys 2) relative to the lysine
sum. Ranges within one or more of any of the preceding percentages
are also included. In one embodiment, a modulated lysine variant
species composition of the invention may comprise more than about
20% to about 30%, about 30% to about 40%, about 40% to about 50%,
about 60% to about 70%, or about 70% to about 80%, about 80% to
about 90%, or about 90% to about 100% Lys 2 antibodies, or
antigen-binding portions thereof relative to the lysine sum. Ranges
within one or more of any of the preceding percentages are also
included.
[0156] In one embodiment, the methods of the invention, alone or in
combination, may reduce the amount or percentage of one or more
C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a protein or
antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,
3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges
within one or more of the preceding, as compared to a non-modulated
lysine variant species composition. In one aspect of this
embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants are
reduced by about 1% to about 25%, about 5% to about 20%, about 5%
to about 15%, or about 5% to about 10%, and ranges within one or
more of the preceding, as compared to a non-modulated lysine
variant species composition.
[0157] In one embodiment, the methods of the invention, alone or in
combination, may increase the amount or percentage of one or more
C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a protein or
antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,
3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges
within one or more of the preceding, as compared to a non-modulated
lysine variant species composition. In one aspect of this
embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants are
increased by about 1% to about 25%, about 5% to about 20%, about 5%
to about 15%, or about 5% to about 10%, and ranges within one or
more of the preceding, as compared to a non-modulated lysine
variant species composition.
[0158] As used herein, the term "non-modulated lysine variant
species composition," refers to a composition in which the levels
of lysine variant species have not been modulated. In one
embodiment, a non-modulated lysine variant species composition is a
composition comprising an antibody, or antigen-binding portion
thereof, that was not produced using one or more methods disclosed
herein for producing a modulated lysine variant species composition
(e.g., a control composition). For example, the control composition
can be a starting composition of antibodies whose lysine variant
species need to be modulated, e.g., in order to increase
therapeutic efficacy and/or stability. For example, the starting
composition of antibodies may be an adalimumab composition, e.g., a
commercial adalimumab (HUMIRA.RTM.) formulation, as described in
the "Highlights of Prescribing Information" for HUMIRA.RTM.
(adalimumab) Injection (Revised January 2008), the contents of
which are hereby incorporated herein by reference.
[0159] As used herein, the phrases "antibody resistant to
C-terminal processing" or "antibody resistant to C-terminal
processing by a carboxypeptidase" refer to an antibody, or
antigen-binding portion thereof, that is resistant to processing of
the C-terminus of its heavy chains by a carboxypeptidase enzyme,
e.g., carboxypeptidase B or carboxypeptidase U. An "antibody
resistant to C-terminal processing" exhibits decreased removal of a
C-terminal lysine of its heavy chains by a carboxypeptidase enzyme,
e.g., carboxypeptidase B or carboxypeptidase U. The antibody, or
antigen-binding portion thereof, may be modified as described in
Attorney Docket No. 117813-73802 (U.S. Patent Application Ser. No.
61/892,710, filed Oct. 18, 2013), the contents of which are
expressly incorporated herein by reference, to exhibit decreased
removal of a C-terminal lysine as compared to an antibody, or
antigen-binding portion thereof, that has not been modified. In one
embodiment, the antibody, or antigen-binding portion thereof,
retains both C-terminal lysines ("Lys 2") and, thus, exhibits no
removal (i.e., exhibits no C-terminal processing) of the C-terminal
lysines of the heavy chains by a carboxypeptidase. C-terminal
processing by a carboxypeptidase may be measured using assays that
are well-known in the art including, but not limited to, the
peptidase assays described in Attorney Docket No. 117813-73802
(U.S. Patent Application Ser. No. 61/892,710, filed Oct. 18,
2013).
[0160] As used herein, the term "carboxypeptidase" refers to a
protease enzyme that hydrolyzes a peptide bond at the
carboxy-terminal ("C-terminal") region of a protein or antibody.
Carboxypeptidases are well-known in the art and are involved in
post-translational modification of proteins. Specifically,
"Carboxypeptidase B" (EC 3.4.17.2) refers to a carboxypeptidase
that preferentially cleaves positively charged, or basic, amino
acids, such as arginine and lysine from the c-terminus of proteins
and antibodies. "Carboxypeptidase U" or "unstable carboxypeptidase"
(EC 3.4.17.20) refers to a carboxypeptidase that is activated by
thrombin or plasmin during clotting.
[0161] The term "modify," "modifying" or "modified," as used
herein, is intended to refer to changing one or more amino acids in
an antibody, or antigen-binding portion thereof. The change can be
produced by adding, substituting or deleting an amino acid at one
or more positions. The change can be produced using standard
techniques known in the art and described in more detail herein,
such as PCR mutagenesis and site-directed mutagenesis.
[0162] As used herein, the phrase "increased cartilage tissue
penetration" refers to the property of an antibody, or
antigen-binding portion thereof, of the invention showing increased
penetration of cartilage tissue. This property can be measured or
determined by, for example, using an in vitro or an in vivo
cartilage model. One non-limiting example of an art-recognized
mouse model of arthritis is the human TNF transgenic 197 mouse
model of arthritis (TNF-Tg197) (see Keffer, J. et al., EMBO J
(1991) 10:4025-4031, the contents of which are expressly
incorporated herein by reference, for further description of the
TNF-Tg197 model of arthritis). Cartilage penetration can be
measured using assays that are well-known in the art including, but
not limited to, the assays described in the Examples section
below.
[0163] As used herein, the terms "acidic species" and "AR," refer
to the variants of a protein, e.g., an antibody or antigen-binding
portion thereof, which are characterized by an acidic charge.
Acidic species are the component molecular entities that comprise
AR. For example, in monoclonal antibody (mAb) preparations, such
acidic species can be detected by various methods, such as, for
example, WCX-10 HPLC (a weak cation exchange chromatography), or
IEF (isoelectric focusing).
[0164] Acidic species of an antibody include charge variants,
structure variants, and/or fragmentation variants. Exemplary charge
variants include, but are not limited to, deamidation variants,
afucosylation variants, methylglyoxal (MGO) variants, glycation
variants, and citric acid variants. Exemplary structure variants
include, but are not limited to, glycosylation variants and
acetonation variants. Exemplary fragmentation variants include any
truncated protein species from the target molecule due to
dissociation of peptide chain, enzymatic and/or chemical
modifications, including, but not limited to, Fc and Fab fragments,
fragments missing a Fab, fragments missing a heavy chain variable
domain, C-terminal truncation variants, variants with excision of
N-terminal Asp in the light chain, and variants having N-terminal
truncation of the light chain. Other acidic species variants
include variants containing unpaired disulfides, host cell
proteins, and host nucleic acids, chromatographic materials, and
media components.
[0165] In certain embodiments, a protein composition may comprise
more than one type of acidic species variant. For example, but not
by way of limitation, the total acidic species can be divided based
on chromatographic residence time. For example, the total acidic
species associated with the expression of adalimumab may be divided
into a first acidic species region (AR1) and a second acidic
species region (AR2). AR1 may comprise, for example, charge
variants such as deamidation variants, MGO modified species,
glycation variants, and citric acid variants, structural variants
such as glycosylation variants and acetonation variants, and/or
fragmentation variants. Other acidic variants such as host cells
and unknown species may also be present. AR2 may comprise, for
example, charge variants such as glycation variants and deamidation
variants.
[0166] In one embodiment, a modulated lysine variant species
composition of the invention comprises about 0.0% to about 10%
acidic species (AR), about 0.0% to about 5% AR, about 0.0% to about
4% AR, about 0.0% to about 3% AR, about 0.0% to about 2% AR, about
3% to about 5% AR, about 5% to about 8% AR, or about 8% to about
10% AR, or about 10% to about 15% AR, and ranges within one or more
of the preceding. In another embodiment, a modulated lysine variant
species composition of the invention comprises, e.g., about 15% or
less AR. In one aspect of this embodiment, a modulated lysine
variant species composition comprises about 14% or less AR, 13% or
less AR, 12% or less AR, 11% or less AR, 10% or less AR, 9% or less
AR, 8% or less AR, 7% or less AR, 6% or less AR, 5% or less AR,
4.5% or less AR, 4% or less AR, 3.5% or less AR, 3% or less AR,
2.5% or less AR, 2% or less AR, 1.9% or less AR, 1.8% or less AR,
1.7% or less AR, 1.6% or less AR, 1.5% or less AR, 1.4% or less AR,
1.3% or less AR, 1.2% or less AR, 1.1% or less AR, 1% or less AR,
0.9% or less AR, 0.8% or less AR, 0.7% or less AR, 0.6% or less AR,
0.5% or less AR, 0.4% or less AR, 0.3% or less AR, 0.2% or less AR,
0.1% or less AR, or 0.0% AR, and ranges within one or more of the
preceding.
[0167] In another embodiment, a modulated lysine variant species
composition comprises about 0.1% or less AR1 and about 3% or less
AR2. In another aspect of this embodiment, the composition
comprises about 0.0% AR1 and about 1.4% AR2. In another aspect of
this embodiment, the composition comprises about 0% to about 3% AR,
e.g., about 3%, 2.5%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1%, 0.9%, 0.8%,
0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less AR1. In another
aspect of this embodiment, the composition comprises about 0% to
about 3% AR, e.g., about 3%, 2.5%, 2%, 1.5%, 1.4%, 1.3%, 1.2%,
1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or
less AR2.
[0168] The term "antibody", as used herein, broadly refers to any
immunoglobulin (Ig) molecule comprised of four polypeptide chains,
two heavy (H) chains and two light (L) chains, or any functional
fragment, mutant, variant, or derivative thereof, which retains the
essential epitope binding features of an Ig molecule. Such mutant,
variant, or derivative antibody formats are known in the art and
non-limiting embodiments of which are discussed herein.
[0169] In a full-length antibody, each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, CH1, CH2 and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA1 and IgA2)
or subclass. The present invention is particularly useful for
IgG.sub.1 antibodies.
[0170] As used herein, the term "adalimumab," also known by its
trade name HUMIRA.RTM. (AbbVie) refers to a human IgG.sub.1
antibody that binds human tumor necrosis factor .alpha.
(TNF.alpha.). In general, the heavy chain constant domain 2 (CH2)
of the adalimumab IgG-Fc region is glycosylated through covalent
attachment of oligosaccharide at asparagine 297 (Asn-297). The
light chain variable region of adalimumab is provided herein as SEQ
ID NO:1, and the heavy chain variable region of adalimumab is
provided herein as SEQ ID NO:2. Adalimumab comprises a light chain
variable region comprising a CDR1 of SEQ ID NO:7, a CDR2 of SEQ ID
NO:5, and a CDR3 of SEQ ID NO:3. Adalimumab comprises a heavy chain
variable region comprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID
NO:6 and CDR3 of SEQ ID NO:4. The nucleic acid sequence of the
light chain variable region is set forth in SEQ ID NO:9. The
nucleic acid sequence of the heavy chain variable region is set
forth in SEQ ID NO:10. The full length amino acid sequence of the
light chain is set forth as SEQ ID NO:11 and the full length amino
acid sequence of the heavy chain is set forth as SEQ ID NO:12.
Adalimumab is described in U.S. Pat. Nos. 6,090,382; 6,258,562;
6,509,015; 7,223,394; 7,541,031; 7,588,761; 7,863,426; 7,919,264;
8,197,813; 8,206,714; 8,216,583; 8,420,081; 8,092,998; 8,093,045;
8,187,836; 8,372,400; 8,034,906; 8,436,149; 8,231,876; 8,414,894;
8,372,401, the entire contents of each which are expressly
incorporated herein by reference in their entireties. Adalimumab is
also described in "Highlights of Prescribing Information" for
HUMIRA.RTM. (adalimumab) Injection (Revised January 2008) the
contents of which are hereby incorporated herein by reference.
[0171] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., TNF.alpha.) and still contain at least
one heavy chain. It has been shown that the antigen-binding
function of an antibody can be performed by fragments of a
full-length antibody. Such antibody embodiments may also be
bispecific, dual specific, or multi-specific formats; specifically
binding to two or more different antigens. Examples of binding
fragments encompassed within the term "antigen-binding portion" of
an antibody include a Fv fragment consisting of the VL and VH
domains of a single arm of an antibody or a halfbody (as described
in, for example, PCT Publication No. WO12/088302, the entire
contents of which are incorporated herein by reference).
Furthermore, although the two domains of the Fv fragment, VL and
VH, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the VL and VH regions pair
to form monovalent molecules (known as single chain Fv (scFv); see
e.g., Bird et al. (1988) Science 242:423-426; and Huston et al.
(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding portion" of an antibody. Other forms of single
chain antibodies, such as diabodies are also encompassed. Diabodies
are bivalent, bispecific antibodies in which VH and VL 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. (1993) Proc. Natl. Acad. Sci. USA
90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).
Such antibody binding portions are known in the art (Kontermann and
Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York.
790 pp. (ISBN 3-540-41354-5).
[0172] The term "antibody construct" as used herein refers to a
polypeptide comprising one or more of the antigen binding portions
of the invention linked to a linker polypeptide or an
immunoglobulin constant domain. Linker polypeptides comprise two or
more amino acid residues joined by peptide bonds and are used to
link one or more antigen binding portions. Such linker polypeptides
are well known in the art (see e.g., Holliger, P., et al. (1993)
Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.
(1994) Structure 2:1121-1123). An immunoglobulin constant domain
refers to a heavy or light chain constant domain. Human IgG heavy
chain and light chain constant domain amino acid sequences are
known in the art.
[0173] An antibody or antigen-binding portion thereof may be part
of a larger immunoadhesion molecule, formed by covalent or
noncovalent association of the antibody or antibody portion with
one or more other proteins or peptides. Examples of such
immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov, S. M., et
al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a
cysteine residue, a marker peptide and a C-terminal polyhistidine
tag to make bivalent and biotinylated scFv molecules (Kipriyanov,
S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody
portions, such as Fab and F(ab').sub.2 fragments, can be prepared
from whole antibodies using conventional techniques, such as papain
or pepsin digestion, respectively, of whole antibodies. Moreover,
antibodies, antibody portions and immunoadhesion molecules can be
obtained using standard recombinant DNA techniques, as described
herein.
[0174] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds human TNF.alpha.). An isolated
antibody that specifically binds TNF.alpha. may, however, have
cross-reactivity to other antigens, such as the TNF.alpha.
molecules from other species. Alternatively, an isolated antibody,
or antigen-binding portion thereof, may not cross-react with the
TNF.alpha. molecules from other species. Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals.
[0175] The term "human antibody", as used herein, 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. In one embodiment, the
human monoclonal antibodies are produced by a hybridoma which
includes a B cell obtained from a transgenic nonhuman animal, e.g.,
a transgenic mouse, having a genome comprising a human heavy chain
transgene and a light chain transgene fused to an immortalized
cell. In another embodiment, the human monoclonal antibodies are
produced by phage display technologies as described, for example,
in the Examples section below.
[0176] The term "recombinant human antibody", as used herein, 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 (Hoogenboom H.
R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E.,
(2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J.
W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P.
(2000) Immunology Today 21:371-378), antibodies isolated from an
animal (e.g., a mouse) that is transgenic for human immunoglobulin
genes (see e.g., U.S. Pat. No. 6,713,610; Taylor, L. D., et al.
(1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.
L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et
al (2000) Immunology Today 21:364-370) 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.
[0177] The term "human antibody derivatives" refers to any modified
form of the human antibody, e.g., a conjugate of the antibody and
another agent or antibody.
[0178] The term "chimeric antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions.
[0179] The term "CDR-grafted antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species but in which the sequences of one or more of the CDR
regions of VH and/or VL are replaced with CDR sequences of another
species, such as antibodies having murine heavy and light chain
variable regions in which one or more of the murine CDRs (e.g.,
CDR3) has been replaced with human CDR sequences.
[0180] The term "humanized antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from a
non-human species (e.g., a mouse) but in which at least a portion
of the VH and/or VL sequence has been altered to be more
"human-like", i.e., more similar to human germline variable
sequences. One type of humanized antibody is a CDR-grafted
antibody, in which human CDR sequences are introduced into
non-human VH and VL sequences to replace the corresponding nonhuman
CDR sequences. Such antibodies were generated by obtaining murine
anti-TNF.alpha. monoclonal antibodies using traditional hybridoma
technology followed by humanization using in vitro genetic
engineering.
[0181] The term "antibody mimetic" or "antibody mimic" is intended
to refer to molecules capable of mimicking an antibody's ability to
bind an antigen, but which are not limited to native antibody
structures. Examples of such antibody mimetics include, but are not
limited to, Adnectins (i.e., fibronectin based binding molecules),
Affibodies, DARPins, Anticalins, Avimers, and Versabodies all of
which employ binding structures that, while they mimic traditional
antibody binding, are generated from and function via distinct
mechanisms. The embodiments of the instant invention, as they are
directed to antibodies, or antigen binding portions thereof, also
apply to the antibody mimetics described above.
[0182] As used herein, "isotype" refers to an antibody class (e.g.,
IgM or IgG1) that is encoded by the heavy chain constant region
genes.
[0183] The terms "Kabat numbering", "Kabat definitions and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid
residues which are more variable (i.e., hypervariable) than other
amino acid residues in the heavy and light chain variable regions
of an antibody, or an antigen binding portion thereof (Kabat et al.
(1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242). For the heavy chain variable region, the
hypervariable region ranges from amino acid positions 31 to 35 for
CDR1, amino acid positions 50 to 65 for CDR2, and amino acid
positions 95 to 102 for CDR3. For the light chain variable region,
the hypervariable region ranges from amino acid positions 24 to 34
for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid
positions 89 to 97 for CDR3.
[0184] As used herein, the terms "acceptor" and "acceptor antibody"
refer to the antibody or nucleic acid sequence providing or
encoding at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100% of the
amino acid sequences of one or more of the framework regions. In
some embodiments, the term "acceptor" refers to the antibody amino
acid or nucleic acid sequence providing or encoding the constant
region(s). In yet another embodiment, the term "acceptor" refers to
the antibody amino acid or nucleic acid sequence providing or
encoding one or more of the framework regions and the constant
region(s). In a specific embodiment, the term "acceptor" refers to
a human antibody amino acid or nucleic acid sequence that provides
or encodes at least 80%, at least 85%, at least 90%, at least 95%,
at least 98%, or 100% of the amino acid sequences of one or more of
the framework regions. In accordance with this embodiment, an
acceptor may contain at least 1, at least 2, at least 3, least 4,
at least 5, or at least 10 amino acid residues that does (do) not
occur at one or more specific positions of a human antibody. An
acceptor framework region and/or acceptor constant region(s) may
be, e.g., derived or obtained from a germline antibody gene, a
mature antibody gene, a functional antibody (e.g., antibodies
well-known in the art, antibodies in development, or antibodies
commercially available).
[0185] As used herein, the term "CDR" refers to the complementarity
determining region within antibody variable sequences. There are
three CDRs in each of the variable regions of the heavy chain and
the light chain, which are designated CDR1, CDR2 and CDR3, for each
of the variable regions. The term "CDR set" as used herein refers
to a group of three CDRs that occur in a single variable region
capable of binding the antigen. The exact boundaries of these CDRs
have been defined differently according to different systems. The
system described by Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987) and (1991)) not only provides an unambiguous residue
numbering system applicable to any variable region of an antibody,
but also provides precise residue boundaries defining the three
CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and
coworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and
Chothia et al., Nature 342:877-883 (1989)) found that certain
sub-portions within Kabat CDRs adopt nearly identical peptide
backbone conformations, despite having great diversity at the level
of amino acid sequence. These sub-portions were designated as L1,
L2 and L3 or H1, H2 and H3 where the "L" and the "H" designates the
light chain and the heavy chains regions, respectively. These
regions may be referred to as Chothia CDRs, which have boundaries
that overlap with Kabat CDRs. Other boundaries defining CDRs
overlapping with the Kabat CDRs have been described by Padlan
(FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45
(1996)). Still other CDR boundary definitions may not strictly
follow one of the above systems, but will nonetheless overlap with
the Kabat CDRs, although they may be shortened or lengthened in
light of prediction or experimental findings that particular
residues or groups of residues or even entire CDRs do not
significantly impact antigen binding. The methods used herein may
utilize CDRs defined according to any of these systems, although
some embodiments use Kabat or Chothia defined CDRs.
[0186] As used herein, the term "canonical" residue refers to a
residue in a CDR or framework that defines a particular canonical
CDR structure as defined by Chothia et al. (J. Mol. Biol.
196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992),
both are incorporated herein by reference). According to Chothia et
al., critical portions of the CDRs of many antibodies have nearly
identical peptide backbone confirmations despite great diversity at
the level of amino acid sequence. Each canonical structure
specifies primarily a set of peptide backbone torsion angles for a
contiguous segment of amino acid residues forming a loop.
[0187] As used herein, the terms "donor" and "donor antibody" refer
to an antibody providing one or more CDRs. In one embodiment, the
donor antibody is an antibody from a species different from the
antibody from which the framework regions are obtained or derived.
In the context of a humanized antibody, the term "donor antibody"
refers to a non-human antibody providing one or more CDRs.
[0188] As used herein, the term "framework" or "framework sequence"
refers to the remaining sequences of a variable region minus the
CDRs. Because the exact definition of a CDR sequence can be
determined by different systems, the meaning of a framework
sequence is subject to correspondingly different interpretations.
The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of light chain and CDR-H1,
CDR-H2, and CDR-H3 of heavy chain) also divide the framework
regions on the light chain and the heavy chain into four
sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is
positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3
between FR3 and FR4. Without specifying the particular sub-regions
as FR1, FR2, FR3 or FR4, a framework region, as referred by others,
represents the combined FR's within the variable region of a
single, naturally occurring immunoglobulin chain. As used herein, a
FR represents one of the four sub-regions, and FRs represents two
or more of the four sub-regions constituting a framework
region.
[0189] Human heavy chain and light chain acceptor sequences are
known in the art.
[0190] As used herein, the term "germline antibody gene" or "gene
fragment" refers to an immunoglobulin sequence encoded by
non-lymphoid cells that have not undergone the maturation process
that leads to genetic rearrangement and mutation for expression of
a particular immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev.
Immunol. 22(3): 183-200 (2002); Marchalonis et al., Adv Exp Med.
Biol. 484:13-30 (2001)). One of the advantages of germ line
antibody genes stems from the recognition that germline antibody
genes are more likely than mature antibody genes to conserve
essential amino acid sequence structures characteristic of
individuals in the species, hence less likely to be recognized as
from a foreign source when used therapeutically in that
species.
[0191] As used herein, the term "key" residues refer to certain
residues within the variable region that have more impact on the
binding specificity and/or affinity of an antibody, in particular a
humanized antibody. A key residue includes, but is not limited to,
one or more of the following: a residue that is adjacent to a CDR,
a potential glycosylation site (can be either N- or O-glycosylation
site), a rare residue, a residue capable of interacting with the
antigen, a residue capable of interacting with a CDR, a canonical
residue, a contact residue between heavy chain variable region and
light chain variable region, a residue within the Vernier zone, and
a residue in the region that overlaps between the Chothia
definition of a variable heavy chain CDR1 and the Kabat definition
of the first heavy chain framework.
[0192] As used herein, the term "humanized antibody" is an antibody
or a variant, derivative, analog or fragment thereof which binds to
an antigen of interest and which comprises a framework (FR) region
having substantially the amino acid sequence of a human antibody
and a complementary determining region (CDR) having substantially
the amino acid sequence of a non-human antibody. As used herein,
the term "substantially" in the context of a CDR refers to a CDR
having an amino acid sequence at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98% or at
least 99% identical to the amino acid sequence of a non-human
antibody CDR. A humanized antibody comprises substantially all of
at least one, and typically two, variable domains (Fab, Fab',
F(ab').sub.2, FabC, Fv) in which all or substantially all of the
CDR regions correspond to those of a non-human immunoglobulin
(i.e., donor antibody) and all or substantially all of the
framework regions are those of a human immunoglobulin consensus
sequence. In one embodiment, a humanized antibody also comprises at
least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. In some embodiments, a
humanized antibody contains both the light chain as well as at
least the variable domain of a heavy chain. The antibody also may
include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy
chain. In some embodiments, a humanized antibody only contains a
humanized light chain. In some embodiments, a humanized antibody
only contains a humanized heavy chain. In specific embodiments, a
humanized antibody only contains a humanized variable domain of a
light chain and/or humanized heavy chain.
[0193] The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including without limitation IgG1, IgG2, IgG3 and IgG4.
The humanized antibody may comprise sequences from more than one
class or isotype, and particular constant domains may be selected
to optimize desired effector functions using techniques well-known
in the art.
[0194] The framework and CDR regions of a humanized antibody need
not correspond precisely to the parental sequences, e.g., the donor
antibody CDR or the consensus framework may be mutagenized by
substitution, insertion and/or deletion of at least one amino acid
residue so that the CDR or framework residue at that site does not
correspond to either the donor antibody or the consensus framework.
Such mutations, however, will not be extensive. Usually, at least
80%, at least 85%, at least 90%, or at least 95% of the humanized
antibody residues will correspond to those of the parental FR and
CDR sequences. As used herein, the term "consensus framework"
refers to the framework region in the consensus immunoglobulin
sequence. As used herein, the term "consensus immunoglobulin
sequence" refers to the sequence formed from the most frequently
occurring amino acids (or nucleotides) in a family of related
immunoglobulin sequences (See e.g., Winnaker, From Genes to Clones
(Verlagsgesellschaft, Weinheim, Germany 1987). In a family of
immunoglobulins, each position in the consensus sequence is
occupied by the amino acid occurring most frequently at that
position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence.
[0195] As used herein, "Vernier" zone refers to a subset of
framework residues that may adjust CDR structure and fine-tune the
fit to antigen as described by Foote and Winter (1992, J. Mol.
Biol. 224:487-499, which is incorporated herein by reference).
Vernier zone residues form a layer underlying the CDRs and may
impact on the structure of CDRs and the affinity of the
antibody.
[0196] The term "multivalent binding protein" is used in this
specification to denote a binding protein comprising two or more
antigen binding sites. In one embodiment, the multivalent binding
protein is engineered to have the three or more antigen binding
sites, and is generally not a naturally occurring antibody. The
term "multispecific binding protein" refers to a binding protein
capable of binding two or more related or unrelated targets. Dual
variable domain (DVD) binding proteins as used herein, are binding
proteins that comprise two or more antigen binding sites and are
tetravalent or multivalent binding proteins. Such DVDs may be
monospecific, i.e. capable of binding one antigen or multispecific,
i.e. capable of binding two or more antigens. DVD binding proteins
comprising two heavy chain DVD polypeptides and two light chain DVD
polypeptides are referred to a DVD Ig. Each half of a DVD Ig
comprises a heavy chain DVD polypeptide, and a light chain DVD
polypeptide, and two antigen binding sites. Each binding site
comprises a heavy chain variable domain and a light chain variable
domain with a total of 6 CDRs involved in antigen binding per
antigen binding site.
[0197] As used herein, the term "neutralizing" refers to
neutralization of biological activity of TNF.alpha.. In one
embodiment, a neutralizing binding protein is a neutralizing
antibody whose binding to TNF.alpha. and/or a mutant TNF.alpha.
protein results in inhibition of a biological activity of
TNF.alpha. and/or the mutant TNF.alpha.. The neutralizing binding
protein can bind TNF.alpha. and/or a mutant TNF.alpha. protein and
reduces a biologically activity of TNF.alpha. and/or a mutant
TNF.alpha. protein by at least about 20%, 30%, 40%, 50%, 60%, 70%,
80%, 85%, 90%, 95% or more. Inhibition of a biological activity of
TNF.alpha. and/or a mutant TNF.alpha. protein by a neutralizing
binding protein can be assessed by measuring one or more indicators
of TNF.alpha. and/or mutant TNF.alpha. biological activity well
known in the art.
[0198] The term "activity" includes activities such as the binding
specificity/affinity of an antibody for an antigen, for example, an
anti-TNF.alpha. antibody that binds to a TNF.alpha. antigen and/or
the neutralizing potency of an antibody, for example, an
anti-TNF.alpha. antibody whose binding to TNF.alpha. inhibits the
biological activity of TNF.alpha..
[0199] The term "epitope" includes any polypeptide determinant
capable of specific binding to an immunoglobulin or T-cell
receptor. In certain embodiments, epitope determinants include
chemically active surface groupings of molecules such as amino
acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain
embodiments, may have specific three dimensional structural
characteristics, and/or specific charge characteristics. An epitope
is a region of an antigen that is bound by an antibody. In certain
embodiments, an antibody is said to specifically bind an antigen
when it preferentially recognizes its target antigen in a complex
mixture of proteins and/or macromolecules.
[0200] The term "surface plasmon resonance", as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the
BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson, U., et
al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)
Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol.
Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.
198:268-277.
[0201] The terms "specific binding" or "specifically binding", as
used herein, in reference to the interaction of an antibody with
another moiety, e.g., TNF.alpha., mean an interaction that is
dependent upon the presence of a particular structure (e.g., an
antigenic determinant or epitope) on the moiety, e.g., TNF.alpha..
For example, an antibody recognizes and binds to a specific protein
structure rather than to proteins, generally. If an antibody is
specific for epitope "A", the presence of a molecule containing
epitope A (or free, unlabeled A), in a reaction containing labeled
"A" and the antibody, will reduce the amount of labeled A bound to
the antibody.
[0202] As used herein, an antibody that "binds" or "specifically
binds" to an antigen, e.g., TNF.alpha., is intended to refer to an
antibody, or antigen-binding portion thereof, that specifically
binds to the antigen. The term "K.sub.on" (also "Kon", "kon"), as
used herein, is intended to refer to the on rate constant for
association of a binding protein of the invention (e.g., an
antibody of the invention) to an antigen to form an association
complex, e.g., antibody/antigen complex, as is known in the art.
The "K.sub.on" also is known by the terms "association rate
constant", or "ka", as used interchangeably herein. This value
indicates the binding rate of an antibody to its target antigen or
the rate of complex formation between an antibody and antigen as is
shown by the equation below:
Antibody ("Ab")+Antigen ("Ag").fwdarw.Ab-Ag.
[0203] The term "K.sub.off" (also "Koff", "koff"), as used herein,
is intended to refer to the off rate constant for dissociation, or
"dissociation rate constant", of a binding protein of the invention
(e.g., an antibody of the invention) from an association complex
(e.g., an antibody/antigen complex) as is known in the art. This
value indicates the dissociation rate of an antibody from its
target antigen or separation of Ab-Ag complex over time into free
antibody and antigen as shown by the equation below:
Ab+Ag.rarw.Ab-Ag.
[0204] The term "K.sub.D" (also "K.sub.d"), as used herein, is
intended to refer to the "equilibrium dissociation constant", and
refers to the value obtained in a titration measurement at
equilibrium, or by dividing the dissociation rate constant (Koff)
by the association rate constant (Kon). The association rate
constant (Kon), the dissociation rate constant (Koff), and the
equilibrium dissociation constant (K are used to represent the
binding affinity of an antibody to an antigen. Methods for
determining association and dissociation rate constants are well
known in the art. Using fluorescence-based techniques offers high
sensitivity and the ability to examine samples in physiological
buffers at equilibrium. Other experimental approaches and
instruments such as a BIAcore.RTM. (biomolecular interaction
analysis) assay can be used (e.g., instrument available from
BIAcore International AB, a GE Healthcare company, Uppsala,
Sweden). Additionally, a KinExA.RTM. (Kinetic Exclusion Assay)
assay, available from Sapidyne Instruments (Boise, Id.) can also be
used.
[0205] The term "antibody conjugate" refers to a binding protein,
such as an antibody, linked, e.g., chemically linked, to a second
chemical moiety, such as a therapeutic or cytotoxic agent. The term
"agent" is used herein to denote a chemical compound, a mixture of
chemical compounds, a biological macromolecule, or an extract made
from biological materials. The therapeutic or cytotoxic agents can
include, but are not limited to, pertussis toxin, taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof.
[0206] The term "polynucleotide" as referred to herein, means a
polymeric form of two or more nucleotides, either ribonucleotides
or deoxynucleotides or a modified form of either type of
nucleotide. The term includes single and double stranded forms of
DNA but typically is double-stranded DNA.
[0207] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or
some combination thereof) that, by virtue of its origin, is not
associated with all or a portion of a polynucleotide with which the
"isolated polynucleotide" is found in nature; is operably linked to
a polynucleotide to which it is not linked in nature; or does not
occur in nature as part of a larger sequence.
[0208] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which exogenous
DNA has been introduced. It should be understood that such terms
are intended to refer not only to the particular subject cell, but,
to 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. Host cells include
prokaryotic and eukaryotic cells selected from any of the Kingdoms
of life. Eukaryotic cells include protist, fungal, plant and animal
cells. Host cells include but are not limited to the prokaryotic
cell line E. Coli; mammalian cell lines CHO, HEK 293 and COS; the
insect cell line Sf9; and the fungal cell Saccharomyces
cerevisiae.
[0209] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be 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. See e.g., Sambrook et al. Molecular Cloning:
A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by
reference for any purpose.
[0210] The terms "regulate" and "modulate" as used interchangeably,
and refer to a change or an alteration in the activity of a
molecule of interest (e.g., the biological activity of TNF.alpha.).
Modulation may be an increase or a decrease in the magnitude of a
certain activity or function of the molecule of interest. Exemplary
activities and functions of a molecule include, but are not limited
to, binding characteristics, enzymatic activity, cell receptor
activation, and signal transduction.
[0211] As used herein, the term "effective amount" refers to the
amount of a therapy which is sufficient to reduce or ameliorate the
severity and/or duration of a disorder or one or more symptoms
thereof, prevent the advancement of a disorder, cause regression of
a disorder, prevent the recurrence, development, onset or
progression of one or more symptoms associated with a disorder,
detect a disorder, or enhance or improve the prophylactic or
therapeutic effect(s) of another therapy (e.g., prophylactic or
therapeutic agent). In one embodiment, an "effective amount" refers
to the amount of an antibody, or antigen-binding portion thereof,
of the invention, e.g., an anti-TNF.alpha. antibody, or
antigen-binding portion thereof, that is sufficient to treat a
disorder in which TNF.alpha. activity is detrimental.
[0212] As used herein, the term "a disorder in which TNF.alpha.
activity is detrimental" is intended to include diseases and other
disorders in which the presence of TNF.alpha. in a subject
suffering from the disorder has been shown to be or is suspected of
being either responsible for the pathophysiology of the disorder or
a factor that contributes to a worsening of the disorder.
Accordingly, a disorder in which TNF.alpha. activity is detrimental
is a disorder in which inhibition of TNF.alpha. activity is
expected to alleviate the symptoms and/or progression of the
disorder. Such disorders may be evidenced, for example, by an
increase in the concentration of TNF.alpha. in a biological fluid
of a subject suffering from the disorder (e.g., an increase in the
concentration of TNF.alpha. in serum, plasma, or synovial fluid of
the subject), which can be detected, for example, using an
anti-TNF.alpha. antibody as described above. There are numerous
examples of disorders in which TNF.alpha. activity is detrimental.
In one embodiment, the disorder in which TNF.alpha. activity is
detrimental is an autoimmune disorder. In one embodiment, the
autoimmune disorder is selected from the group consisting of
rheumatoid arthritis, juvenile idiopathic arthritis, rheumatoid
spondylitis, ankylosing spondylitis, psoriasis, osteoarthritis,
gouty arthritis, an allergy, multiple sclerosis, psoriatic
arthritis, autoimmune diabetes, autoimmune uveitis, nephrotic
syndrome, juvenile rheumatoid arthritis, Crohn's disease, active
axial spondyloarthritis (active axSpA) and non-radiographic axial
spondyloarthritis (nr-axSpA), and ulcerative colitis. Disorders in
which TNF.alpha. activity is detrimental are set forth in U.S. Pat.
No. 6,090,382 and also in the "Highlights of Prescribing
Information" for HUMIRA.RTM. (adalimumab) Injection (Revised
January 2008), the contents of which are hereby incorporated herein
by reference. The use of TNF.alpha. antibodies and antibody
portions obtained using methods of the invention for the treatment
of specific disorders is discussed in further detail below.
[0213] The phrase "clarified harvest" refers to a liquid material
containing a protein of interest, for example, an antibody of
interest such as a monoclonal or polyclonal antibody of interest,
that has been extracted from cell culture, for example, a
fermentation bioreactor, after undergoing centrifugation to remove
large solid particles and subsequent filtration to remove finer
solid particles and impurities from the material.
[0214] As used herein, the term "cell culture" refers to methods
for generating and maintaining a population of host cells capable
of producing a recombinant protein of interest, as well as the
methods and techniques for optimizing the production and collection
of the protein of interest. For example, once an expression vector
has been incorporated into an appropriate host, the host can be
maintained under conditions suitable for expression of the relevant
nucleotide coding sequences, and the collection and purification of
the desired recombinant protein.
[0215] When using the cell culture techniques of the instant
invention, the protein of interest can be produced intracellularly,
in the periplasmic space, or directly secreted into the medium. In
embodiments where the protein of interest is produced
intracellularly, the particulate debris, either host cells or lysed
cells (e.g., resulting from homogenization) can be removed by a
variety of means, including but not limited to, centrifugation or
ultrafiltration. Where the protein of interest is secreted into the
medium, supernatants from such expression systems can be first
concentrated using a commercially available protein concentration
filter, e.g., an Amicon.TM. or Millipore Pellicon.TM.
ultrafiltration unit.
II. MODULATED LYSINE VARIANT SPECIES COMPOSITIONS OF THE
INVENTION
[0216] In one embodiment, the present invention provides modulated
lysine variant species compositions comprising antibodies, or
antigen-binding portions thereof, such as adalimumab, comprising
modulated percentages of Lys 1, Lys 2, and/or Lys 0 variants
relative to the percentage of Lys 1, Lys 2, and Lys 0 proteins in a
population of proteins. In one embodiment, the methods of the
invention are used to produce a protein composition comprising an
increased amount of Lys 1 and/or Lys 2 variants and a decreased
amount of Lys 0 variants in a population of proteins, e.g., as
compared to a non-modulated lysine variant species composition,
e.g., a control composition. In another embodiment, the methods of
the invention are used to produce a protein composition comprising
a decreased amount of Lys 1 and/or Lys 2 variants, and an increased
amount of Lys 0 variants in a population of proteins, e.g., as
compared to a non-modulated lysine variant species composition,
e.g., a control composition.
[0217] For example, a modulated lysine variant species composition
of the invention may comprise more than about 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100% Lys 1 and/or Lys 2 antibodies, or
antigen-binding portions thereof relative to the lysine sum (i.e.,
the sum of Lys 0, Lys 1 and Lys 2 in the composition). In one
embodiment, a modulated lysine variant species composition of the
invention may comprise more than about 20% to about 30%, about 30%
to about 40%, about 40% to about 50%, about 60% to about 70%, or
about 70% to about 80%, about 80% to about 90%, or about 90% to
about 100% Lys 1 and/or Lys 2 antibodies, or antigen-binding
portions thereof relative to the lysine sum. Ranges within one or
more of any of the preceding percentages are also included.
[0218] A modulated lysine variant species composition of the
invention may also comprise less than about 80%, 75%, 70%, 65%,
60%, 55%, 50%, 45%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%,
1%, or zero antibodies, or antigen-binding portions thereof that
are Lys 0, relative to the lysine sum. In one embodiment, a
modulated lysine variant species composition of the invention may
also comprise less than about 0% to about 10%, about 10% to about
20%, about 20% to about 30%, about 30% to about 40%, about 50% to
about 60%, about 60% to about 70%, or about 70% to about 80%,
antibodies or antigen-binding portions thereof that are Lys 0,
relative to the lysine sum. Ranges within one or more of any of the
preceding percentages are also included.
[0219] A modulated lysine variant species composition also includes
a composition comprising an antibody, or antigen-binding portion
thereof, that comprises more than about 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% antibodies, or antigen-binding portions thereof
that have one C-terminal lysine (Lys 1) relative to the lysine sum.
Ranges within one or more of any of the preceding percentages are
also included. In one embodiment, a modulated lysine variant
species composition of the invention may comprise more than about
20% to about 30%, about 30% to about 40%, about 40% to about 50%,
about 60% to about 70%, or about 70% to about 80%, about 80% to
about 90%, or about 90% to about 100% Lys 1 antibodies, or
antigen-binding portions thereof relative to the lysine sum. Ranges
within one or more of any of the preceding percentages are also
included.
[0220] A modulated lysine variant species composition also includes
a composition comprising an antibody, or antigen-binding portion
thereof, that comprises more than about 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% antibodies, or antigen-binding portions thereof
that have two C-terminal lysines (Lys 2) relative to the lysine
sum. Ranges within one or more of any of the preceding percentages
are also included. In one embodiment, a modulated lysine variant
species composition of the invention may comprise more than about
20% to about 30%, about 30% to about 40%, about 40% to about 50%,
about 60% to about 70%, or about 70% to about 80%, about 80% to
about 90%, or about 90% to about 100% Lys 2 antibodies, or
antigen-binding portions thereof relative to the lysine sum. Ranges
within one or more of any of the preceding percentages are also
included.
[0221] In one embodiment, the methods of the invention, alone or in
combination, may reduce the amount or percentage of one or more
C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a protein or
antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,
3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges
within one or more of the preceding, as compared to a non-modulated
lysine variant species composition. In one aspect of this
embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants are
reduced by about 1% to about 25%, about 5% to about 20%, about 5%
to about 15%, or about 5% to about 10%, and ranges within one or
more of the preceding, as compared to a non-modulated lysine
variant species composition.
[0222] In one embodiment, the methods of the invention, alone or in
combination, may increase the amount or percentage of one or more
C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a protein or
antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,
3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges
within one or more of the preceding, as compared to a non-modulated
lysine variant species composition. In one aspect of this
embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants are
increased by about 1% to about 25%, about 5% to about 20%, about 5%
to about 15%, or about 5% to about 10%, and ranges within one or
more of the preceding, as compared to a non-modulated lysine
variant species composition.
[0223] As demonstrated herein, certain modulated lysine variant
species compositions have improved therapeutic efficacy and
improved biological properties, for example, increased cartilage
tissue penetration, reduced cartilage destruction, reduced synovial
proliferation, reduced bone erosion, increased protection against
the development of arthritic scores and/or histopathology scores,
reduced cell infiltration, reduced proteoglycan loss, reduced
chondrocyte death, and/or increased TNF.alpha. affinity, as
compared to non-modulated lysine variant species composition
(Example 5).
[0224] In one embodiment, the protein in the modulated lysine
variant species compositions of the invention is an antibody or
antigen binding portion thereof. For example, the antibody, or
antigen binding portion thereof may be an anti-TNF.alpha. antibody,
or antigen binding portion thereof, such as adalimumab, or an
antigen binding portion thereof. In one aspect of this embodiment,
antibody, or antigen binding portion thereof, can comprise a light
chain variable region comprising the sequence set forth as SEQ ID
NO:1, and a heavy chain variable region comprising the sequence set
forth as SEQ ID NO:2. In another aspect of this embodiment, the
antibody can comprise a light chain variable region comprising a
CDR1 having the sequence set forth as SEQ ID NO:7, a CDR2 having
the sequence set forth as SEQ ID NO:5, and a CDR3 having the
sequence set forth as SEQ ID NO:3. In another aspect of this
embodiment, the antibody can comprise a heavy chain variable region
comprising a CDR1 having the sequence set forth as SEQ ID NO:8, a
CDR2 having the sequence set forth as SEQ ID NO:6 and a CDR3 having
the sequence set forth as SEQ ID NO:4.
[0225] The antibody, or antigen binding portion thereof, used in
the modulated lysine variant species compositions of the invention,
may be a human, humanized, or chimeric antibody.
[0226] The antibodies, or antigen-binding portions thereof, used in
the modulated lysine variant species compositions of the invention,
can be generated by a variety of techniques, including immunization
of an animal with the antigen of interest followed by conventional
monoclonal antibody methodologies e.g., the standard somatic cell
hybridization technique of Kohler and Milstein (1975) Nature 256:
495. Somatic cell hybridization procedures can be used. In
principle, other techniques for producing monoclonal antibody can
be employed as well, including viral or oncogenic transformation of
B lymphocytes.
[0227] One animal system for preparing hybridomas is the murine
system. Hybridoma production is a very well-established procedure.
Immunization protocols and techniques for isolation of immunized
splenocytes for fusion are known in the art. Fusion partners (e.g.,
murine myeloma cells) and fusion procedures are also known.
[0228] An antibody used in the modulated lysine variant species
compositions of the invention can be a human, a chimeric, or a
humanized antibody. Chimeric or humanized antibodies used in the
modulated lysine variant species compositions of the invention can
be prepared based on the sequence of a non-human monoclonal
antibody prepared as described above. DNA encoding the heavy and
light chain immunoglobulins can be obtained from the non-human
hybridoma of interest and engineered to contain non-murine (e.g.,
human) immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, murine
variable regions can be linked to human constant regions using
methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to
Cabilly et al.). To create a humanized antibody, murine CDR regions
can be inserted into a human framework using methods known in the
art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.).
[0229] In one non-limiting embodiment, the antibodies, or
antigen-binding portions thereof, used in the modulated lysine
variant species compositions of the invention are human monoclonal
antibodies. Such human monoclonal antibodies can be generated using
transgenic or transchromosomic mice carrying parts of the human
immune system rather than the mouse system. These transgenic and
transchromosomic mice include mice referred to herein as the HuMAb
Mouse.RTM. (Medarex, Inc.), KM Mouse.RTM. (Medarex, Inc.), and
XenoMouse.RTM. (Amgen). The antibodies, or antigen-binding portions
thereof, used in the modulated lysine variant species compositions
of the invention can also be produced using the methods described
in U.S. Pat. No. 6,090,382, the entire contents of which is
expressly incorporated herein by reference.
[0230] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise antibodies of the disclosure. For example,
mice carrying both a human heavy chain transchromosome and a human
light chain transchromosome, referred to as "TC mice" can be used;
such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad.
Sci. USA 97:722-727. Furthermore, cows carrying human heavy and
light chain transchromosomes have been described in the art (e.g.,
Kuroiwa et al. (2002) Nature Biotechnology 20:889-894 and PCT
application No. WO 2002/092812) and can be used to raise antibodies
of this disclosure.
[0231] Recombinant human antibodies, or antigen-binding portions
thereof, used in the modulated lysine variant species compositions
of the invention can be isolated by screening of a recombinant
combinatorial antibody library, e.g., a scFv phage display library,
prepared using human VL and VH cDNAs prepared from mRNA derived
from human lymphocytes. Methodologies for preparing and screening
such libraries are known in the art. In addition to commercially
available kits for generating phage display libraries (e.g., the
Pharmacia Recombinant Phage Antibody System, catalog no.
27-9400-01; and the Stratagene SurfZAP.TM. phage display kit,
catalog no. 240612, the entire teachings of which are incorporated
herein), examples of methods and reagents particularly amenable for
use in generating and screening antibody display libraries can be
found in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.
PCT Publication No. WO 92/18619; Dower et al. PCT Publication No.
WO 91/17271; Winter et al. PCT Publication No. WO 92/20791;
Markland et al. PCT Publication No. WO 92/15679; Breitling et al.
PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication
No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690;
Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992)
Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; McCafferty et al., Nature (1990) 348:552-554;
Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982; the
entire teachings of which are incorporated herein.
[0232] The antibodies, or antigen-binding portions thereof, used in
the modulated lysine variant species compositions of the invention
can also be prepared using SCID mice into which human immune cells
have been reconstituted such that a human antibody response can be
generated upon immunization. Such mice are described in, for
example, U.S. Pat. Nos. 5,476,996 and 5,698,767 to Wilson et
al.
[0233] In certain embodiments, the antibodies, or antigen-binding
portions thereof, used in the modulated lysine variant species
compositions of the invention include anti-TNF.alpha. antibodies
and antigen-binding portions thereof, anti-TNF.alpha.-related
antibodies and antigen-binding portions thereof, and human
antibodies and antigen-binding portions thereof with equivalent
properties to anti-TNF.alpha., such as high affinity binding to
hTNF.alpha. with low dissociation kinetics and high neutralizing
capacity. In one aspect, the modulated lysine variant species
compositions of the invention, include an isolated human antibody,
or an antigen-binding portion thereof, that dissociates from
hTNF.alpha. with a Kd of about 1.times.10.sup.-8 M or less and a
Koff rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance. In specific non-limiting
embodiments, an anti-TNF.alpha. antibody, or antigen-binding
portions thereof, purified according to the invention competitively
inhibits binding of adalimumab to TNF.alpha. under physiological
conditions. In one embodiment, the modulated lysine variant species
compositions of the invention comprise adalimumab or an antigen
binding portion thereof.
[0234] Antibodies or antigen-binding portions thereof used in the
modulated lysine variant species compositions of the invention can
be altered wherein the constant region of the antibody is modified
to reduce at least one constant region-mediated biological effector
function relative to an unmodified antibody. To modify an antibody
used in the modulated lysine variant species compositions of the
invention such that it exhibits reduced binding to the Fc receptor,
the immunoglobulin constant region segment of the antibody can be
mutated at particular regions necessary for Fc receptor (FcR)
interactions (see, e.g., Canfield and Morrison (1991) J. Exp. Med.
173:1483-1491; and Lund et al. (1991) J. of Immunol. 147:2657-2662,
the entire teachings of which are incorporated herein). Reduction
in FcR binding ability of the antibody may also reduce other
effector functions which rely on FcR interactions, such as
opsonization and phagocytosis and antigen-dependent cellular
cytotoxicity.
III. PROTEIN PRODUCTION
[0235] To express an antibody, or antigen-binding portion thereof,
used in the modulated lysine variant species compositions of the
invention, such as an antibody or antigen-binding fragment thereof,
DNAs encoding the protein, such as DNAs encoding partial or
full-length light and heavy chains, are inserted into one or more
expression vector such that the genes are operatively linked to
transcriptional and translational control sequences. (See, e.g.,
U.S. Pat. No. 6,090,382, the entire teaching of which is
incorporated herein by reference.) In this context, the term
"operatively linked" is intended to mean that a gene encoding the
protein of interest 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 gene. The expression vector
and expression control sequences are chosen to be compatible with
the expression host cell used. In certain embodiments, the protein
of interest will comprise multiple polypeptides, such as the heavy
and light chains of an antibody. Thus, in certain embodiments,
genes encoding multiple polypeptides, such as antibody light chain
genes and antibody heavy chain genes, can be inserted into a
separate vector or, more typically, the genes are inserted into the
same expression vector. Genes are inserted into expression vectors
by standard methods (e.g., ligation of complementary restriction
sites on the gene fragment and vector, or blunt end ligation if no
restriction sites are present). Prior to insertion of the gene or
genes, the expression vector may already carry additional
polypeptide sequences, such as, but not limited to, antibody
constant region sequences. For example, one approach to converting
the antibody or antibody-related VH and VL sequences to full-length
antibody genes is to insert them into expression vectors already
encoding heavy chain constant and light chain constant regions,
respectively, such that the VH segment is operatively linked to the
CH segment(s) within the vector and the VL segment is operatively
linked to the CL segment within the vector. Additionally or
alternatively, the recombinant expression vector can encode a
signal peptide that facilitates secretion of the protein from a
host cell. The gene can be cloned into the vector such that the
signal peptide is linked in-frame to the amino terminus of the
gene. The signal peptide can be an immunoglobulin signal peptide or
a heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0236] In addition to protein coding genes, a recombinant
expression vector of the invention can carry one or more regulatory
sequence that controls the expression of the protein coding genes
in a host cell. The term "regulatory sequence" is intended to
include promoters, enhancers and other expression control elements
(e.g., polyadenylation signals) that control the transcription or
translation of the protein coding genes. Such regulatory sequences
are described, e.g., in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990), the entire teaching of which is incorporated herein by
reference. 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. Suitable 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 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)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see, e.g., U.S. Pat.
No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al.
and U.S. Pat. No. 4,968,615 by Schaffner et al., the entire
teachings of which are incorporated herein by reference.
[0237] In addition to the protein coding genes and regulatory
sequences, a recombinant expression vector of the invention may
carry one or more additional sequences, such as a sequence that
regulates replication of the vector in host cells (e.g., origins of
replication) and/or a selectable marker gene. 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, all by Axel et al., the entire teachings of which are
incorporated herein by reference). 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. Suitable selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0238] An antibody, or antigen-binding portion thereof, used in the
modulated lysine variant species compositions of the invention can
be prepared by recombinant expression of immunoglobulin light and
heavy chain genes in a host cell. To express an antibody
recombinantly, a host cell is transfected with one or more
recombinant expression vectors carrying DNA fragments encoding the
immunoglobulin light and heavy chains of the antibody such that the
light and heavy chains are expressed in the host cell and secreted
into the medium in which the host cells are cultured, from which
medium the antibodies can be recovered. Standard recombinant DNA
methodologies are used to obtain antibody heavy and light chain
genes, incorporate these genes into recombinant expression vectors
and introduce the vectors into host cells, such as those described
in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989), Ausubel et al. (eds.) Current Protocols in Molecular
Biology, Greene Publishing Associates, (1989) and in U.S. Pat. Nos.
4,816,397 & 6,914,128, the entire teachings of which are
incorporated herein.
[0239] For expression of protein, for example, the light and heavy
chains of an antibody, the expression vector(s) encoding the
protein is (are) transfected into a host cell by standard
techniques. The various forms of the term "transfection" are
intended to encompass a wide variety of techniques commonly used
for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation, DEAE-dextran transfection and the like. Although it
is theoretically possible to express the proteins of the invention
in either prokaryotic or eukaryotic host cells, expression of
antibodies in eukaryotic cells, such as mammalian host cells, is
suitable because such eukaryotic cells, and in particular mammalian
cells, are more likely than prokaryotic cells to assemble and
secrete a properly folded and immunologically active protein.
Prokaryotic expression of protein genes has been reported to be
ineffective for production of high yields of active protein (Boss
and Wood (1985) Immunology Today 6:12-13, the entire teaching of
which is incorporated herein by reference).
[0240] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, e.g.,
Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One suitable E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0241] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for polypeptide encoding vectors. Saccharomyces cerevisiae, or
common baker's yeast, is the most commonly used among lower
eukaryotic host microorganisms. However, a number of other genera,
species, and strains are commonly available and useful herein, such
as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger.
[0242] Suitable host cells for the expression of glycosylated
proteins, for example, glycosylated antibodies, are derived from
multicellular organisms. Examples of invertebrate cells include
plant and insect cells. Numerous baculoviral strains and variants
and corresponding permissive insect host cells from hosts such as
Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),
Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly),
and Bombyx mori have been identified. A variety of viral strains
for transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells. Plant cell cultures of cotton, corn, potato,
soybean, petunia, tomato, and tobacco can also be utilized as
hosts.
[0243] Suitable mammalian host cells for expressing the recombinant
proteins of the invention include Chinese Hamster Ovary (CHO cells)
(including dhfr-CHO cells, described in Urlaub and Chasin, (1980)
PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., as
described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621, the
entire teachings of which are incorporated herein by reference),
NS0 myeloma cells, COS cells and SP2 cells. When recombinant
expression vectors encoding protein genes are introduced into
mammalian host cells, the antibodies are produced by culturing the
host cells for a period of time sufficient to allow for expression
of the antibody in the host cells or secretion of the antibody into
the culture medium in which the host cells are grown. Other
examples of useful mammalian host cell lines are monkey kidney CV1
line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic
kidney line (293 or 293 cells subcloned for growth in suspension
culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster
kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR
(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2), the entire teachings of which
are incorporated herein by reference.
[0244] Host cells are transformed with the above-described
expression or cloning vectors for protein production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0245] The host cells used to produce a protein may be cultured in
a variety of media. Commercially available media such as Ham's
F10.TM. (Sigma), Minimal Essential Medium.TM. (MEM), (Sigma),
RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium.TM.
(DMEM), (Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used
as culture media for the host cells, the entire teachings of which
are incorporated herein by reference. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as gentamycin drug), trace elements
(defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, dissolved oxygen (DO) concentration, phosphate
concentration, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0246] Host cells can also be used to produce portions of intact
proteins, for example, antibodies, including Fab fragments or scFv
molecules. It is understood that variations on the above procedure
are within the scope of the present invention. For example, in
certain embodiments it may be desirable to transfect a host cell
with DNA encoding either the light chain or the heavy chain (but
not both) of an antibody. Recombinant DNA technology may also be
used to remove some or all of the DNA encoding either or both of
the light and heavy chains that is not necessary for binding to an
antigen. The molecules expressed from such truncated DNA molecules
are also encompassed by the antibodies of the invention. In
addition, bifunctional antibodies may be produced in which one
heavy and one light chain are an antibody of the invention and the
other heavy and light chain are specific for an antigen other than
the target antigen, depending on the specificity of the antibody of
the invention, by crosslinking an antibody of the invention to a
second antibody by standard chemical crosslinking methods.
[0247] In a suitable system for recombinant expression of a
protein, for example, an antibody, or antigen-binding portion
thereof, a recombinant expression vector encoding the protein, for
example, both an antibody heavy chain and an antibody light chain,
is introduced into dhfr-CHO cells by calcium phosphate-mediated
transfection. Within the recombinant expression vector, the protein
gene(s) are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
gene(s). The recombinant expression vector also carries a DHFR
gene, which allows for selection of CHO cells that have been
transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the protein, for example, the
antibody heavy and light chains, and intact protein, for example,
an antibody, is recovered from the culture medium. Standard
molecular biology techniques are used to prepare the recombinant
expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the protein from
the culture medium.
[0248] When using recombinant techniques, the protein, for example,
antibodies or antigen binding fragments thereof, can be produced
intracellularly, in the periplasmic space, or directly secreted
into the medium. In one aspect, if the protein is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed cells (e.g., resulting from homogenization),
can be removed, e.g., by centrifugation or ultrafiltration. Where
the protein is secreted into the medium, supernatants from such
expression systems can be first concentrated using a commercially
available protein concentration filter, e.g., an Amicon.TM. or
Millipore Pellicon.TM. ultrafiltration unit.
[0249] Some proteins can be secreted directly from the cell into
the surrounding growth media; others are made intracellularly. For
the latter proteins, the first step of a purification process
typically involves: lysis of the cell, which can be done by a
variety of methods, including mechanical shear, osmotic shock, or
enzymatic treatments. Such disruption releases the entire contents
of the cell into the homogenate, and in addition produces
subcellular fragments that are difficult to remove due to their
small size. These are generally removed by differential
centrifugation or by filtration. Where the protein is secreted,
supernatants from such expression systems are generally first
concentrated using a commercially available protein concentration
filter, e.g., an Amicon.TM. or Millipore Pellicon.TM.
ultrafiltration unit. Where the protein is secreted into the
medium, the recombinant host cells can also be separated from the
cell culture medium, e.g., by tangential flow filtration. Protein
can be further recovered from the culture medium using the protein
purification methods of the invention.
[0250] Numerous populations of proteins expressed by host cells,
including, but not limited to, host cells expressing antibodies,
such as adalimumab, may comprise a number of lysine variants, for
example, combinations of two or more of Lys 0, Lys 1 and Lys 2, and
are therefore amenable to the methods described herein for
modification of C-terminal lysine variant heterogeneity. For
example, ion chromatography analysis, such as weak cation-exchange
chromatography (WCX) analysis, of adalimumab has shown the presence
of the three lysine variants corresponding to Lys 0, Lys 1 and Lys
2. Therefore, WCX provides an exemplary system for identifying
modification of Lys 0, Lys 1, and Lys 2 in a composition comprising
an antibody, or antigen binding portion thereof, and therefore
allowing for identification of particular cell culture conditions
that allow for control over lysine variant heterogeneity.
[0251] The production of C-terminal lysine variants can be
dependent upon changes in process parameters. (Lawrence, D. (2008),
C-terminal Lysine Variants in Fully Human Monoclonal Antibodies:
Investigation of Test Methods and Possible Causes. Biotechnology
and Bioengineering. 100: 1132-1143). Native glycoproteins and/or
recombinant glycoproteins (e.g., natural antibodies and/or
therapeutic antibodies) that are translated at the endoplasmic
reticulum (ER) must fold properly and often assemble into
multimeric complexes. There are several proteins that help these
proteins to fold properly. Some of these proteins only need the
cleavage of the ER N-terminal sequence of the protein to become a
mature protein. However, other glycoproteins can require further
processing ("posttranslational modifications") to become a mature
and fully-functional. Some of these posttranslational modifications
include glycosylation, formation of disulfide bonds, N-terminal
pyroglutamate, methionine oxidation, asparagine deamination,
phosphorylation, acetylation, and enzymatic removal of C-terminal
lysine or arginine residues. (Ahrer et al., (2006), Chromatographic
and Electrophoretic Characterization of Protein Variants. Journal
of Chromatography. 841:110-122; Li et al. (2005), Current
Therapeutic Antibody Production and Process Optimization.
Bioprocessing Journal; Harris, J. (1995), Processing of C-terminal
Lysine and Arginine Residues of Proteins Isolated from Mammalian
Cell Culture. Journal of Chromatography. 705: 129-134; and Parkins,
M., Theiler, R., et al. (2000), Determination of the Origin of
Charge Heterogeneity in a Murine Monoclonal Antibody.
Pharmaceutical Research. 17: 1110-1117).
[0252] Without being bound by theory, studies have demonstrated
that the enzymatic removal of the C-terminal Lys residues is a
primary contributor to the heterogeneity of recombinant monoclonal
antibodies, including, but not limited to, the adalimumab
glycoprotein. (Harris et al. (2004), Commercial Manufacturing Scale
Formulation and Analytical Characterization of Therapeutic
Recombinant Antibodies. Drug Development Research. 61: 137-154).
Additional studies have determined that the charge heterogeneity
can be generated by: 1) removal of the lysine or arginine
C-terminal residue in the IgG heavy chains; 2) conversion of
N-terminal glutamate to pyroglutamate; 3) dehydration of aspartate
residues; and 4) alternate cleavage of a signal peptide that
results in the presence of basic residues. The adalimumab heavy
chain terminal sequence is proline-glycine-lysine. However, as
noted above, the lysine residues are partially removed during the
manufacturing process, alternatively, they appear to be cleaved
subsequent to administration to a mammalian subject. Thus,
adalimumab can comprise a mixture of antibodies bearing zero, one,
or two C-terminal lysine residues. The specific sequence can be
detected by cation exchange chromatography. It has been observed
that this charge heterogeneity, caused by the incomplete
posttranslational cleavage, may not affect the potency of the
protein to bind TNF-.alpha.. (Santora et al., (2001),
Characterization of Noncovalent Complexes of Recombinant Human
Monoclonal Antibody and Antigen Using Cation Exchange, Size
Exclusion Chromatography, and BIAcore. Analytical Biochemistry 299:
119-129). However, even though the removal of C-terminal lysine
residues may not couple with the biological function of the
antibody, it is a factor to consider in maintaining batch-to-batch
consistency and efficacy. (Parkins et al. (2000), Determination of
the Origin of Charge Heterogeneity in a Murine Monoclonal Antibody.
Pharmaceutical Research. 17: 1110-1117).
[0253] Experiments have shown that the charge heterogeneity of an
antibody, associated with the incomplete removal of C-terminal
lysine residues, is due to carboxypeptidase activity/expression
differences due to process parameter variability or changes. Since
the penultimate residue, glycine, is not removed, this suggests
that the carboxypeptidase is specific for basic residues, such as
lysine and arginine. (Lawrence, D. (2008), C-terminal Lysine
Variants in Fully Human Monoclonal Antibodies: Investigation of
Test Methods and Possible Causes. Biotechnology and Bioengineering.
100: 1132-1143; and Harris, J. (1995), Processing of C-terminal
Lysine and Arginine Residues of Proteins Isolated from Mammalian
Cell Culture. Journal of Chromatography. 705: 129-134).
[0254] Antibodies resistant to C-terminal processing by a
carboxypeptidase, which are resistant to removal of a C-terminal
lysine of a heavy chains and exhibit decreased removal of a
C-terminal lysine by a carboxypeptidase enzyme, e.g.,
carboxypeptidase B or carboxypeptidase U, can be produced using the
methods described in Attorney Docket No. 117813-73802 (U.S. Patent
Application Ser. No. 61/892,710, filed Oct. 18, 2013), the contents
of which are expressly incorporated herein by reference. In one
embodiment, the antibody, or antigen-binding portion thereof,
retains both C-terminal lysines ("Lys 2") and, thus, exhibits no
removal (i.e., exhibits no C-terminal processing) of the C-terminal
lysines of the heavy chains by a carboxypeptidase. C-terminal
processing by a carboxypeptidase may be measured using assays that
are well-known in the art including, but not limited to, the
peptidase assays described in Attorney Docket No. 117813-73802
(U.S. Patent Application Ser. No. 61/892,710, filed Oct. 18,
2013).
[0255] The experiments outlined in the Examples below demonstrate
strategies for modifying the lysine distribution in compositions
comprising an antibody or antigen binding portion thereof. In
particular, the methods described herein can be used to increase
the Lys 1 and/or Lys 2 variants in a composition, and decrease the
Lys 0 variants in the composition, not to mention decrease the AR
contribution, to thereby produce a high Lys 1 and/or Lys 2
composition.
[0256] The experiments disclosed herein demonstrate that, in
certain embodiments, variation in raw materials used in cell
culture, and particularly in the context of media preparation, can
vary C-terminal Lys variant distribution. For example, as outlined
herein, control over the amount of zinc present in cell culture
media can allow for the modulation of C-terminal lysine variant
heterogeneity. zinc has been previously reported in literature to
be a cofactor of the enzyme carboxypeptidase (Valee B et. al
(1960), The role of zinc in carboxypeptidase, Journal of Biological
Chemistry, 235, 1, 64-69). However, it was not appreciated until
the filing of the instant application that adjusting the levels of
zinc in culture media could allows for modulation of the lysine
variants.
[0257] Adjusting Zinc Concentration to Control Lysine Variation
[0258] In certain embodiments of the instant invention, control of
C-terminal lysine variants in a composition comprising an antibody,
or antigen binding portion thereof, can be attained by adjustment
of the zinc concentration in a media employed in the cell culture
run. In one non-limiting embodiment, such adjustment will be to
decrease the amount of zinc in the media, while in other
non-limiting embodiments, the necessary adjustment to achieve the
desired control over lysine variant heterogeneity will involve an
increase in the amount of zinc in the media. Such increases or
decreases in the amount of zinc can be of a magnitude of about 1%,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more
of the preceding, of the original amount.
[0259] In certain embodiments, cell culture media containing no
zinc is supplemented with zinc to achieve a final zinc
concentration in the cell culture media of less than 60 .mu.M. In
certain embodiments, the cell culture will contain a total
concentration of zinc of between about 0.025 and about 10 .mu.M,
between about 0.05 and 10 .mu.M, between about 0.1 and 10 .mu.M,
between about 0.2 and 10 .mu.M, between about 0.25 and 10 .mu.M,
between about 0.5 and 10 .mu.M, between about 1 and 10 .mu.M,
between about 1.5 and 9.5 .mu.M, between about 2 and 9 .mu.M,
between about 2.5 and 8.5 .mu.M, between about 3 and 8 .mu.M,
between about 3.5 and 7.5 .mu.M, between about 4 and 7 .mu.M,
between about 4.5 and 6.5 .mu.M, between about 5 and 6 .mu.M. In
certain embodiments, the cell culture media containing no zinc is
supplemented with zinc to achieve a final zinc concentration in the
cell culture media of about 3.4 .mu.M or about 6.7 .mu.M.
[0260] In certain embodiments, the cell culture media contains zinc
in an amount effective to reduce the amount of one or more
C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a protein or
antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,
3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges
within one or more of the preceding, as compared to a non-modulated
lysine variant species composition. In one aspect of this
embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants are
reduced by about 1% to about 25%, about 5% to about 20%, about 5%
to about 15%, or about 5% to about 10%, and ranges within one or
more of the preceding, as compared to a non-modulated lysine
variant species composition.
[0261] In certain embodiments, the cell culture media contains zinc
in an amount effective to increase the amount of one or more
C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a protein or
antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,
3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges
within one or more of the preceding, as compared to a non-modulated
lysine variant species composition. In one aspect of this
embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants are
increased by about 1% to about 25%, about 5% to about 20%, about 5%
to about 15%, or about 5% to about 10%, and ranges within one or
more of the preceding, as compared to a non-modulated lysine
variant species composition,
[0262] In certain embodiments, the cell culture media contains zinc
in an amount effective to reduce the amount of a Lys 0 lysine
variant, and to increase the amount of a Lys 1 and/or Lys 2 lysine
variant expressed by the cell culture.
[0263] For example, and not by way of limitation, as detailed in
Example 1, below, certain embodiments include reducing the zinc
concentration of the cell culture medium employed from a control
concentration of about 10 .mu.M to about 3.4 .mu.M, and the % Lys 0
of an adalimumab sample purified from the supplemented culture is
reduced to 67.9% from a control amount of 92.7%. Additionally, the
amounts of Lys 1 and Lys 2 in the adalimumab sample are increased
to 19.9% (Lys 1) and 12.2% (Lys 2) from an adalimumab sample
purified from the control culture (10 .mu.M zinc) having 6.3% Lys 1
and 1.0% Lys 2. Furthermore, as detailed in Example 1, below,
certain embodiments include reducing the zinc concentration of the
cell culture medium employed from a control concentration of about
10 .mu.M to about 3.4 .mu.M or about 6.7 .mu.M, and the % Lys 0 of
an adalimumab sample purified from the supplemented culture is
reduced to 69.0% (3.4 .mu.M) or 89.9% (6.7 .mu.M) from a control
amount of 92.8%. Additionally, the amounts of Lys 1 and Lys 2 in
the adalimumab sample are increased to 21.8% (Lys 1-3.4 .mu.M),
8.7% (Lys 1-6.7 .mu.M), 9.1% (Lys 2-3.4 .mu.M), and 1.4% (Lys 2-6.7
.mu.M) from an adalimumab sample purified from the control culture
(10 .mu.M zinc) having 6.2% Lys 1 and 1.1% Lys 2.
[0264] In certain embodiments, the cell culture contains zinc as
well as arginine, lysine, and histidine, each at a concentration
sufficient to reduce the amount of a Lys 0 lysine variant, and to
increase the amount of a Lys 1 and/or Lys 2 lysine variant
expressed by the cell culture. For example, but not by way of
limitation, the concentration range for arginine and lysine can be
between about 1 to about 3 g/L, while the concentration range for
histidine is between about 0 to about 1 g/L and the concentration
range for zinc is about 30 .mu.M to about 60 .mu.M.
[0265] Adjusting Amino Acid Concentration to Control C-Terminal
Lysine Variation
[0266] In certain embodiments of the instant invention, control of
C-terminal lysine variants in a composition comprising an antibody,
or antigen binding portion thereof, can be attained by adjustment
of the amino acid composition of the cell culture media. In certain
embodiments, the amount of one or more amino acids in the media is
increased, while in other embodiments the amount of one or more
amino acids in the media is decreased. Such increases or decreases
in the amount of the one or more amino acids can be of a magnitude
of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or
more of the preceding, of the original amount.
[0267] In certain embodiments, the cell culture media is
supplemented with one or more amino acids wherein each of the one
or more amino acids is supplemented in an amount of between about
0.025 and 20 g/L, or between about 0.05 and 15 g/L, or between
about 0.1 and 14 g/L, or between about 0.2 and 13 g/L, or between
about 0.25 and 12 g/L, or between about 0.5 and 11 g/L, or between
about 1 and 10 g/L, or between about 1.5 and 9.5 g/L, or between
about 2 and 9 g/L, or between about 2.5 and 8.5 g/L, or between
about 3 and 8 g/L, or between about 3.5 and 7.5 g/L, or between
about 4 and 7 g/L, or between about 4.5 and 6.5 g/L, or between
about 5 and 6 g/L. In certain embodiments, the cell culture media
is supplemented with one or more amino acids wherein each of the
one or more amino acids is supplemented in an amount of about 0.5
g/L, or about 1 g/L, or about 2 g/L, or about 4 g/L, or about 8
g/L.
[0268] In certain embodiments, the cell culture media is
supplemented with one or more amino acids wherein each of the one
or more amino acids is supplemented in an amount effective to
reduce the amount of one or more lysine variants (Lys 0, Lys 1, or
Lys 2) in a protein or antibody sample by about 1%, 1.2%, 1.5%, 2%,
2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 100%, and ranges within one or more of the preceding, as
compared to a non-modulated lysine variant species composition. In
one aspect of this embodiment, the percentage of Lys 0, Lys 1, or
Lys 2 variants are reduced by about 1% to about 25%, about 5% to
about 20%, about 5% to about 15%, or about 5% to about 10%, and
ranges within one or more of the preceding, as compared to a
non-modulated lysine variant species composition.
[0269] In certain embodiments, the cell culture media is
supplemented with one or more amino acids wherein each of the one
or more amino acids is supplemented in an amount effective to
increase the amount of one or more C-terminal lysine variants (Lys
0, Lys 1, or Lys 2) in a protein or antibody sample by about 1%,
1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the
preceding, as compared to a non-modulated lysine variant species
composition. In one aspect of this embodiment, the percentage of
Lys 0, Lys 1, or Lys 2 variants are increased by about 1% to about
25%, about 5% to about 20%, about 5% to about 15%, or about 5% to
about 10%, and ranges within one or more of the preceding, as
compared to a non-modulated lysine variant species composition.
[0270] In certain embodiments, the cell culture media is
supplemented with one or more amino acids wherein each of the one
or more amino acids is supplemented in an amount effective to
reduce the amount of a Lys 0 lysine variant, and to increase the
amount of a Lys 1 and/or Lys 2 lysine variant expressed by the cell
culture.
[0271] In certain embodiments, the one or more amino acids used to
supplement the cell culture media is arginine, lysine, histidine,
or combinations of arginine or lysine with ornithine. In certain
embodiments, the amino acids are provided as single peptides, as
dipeptides, as tripeptides or as longer oligopeptides. In certain
embodiments, the di-, tri-, and/or oligopeptides are individually
composed of a single amino acid, while in alternative embodiments,
the di-, tri-, and/or oligopeptides are individually composed of
two or more particular amino acids.
[0272] For example, and not by way of limitation, as detailed in
Example 2, below, when the production medium employed in the
example was supplemented with 3 g/L arginine and 3 g/L lysine, the
% Lys 0 of an adalimumab sample purified from the supplemented
culture was reduced to 72.1% from a control amount of 86.7%.
Additionally, the amounts of Lys 1 and Lys 2 in the adalimumab
sample was increased to 20.8% (Lys 1) and 7.1% (Lys 2) from an
adalimumab sample purified from a control culture having 11.1% Lys
1 and 2.2% Lys 2.
[0273] Similarly, although the percentage of Lys 0 relative to
lysine sum in the control sample was 82.9% on day 10, in the sample
with the highest concentration of arginine in this experiment (9
g/L), the percentage of relative Lys 0 was reduced to 73.4%. This
relative modulation of Lys 0, Lys 1 and Lys 2 was directly related
to the concentration of arginine in the media. Thus, in certain
embodiments, the relative level of Lys 0 to lysine sum can be
modulated, i.e., increased or decreased, by adjusting the
concentration of arginine in the culture media.
[0274] In certain embodiments, a lysine supplementation of cell
culture is employed to modulate the ratio of Lys 0 to lysine sum.
As detailed Example 2, below, in the sample with the highest
concentration of lysine (11 g/L), the percentage of relative levels
of Lys 0 was reduced to 67.7%, which contrasts with the percentage
of Lys 0 relative to lysine sum (sum of the peak areas
corresponding to Lys 0, Lys 1 and Lys 2) in the control sample of
92.5%. A dose dependent decrease in relative Lys 0, and a dose
dependent increase in relative Lys 1 and Lys 2 regions was observed
in test conditions with increased lysine concentration. Thus, in
certain embodiments, the relative level of Lys 0 to lysine sum can
be modulated, i.e., increased or decreased, by adjusting the
concentration of lysine in the culture media.
[0275] In certain embodiments, a histidine supplementation of cell
culture is employed to modulate the ratio of Lys 0 to lysine sum.
As detailed Example 2, below, in the sample with the highest
concentration of histidine (10 g/L), the percentage of relative Lys
0 was reduced to 80.6% from a control sample percentage of 92.5%.
The decrease in Lys 0 corresponded with the increase in relative
levels of both Lys 1 and Lys 2. In an alternative example, the
percentage of Lys 0 relative to lysine sum in the control sample
was 94.2%. In the sample with the highest concentration of
histidine in this experiment (8 g/L), the percentage of relative
Lys 0 was reduced to 81.5%. The decrease in Lys 0 corresponded with
the increase in relative levels of both Lys 1 and Lys 2. A dose
dependent decrease in relative Lys 0, and a dose dependent increase
in relative Lys 1 and Lys 2 regions was observed in test conditions
with increased histidine concentration. Thus, in certain
embodiments, the relative level of Lys 0 to lysine sum can be
modulated, i.e., increased or decreased, by adjusting the
concentration of histidine in the culture media.
[0276] In certain embodiments, an ornithine/arginine or
ornithine/lysine supplementation of cell culture is employed to
modulate the ratio of Lys 0 to lysine sum. As detailed Example 2,
below, the combination of ornithine with arginine or lysine reduced
the relative level of Lys 0 to 81.9% in comparison with the
condition with just arginine and lysine increase with a relative
level Lys 0 of 84.7%. Thus, the increase of ornithine may exhibit
synergistic effects in modulating lysine variant distribution when
added in combination with arginine and lysine. Thus, in certain
embodiments, the relative level of Lys 0 to lysine sum can be
modulated, i.e., increased or decreased, by adjusting the
concentration of a combination of ornithine and either arginine
and/or lysine in the culture media.
[0277] In certain embodiments, an
arginine/lysine/histidine/ornithine combination supplementation of
cell culture is employed to modulate the ratio of Lys 0 to lysine
sum. As detailed in Example 2, below, in comparison to the lower
concentrations, or conditions where amino acids were supplemented
individually, a further reduction in Lys 0 relative to lysine sum
was observed in conditions where combinations of amino acids were
increased in the media. A progressive decrease was observed in
relative Lys 0 when more amino acids were increased in combination.
The percentage of relative Lys 0 was reduced from 94.9% in the
control sample to 73.9% in the sample with all four amino acid
concentrations increased. Thus, in certain embodiments, the
relative level of Lys 0 to lysine sum can be modulated, i.e.,
increased or decreased, by adjusting the concentration of a
combination of all four amino acids (arginine, lysine, histidine,
and ornithine) in the culture media.
[0278] In certain embodiments, the three amino acid monomers
arginine, lysine, and histidine, the dipeptides lys-lys and
arg-lys, or the tripeptides lys-lys-lys, his-arg-lys, and
arg-his-lys are used as supplements to cell culture in order to
modulate the ratio of Lys 0 to lysine sum. As detailed in Example
2, below, in comparison to the control, a reduction in Lys 0
relative to lysine sum was also observed in conditions where
dipeptides and tripeptides were supplemented to the media.
Specifically, a decrease was observed in relative Lys 0 when
polypeptides were supplemented. The percentage of relative Lys 0
was reduced from 88.0% in the control sample to 71.9% in a sample
supplemented with tripeptide arg-his-lys (4 g/L), and to 74.0% in a
sample supplemented with his-arg-lys (2 g/L).
[0279] In certain embodiments, the medium supplements described
herein are such that they can be included in the medium at the
start of culture, or can be added in a fed-batch or in a continuous
manner. The medium supplements could be supplemented to chemically
defined or hydrolysate based basal media. The methods described in
this invention may be used in combination with different
cultivation methods including, but not limited to, batch,
fed-batch, chemostat and perfusion, and with various cell culture
equipment including, but not limited to, shake flasks with or
without suitable agitation, spinner flasks, stirred bioreactors,
airlift bioreactors, membrane bioreactors, reactors with cells
retained on a solid support or immobilized/entrapped as in
microporous beads, and any other configuration appropriate for
optimal growth and productivity of the desired cell line. The
methods described here may also be combined with the appropriate
choice of process parameters as described herein.
Adjusting Phosphate Concentration to Control Lysine Variation
[0280] In certain embodiments of the instant invention, control of
C-terminal lysine variants in a composition comprising an antibody,
or antigen binding portion thereof, can be attained by adjustment
of the phosphate concentration of the media employed in the cell
culture run. In one non-limiting embodiment, such adjustment will
be to increase the amount of phosphate in the media, while in other
non-limiting embodiments, the necessary adjustment to achieve the
desired control over lysine variant heterogeneity will involve an
decrease in the amount of phosphate in the media. Such increases or
decreases in the amount of phosphate can be of a magnitude of about
1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more
of the preceding, of the original amount.
[0281] In certain embodiments, the cell culture media is
supplemented with phosphate in an amount of between about 6 and 24
mM, or between about 0.05 and 22 mM, or between about 0.1 and 20
mM, or between about 0.2 and 18 mM, or between about 0.25 and 16
mM, or between about 0.5 and 14 mM, or between about 1 and 12 mM,
or between about 1.5 and 10 mM, or between about 2 and 8 mM, or
between about 2.5 and 6 mM, or between about 3 and 4 mM,
[0282] In certain embodiments, the cell culture media contains
phosphate in an amount effective to reduce the amount of one or
more C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a
protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%,
3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,
and ranges within one or more of the preceding, as compared to a
non-modulated lysine variant species composition. In one aspect of
this embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants
are reduced by about 1% to about 25%, about 5% to about 20%, about
5% to about 15%, or about 5% to about 10%, and ranges within one or
more of the preceding, as compared to a non-modulated lysine
variant species composition.
[0283] In certain embodiments, the cell culture media contains
phosphate in an amount effective to increase the amount of one or
more C-terminal lysine variants (Lys 0, Lys 1, or Lys 2) in a
protein or antibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%,
3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,
and ranges within one or more of the preceding, as compared to a
non-modulated lysine variant species composition. In one aspect of
this embodiment, the percentage of Lys 0, Lys 1, or Lys 2 variants
are increased by about 1% to about 25%, about 5% to about 20%,
about 5% to about 15%, or about 5% to about 10%, and ranges within
one or more of the preceding, as compared to a non-modulated lysine
variant species composition.
[0284] In certain embodiments, the cell culture media contains
phosphate in an amount effective to reduce the amount of a Lys 0
lysine variant, and to increase the amount of a Lys 1 and/or Lys 2
lysine variant expressed by the cell culture.
[0285] In certain embodiments, a phosphate supplementation of cell
culture is employed to modulate the ratio of Lys 0 to lysine sum.
As detailed Example 4, below, in the sample with the highest
concentration of phosphate supplementation (24 mM), the percentage
of relative levels of Lys 0 was reduced to 85.7%, which contrasts
with the percentage of Lys 0 relative to lysine sum (sum of the
peak areas corresponding to Lys 0, Lys 1 and Lys 2) in the control
sample of 91.3%. A dose dependent decrease in relative Lys 0, and a
dose dependent increase in relative Lys 1 and Lys 2 regions was
observed in test conditions with increased phosphate concentration.
Thus, in certain embodiments, the relative level of Lys 0 to lysine
sum can be modulated, i.e., increased or decreased, by adjusting
the concentration of phosphate in the culture media.
[0286] In certain embodiments, the cell culture contains phosphate
as well as arginine, lysine, and zinc, each at a concentration
sufficient to reduce the amount of a Lys 0 lysine variant, and to
increase the amount of a Lys 1 and/or Lys 2 lysine variant
expressed by the cell culture. For example, but not by way of
limitation, the concentration range for arginine can be between 6.0
mM to about 19.9 mM, and the concentration range for lysine can be
between about 4.0 to about 15.5 mM, while the concentration range
for zinc is between about 10 to about 40 .mu.M and the
concentration range for phosphate is about 5.8 to 17.8 mM.
[0287] Adjusting Process Parameters to Control Lysine Variation
[0288] The variation in the process parameters, such as the
temperature, dissolved oxygen (DO) concentration, and/or pH, at
which cells are cultured, can vary product quality significantly.
In certain embodiments of the instant invention, control of
C-terminal lysine variant heterogeneity can be attained by
adjustment of the temperature, DO concentration, phosphate
concentration, and/or pH of the cell culture run. In certain
embodiments, such adjustment will be to increase the temperature,
DO concentration, phosphate concentration, and/or pH at which a
cell culture is cultured, while in other embodiments the necessary
adjustment to achieve the desired control over lysine variant
heterogeneity will involve a modulation of the temperature, DO
concentration, phosphate concentration, and/or pH at which a cell
culture is cultured. Such increases or decreases in cell culture
temperature, DO concentration, phosphate concentration, and/or pH
can be of a magnitude of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and
ranges within one or more of the preceding, of the original
temperature DO concentration, and/or pH.
[0289] In certain embodiments, the cell culture is cultured at a
temperature of between about 25 and 50.degree. C., or between about
30.degree. C. and 40.degree. C., or between about 31.degree. C. and
39.degree. C., or between about 31.5.degree. C. and 38.5.degree.
C., or between about 32.degree. C. and 38.degree. C., or between
about 32.5.degree. C. and 37.5.degree. C., or between about
33.degree. C. and 37.degree. C., or between about 33.5.degree. C.
and 36.5.degree. C., or between about 34.degree. C. and 36.degree.
C., or between about 34.5.degree. C. and 35.5.degree. C. In certain
embodiments, the cell culture is cultured at a temperature of about
30.degree. C., 31.degree. C., 32.degree. C., 33.degree. C.,
34.degree. C., 35.degree. C., 36.degree. C., or 37.degree. C.
[0290] For example, and not by way of limitation, as detailed in
Example 3, below, when the temperature of a cell culture run was
decreased from 37.degree. C. to 31.degree. C., the % Lys 0 of an
adalimumab sample purified from the culture was reduced from 84.9%
to 72.8%. Additionally, the amounts of Lys 1 and Lys 2 in the
adalimumab sample was increased from 13.4% (Lys 1) and 1.7% (Lys
2), to 22.2% (Lys 1) and 5.0% (Lys 2). Thus, in certain
embodiments, the relative level of Lys 0 to lysine sum can be
modulated, i.e., increased or decreased, by adjusting the
temperature of the cell culture run.
[0291] In certain embodiments, but not by way of limitation, DO
concentrations maintained above 15%, e.g., between about 30% and
about 50% or at about 80%, can be used to achieve the desired
change in lysine sum distribution.
[0292] For example, and not by way of limitation, as detailed in
Example 3, below, when the DO concentration in a cell culture run
was assessed at 20% and 50%, at a temperature of 35.degree. C., the
relative fraction of Lys 0 decreased from 84.8% at 20% DO to 81.0%
at 50% DO, with a corresponding 3.2% increase in relative levels of
Lys 1 and 0.7% increase in Lys 2. Thus, in certain embodiments, the
relative level of Lys 0 to lysine sum can be modulated, i.e.,
increased or decreased, by adjusting the DO concentration of the
cell culture run.
[0293] In certain embodiments, pH is either increased or decreased
in order to increase or decrease the amount of Lys 0 relative to
the lysine sum. For example, but not by way of limitation, a
reduction in pH to 6.7 from a control pH of 7.1 can be employed to
increase the amount of Lys 0 relative to the lysine sum.
[0294] For example, and not by way of limitation, as detailed in
Example 3, below, when the pH of a cell culture run was decreased
from 7.1 to 6.7, the % Lys 0 of an adalimumab sample purified from
the culture was increased from 82.0% to 88.7%. In certain
embodiments the pH is increased from 6.7 to 6.8, 6.9, 7.0, or 7.1
in order to achieve a decrease in the amount of Lys 0 relative to
the lysine sum.
[0295] In certain embodiments, the temperature, DO concentration,
and/or pH of the cell culture is decreased or increased in an
amount effective to reduce the amount of one or more lysine
variants in a protein or antibody sample by about 1%, 1.2%, 1.5%,
2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 100%, and ranges within one or more of the preceding, as
compared to a non-modulated lysine variant species composition. In
one aspect of this embodiment, the percentage of Lys 0, Lys 1, or
Lys 2 variants are reduced by about 1% to about 25%, about 5% to
about 20%, about 5% to about 15%, or about 5% to about 10%, and
ranges within one or more of the preceding, as compared to a
non-modulated lysine variant species composition.
[0296] In certain embodiments, the temperature, DO concentration,
and/or pH of the cell culture is decreased or increased in an
amount effective to increase the amount of one or more lysine
variants in a protein or antibody sample by about 1%, 1.2%, 1.5%,
2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 100%, and ranges within one or more of the preceding, as
compared to a non-modulated lysine variant species composition. In
one aspect of this embodiment, the percentage of Lys 0, Lys 1, or
Lys 2 variants are increased by about 1% to about 25%, about 5% to
about 20%, about 5% to about 15%, or about 5% to about 10%, and
ranges within one or more of the preceding, as compared to a
non-modulated lysine variant species composition.
[0297] In certain embodiments, the temperature, DO concentration,
and/or pH of the cell culture is decreased or increased in an
amount effective to reduce the amount of a Lys 0 lysine variant,
and to increase the amount of a Lys 1 and/or Lys 2 lysine variant
expressed by the cell culture.
[0298] Additional Exemplary Strategies
[0299] In addition to the above-described embodiments, the present
invention is also directed to embodiments wherein the medium
supplements described herein are added in a batch-wise fashion, a
continuous feeding fashion, or a combination of both during cell
culture. In addition, certain embodiments will involve the adding
such media supplements one at a time and/or addition at multiple
time points during the cell culture process. In certain
embodiments, the cell culture process will involve preloading the
culture media with excess of one or more medium supplements. In
certain embodiments, the addition of one or more supplements will
be based on measurements taken on-line, in-line, and/or at line. In
certain embodiments, the addition of one or more supplements will
occur with other substrates, metal scavengers, and/or combination
with other culture conditions such as temperature, DO
concentration, phosphate concentration, pH, etc. In certain
embodiments, one or more media supplements will be added as
multimers, e.g., arg-arg, his-his, arg-his-orn, etc., and/or as
chemical variants of amino acids or analogs of amino acids, salt
forms of amino acids, controlled release of amino acids by
immobilizing in gels, etc, and/or in fully or partially dissolved
form.
[0300] In certain embodiments, the culture process will occur in
bags, flasks, disposables, hollow fiber, perfusion, and/or air lift
process equipment. In certain embodiments, one or more media
supplements will be added to seed bioreactor before transfer to
achieve a final concentration in the fermentor. In certain
embodiments, achieving a known concentration of one or more of the
media supplements can occur either through an in-situ combination
resulting the generation of the supplement or a
degradation/reaction resulting the generation of the supplement,
i.e., adding a substrate and enzyme/catalyst to produce the
components necessary. In certain embodiments the addition of one or
more media supplement will based on measured amount of lysine
distribution.
IV. PROTEIN PURIFICATION
[0301] Protein Purification Generally
[0302] In certain embodiments, the methods for producing the
modulated lysine variant species compositions of the present
invention can be used in combination with techniques for protein
purification to provide for the production of a purified protein
preparation, for example, a preparation comprising an antibody or
an antigen binding fragment thereof, from a mixture comprising a
protein and at least one process-related impurity or
product-related substance (e.g., acidic species).
[0303] For example, but not by way of limitation, once a clarified
solution or mixture comprising the protein of interest, for
example, an antibody or antigen binding fragment thereof, has been
obtained, separation of the protein of interest from the
process-related impurities and/or product-related substances (e.g.,
acidic species) can be effected using a combination of different
purification techniques, including, but not limited to, affinity
separation steps, ion exchange separation steps, mixed mode
separation steps, and hydrophobic interaction separation steps,
singly or in combination. The separation steps separate mixtures of
proteins on the basis of their charge, degree of hydrophobicity, or
size depending upon the particular form of separation, including
chromatographic separation. In one aspect of the invention,
separation is performed using chromatography, including cationic,
anionic, and hydrophobic interaction. Several different
chromatography resins are commercially available for each of these
techniques, allowing accurate tailoring of the purification scheme
to the particular protein involved. The essence of each of the
separation methods is that proteins can be caused either to
traverse at different rates through a column's resin, achieving a
physical separation that increases as they pass further through the
column, or to adhere selectively to a column's separation resin,
and then differentially eluted using different eluents. In some
cases, the antibody is separated from impurities when the
impurities specifically adhere to the column's resin and the
antibody does not, i.e., the antibody is contained in the eluent,
while in other cases the antibody of interest will adhere to the
column's resin, while impurities and/or product-related substances
are extruded from the column during a wash cycle.
[0304] As noted above, accurate tailoring of a purification scheme
relies on consideration of the protein to be purified. In certain
embodiments, the separation steps employed in connection with the
cell culture methods of the instant invention facilitate the
separation of an antibody from one or more process-related impurity
(acidic or basic species) and/or product-related substance.
Antibodies that can be successfully purified using the methods
described herein include, but are not limited to, human IgA1, IgA2,
IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM antibodies. In certain
embodiments, Protein A affinity chromatography can be useful.
However, IgG3 antibodies bind to Protein A inefficiently. Other
factors that allow for specific tailoring of a purification scheme
include, but are not limited to: the presence or absence of an Fc
region (e.g., in the context of full length antibody as compared to
an Fab fragment thereof) because Protein A binds to the Fc region;
the particular germline sequences employed in generating to
antibody of interest; and the amino acid composition of the
antibody (e.g., the primary sequence of the antibody as well as the
overall charge/hydrophobicity of the molecule). Antibodies sharing
one or more characteristic can be purified using purification
strategies tailored to take advantage of that characteristic.
[0305] Methods for purification of proteins, e.g., antibodies, are
described in detail in Attorney Docket No. 117813-73901 (U.S.
Patent Application Ser. No. 61/893,068), filed on Oct. 18, 2013,
the contents of which are expressly incorporated herein by
reference.
[0306] Primary Recovery and Virus Inactivation
[0307] In certain embodiments, it will be advantageous to subject a
sample produced by the techniques of the instant invention to at
least a first phase of clarification and primary recovery. In
addition, the primary recovery process can also be a point at which
to reduce or inactivate viruses that can be present in the sample
mixture. For example, any one or more of a variety of methods of
viral reduction/inactivation can be used during the primary
recovery phase of purification including heat inactivation
(pasteurization), pH inactivation, solvent/detergent treatment, UV
and .gamma.-ray irradiation and the addition of certain chemical
inactivating agents such as .beta.-propiolactone or e.g., copper
phenanthroline as in U.S. Pat. No. 4,534,972, the entire contents
of which are incorporated herein by reference.
[0308] The primary recovery may also include one or more
centrifugation steps to further clarify the sample mixture and
thereby aid in purifying the protein of interest. Centrifugation of
the sample can be run at, for example, but not by way of
limitation, 7,000.times.g to approximately 12,750.times.g. In the
context of large scale purification, such centrifugation can occur
on-line with a flow rate set to achieve, for example, but not by
way of limitation, a turbidity level of 150 NTU in the resulting
supernatant. Such supernatant can then be collected for further
purification.
[0309] In certain embodiments, the primary recovery may also
include the use of one or more depth filtration steps to further
clarify the sample matrix and thereby aid in purifying the
antibodies produced using the cell culture techniques of the
present invention. Depth filters contain filtration media having a
graded density. Such graded density allows larger particles to be
trapped near the surface of the filter while smaller particles
penetrate the larger open areas at the surface of the filter, only
to be trapped in the smaller openings nearer to the center of the
filter. In certain embodiments, the depth filtration step can be a
delipid depth filtration step. Although certain embodiments employ
depth filtration steps only during the primary recovery phase,
other embodiments employ depth filters, including delipid depth
filters, during one or more additional phases of purification.
Non-limiting examples of depth filters that can be used in the
context of the instant invention include the Cuno.TM. model 30/60ZA
depth filters (3M Corp.), and 0.45/0.2 .mu.m Sartopore.TM. bi-layer
filter cartridges.
[0310] Affinity Chromatography
[0311] In certain embodiments, it will be advantageous to subject a
modulated Lysine sample produced by the methods of the instant
invention to affinity chromatography to further purify the protein
of interest away from process-related impurities and/or
product-related substances. In certain embodiments the
chromatographic material is capable of selectively or specifically
binding to the protein of interest. Non-limiting examples of such
chromatographic material include: Protein A, Protein G,
chromatographic material comprising, for example, an antigen bound
by an antibody of interest, and chromatographic material comprising
an Fc binding protein. In specific embodiments, the affinity
chromatography step involves subjecting the primary recovery sample
to a column comprising a suitable Protein A resin. In certain
embodiments, Protein A resin is useful for affinity purification
and isolation of a variety of antibody isotypes, particularly IgG1,
IgG2, and IgG4. Protein A is a bacterial cell wall protein that
binds to mammalian IgGs primarily through their Fc regions. In its
native state, Protein A has five IgG binding domains as well as
other domains of unknown function.
[0312] There are several commercial sources for Protein A resin.
One suitable resin is MabSelect.TM. from GE Healthcare. A
non-limiting example of a suitable column packed with MabSelect.TM.
is an about 1.0 cm diameter.times.about 21.6 cm long column
(.about.17 ml bed volume). This size column can be used for small
scale purifications and can be compared with other columns used for
scale ups. For example, a 20 cm.times.21 cm column whose bed volume
is about 6.6 L can be used for larger purifications. Regardless of
the column, the column can be packed using a suitable resin such as
MabSelect.TM..
[0313] Ion Exchange Chromatography
[0314] In certain embodiments, it will be advantageous to subject a
modulated Lysine sample produced by the methods of the instant
invention to ion exchange chromatography in order to purify the
protein of interest away from process-related impurities and/or
product-related substances. Ion exchange separation includes any
method by which two substances are separated based on the
difference in their respective ionic charges, and can employ either
cationic exchange material or anionic exchange material. For
example, the use of a cationic exchange material versus an anionic
exchange material is based on the localized charges of the protein.
Therefore, it is within the scope of this invention to employ an
anionic exchange step prior to the use of a cationic exchange step,
or a cationic exchange step prior to the use of an anionic exchange
step. Furthermore, it is within the scope of this invention to
employ only a cationic exchange step, only an anionic exchange
step, or any serial combination of the two.
[0315] In performing the separation, the initial protein mixture
can be contacted with the ion exchange material by using any of a
variety of techniques, e.g., using a batch purification technique
or a chromatographic technique.
[0316] Anionic or cationic substituents may be attached to matrices
in order to form anionic or cationic supports for chromatography.
Non-limiting examples of anionic exchange substituents include
diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and
quaternary amine (Q) groups. Cationic substituents include
carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate
(P) and sulfonate (S). Cellulose ion exchange resins such as
DE23.TM., DE32.TM., DE52.TM., CM-23.TM., CM-32.TM., and CM-52.TM.
are available from Whatman Ltd. Maidstone, Kent, U.K.
SEPHADEX.RTM.-based and -locross-linked ion exchangers are also
known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX.RTM. and
DEAE-, Q-, CM- and S-SEPHAROSE.RTM. and SEPHAROSE.RTM. Fast Fe all
available from Pharmacia AB. Further, both DEAE and CM derivitized
ethylene glycol-methacrylate copolymer such as TOYOPEARL.TM.
DEAE-650S or M and TOYOPEARL.TM. CM-650S or M are available from
Toso Haas Co., Philadelphia, Pa.
[0317] Ultrafiltration/Diafiltration
[0318] In certain embodiments, it will be advantageous to subject a
modulated Lysine sample produced by the methods of the instant
invention to ultrafiltration and/or diafiltration in order to
purify the protein of interest away from process-related impurities
and/or product-related substances. Ultrafiltration is described in
detail in: Microfiltration and Ultrafiltration: Principles and
Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New
York, N.Y., 1996); and in: Ultrafiltration Handbook, Munk Cheryan
(Technomic Publishing, 1986; ISBN No. 87762-456-9). One filtration
process is Tangential Flow Filtration as described in the Millipore
catalogue entitled "Pharmaceutical Process Filtration Catalogue"
pp. 177-202 (Bedford, Mass., 1995/96). Ultrafiltration is generally
considered to mean filtration using filters with a pore size of
smaller than 0.1 .mu.m. By employing filters having such small pore
size, the volume of the sample can be reduced through permeation of
the sample buffer through the filter while antibodies are retained
behind the filter.
[0319] Diafiltration is a method of using ultrafilters to remove
and exchange salts, sugars, and non-aqueous solvents, to separate
free from bound species, to remove low molecular-weight material,
and/or to cause the rapid change of ionic and/or pH environments.
Microsolutes are removed most efficiently by adding solvent to the
solution being ultrafiltered at a rate approximately equal to the
ultratfiltration rate. This washes microspecies from the solution
at a constant volume, effectively purifying the retained protein.
In certain embodiments of the present invention, a diafiltration
step is employed to exchange the various buffers used in connection
with the instant invention, optionally prior to further
chromatography or other purification steps, as well as to remove
impurities from the protein preparations.
[0320] Hydrophobic Interaction Chromatography
[0321] In certain embodiments, it will be advantageous to subject a
modulated Lysine sample produced by the methods of the instant
invention to hydrophobic interaction chromatography in order to
purify the protein of interest away from process-related impurities
and/or product-related substances. For example, a first eluate
obtained from an ion exchange column can be subjected to a
hydrophobic interaction material such that a second eluate having a
reduced level of impurity is obtained. Hydrophobic interaction
chromatography (HIC) steps, such as those disclosed herein, are
generally performed to remove protein aggregates, such as antibody
aggregates, and process-related impurities.
[0322] In performing an HIC-based separation, the sample mixture is
contacted with the HIC material, e.g., using a batch purification
technique or using a column. Prior to HIC purification it may be
desirable to remove any chaotropic agents or very hydrophobic
substances, e.g., by passing the mixture through a pre-column.
[0323] Whereas ion exchange chromatography relies on the charges of
the protein to isolate them, hydrophobic interaction chromatography
uses the hydrophobic properties of the protein. Hydrophobic groups
on the protein interact with hydrophobic groups on the column. The
more hydrophobic a protein is the stronger it will interact with
the column. Thus the HIC step removes host cell derived impurities
(e.g., DNA and other high and low molecular weight product-related
species).
[0324] Hydrophobic interactions are strongest at high ionic
strength, therefore, this form of separation is conveniently
performed following salt precipitations or ion exchange procedures.
Adsorption of the protein of interest to a HIC column is favored by
high salt concentrations, but the actual concentrations can vary
over a wide range depending on the nature of the protein and the
particular HIC ligand chosen. Various ions can be arranged in a
so-called soluphobic series depending on whether they promote
hydrophobic interactions (salting-out effects) or disrupt the
structure of water (chaotropic effect) and lead to the weakening of
the hydrophobic interaction. Cations are ranked in terms of
increasing salting out effect as Ba.sup.++; Ca.sup.++; Mg.sup.++;
Li.sup.+; Cs.sup.+; Na.sup.+; K.sup.+; Rb.sup.+; NH4.sup.+, while
anions may be ranked in terms of increasing chaotropic effect as
P0.sup.---; S0.sub.4.sup.--; CH.sub.3CO.sub.3; Cl.sup.-; Br.sup.-;
NO.sub.3.sup.-; ClO.sub.4.sup.-; I.sup.-; SCN.sup.-.
[0325] In general, Na, K or NH.sub.4 sulfates effectively promote
ligand-protein interaction in HIC. Salts may be formulated that
influence the strength of the interaction as given by the following
relationship:
(NH.sub.4).sub.2SO.sub.4>Na.sub.2SO.sub.4>NaCl>NH.sub.4Cl>NaB-
r>NaSCN. In general, salt concentrations of between about 0.75
and about 2 M ammonium sulfate or between about 1 and 4 M NaCl are
useful.
[0326] HIC columns normally comprise a base matrix (e.g.,
cross-linked agarose or synthetic copolymer material) to which
hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. A
suitable HIC column comprises an agarose resin substituted with
phenyl groups (e.g., a Phenyl Sepharose.TM. column). Many HIC
columns are available commercially. Examples include, but are not
limited to, Phenyl Sepharose.TM. 6 Fast Flow column with low or
high substitution (Pharmacia LKB Biotechnology, AB, Sweden); Phenyl
Sepharose.TM. High Performance column (Pharmacia LKB Biotechnology,
AB, Sweden); Octyl Sepharose.TM. High Performance column (Pharmacia
LKB Biotechnology, AB, Sweden); Fractogel.TM. EMD Propyl or
Fractogel.TM. EMD Phenyl columns (E. Merck, Germany);
Macro-Prep.TM. Mehyl or Macro-Prep.TM. t-Butyl Supports (Bio-Rad,
California); WP HI-Propyl (C3).TM. column (J. T. Baker, New
Jersey); and Toyopearl.TM. ether, phenyl or butyl columns
(TosoHaas, PA).
[0327] Multimodal Chromatography
[0328] In certain embodiments, it will be advantageous to subject a
modulated Lysine sample produced by the methods of the instant
invention to multimodal chromatography in order to purify the
protein of interest away from process-related impurities and/or
product-related substances. Multimodal chromatography is
chromatography that utilizes a multimodal media resin. Such a resin
comprises a multimodal chromatography ligand. In certain
embodiments, such a ligand refers to a ligand that is capable of
providing at least two different, but co-operative, sites which
interact with the substance to be bound. One of these sites gives
an attractive type of charge-charge interaction between the ligand
and the substance of interest. The other site typically gives
electron acceptor-donor interaction and/or hydrophobic and/or
hydrophilic interactions. Electron donor-acceptor interactions
include interactions such as hydrogen-bonding, .pi.-.pi.,
cation-.pi., charge transfer, dipole-dipole, induced dipole etc.
Multimodal chromatography ligands are also known as "mixed mode"
chromatography ligands.
[0329] In certain embodiments, the multimodal chromatography resin
is comprised of multimodal ligands coupled to an organic or
inorganic support, sometimes denoted a base matrix, directly or via
a spacer. The support may be in the form of particles, such as
essentially spherical particles, a monolith, filter, membrane,
surface, capillaries, etc. In certain embodiments, the support is
prepared from a native polymer, such as cross-linked carbohydrate
material, such as agarose, agar, cellulose, dextran, chitosan,
konjac, carrageenan, gellan, alginate etc. To obtain high
adsorption capacities, the support can be porous, and ligands are
then coupled to the external surfaces as well as to the pore
surfaces. Such native polymer supports can be prepared according to
standard methods, such as inverse suspension gelation (S Hjerten:
Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the
support can be prepared from a synthetic polymer, such as
cross-linked synthetic polymers, e.g. styrene or styrene
derivatives, divinylbenzene, acrylamides, acrylate esters,
methacrylate esters, vinyl esters, vinyl amides etc. Such synthetic
polymers can be produced according to standard methods, see e.g.
"Styrene based polymer supports developed by suspension
polymerization" (R Arshady: Chimica e L'Industria 70(9), 70-75
(1988)). Porous native or synthetic polymer supports are also
available from commercial sources, such as Amersham Biosciences,
Uppsala, Sweden.
V. METHODS OF TREATMENT USING THE MODULATED LYSINE VARIANT SPECIES
COMPOSITIONS OF THE INVENTION
[0330] The modulated lysine variant species compositions of the
invention may be used to treat any disorder in a subject for which
the therapeutic protein comprised in the composition is appropriate
for treating.
[0331] A "disorder" is any condition that would benefit from
treatment with the protein. This includes chronic and acute
disorders or diseases including those pathological conditions which
predispose the subject to the disorder in question. In the case of
an anti-TNF.alpha. antibody, or antigen binding portion thereof,
such as adalimumab, a therapeutically effective amount of the
modulated lysine variant species composition may be administered to
treat a disorder in which TNF.alpha. activity is detrimental.
[0332] A disorder in which TNF.alpha. activity is detrimental
includes a disorder in which inhibition of TNF.alpha. activity is
expected to alleviate the symptoms and/or progression of the
disorder. Such disorders may be evidenced, for example, by an
increase in the concentration of TNF.alpha. in a biological fluid
of a subject suffering from the disorder (e.g., an increase in the
concentration of TNF.alpha. in serum, plasma, synovial fluid, etc.
of the subject), which can be detected, for example, using an
anti-TNF.alpha. antibody.
[0333] TNF.alpha. has been implicated in the pathophysiology of a
wide variety of a TNF.alpha.-related disorders including sepsis,
infections, autoimmune diseases, transplant rejection and
graft-versus-host disease (see e.g., Moeller, A., et al. (1990)
Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.;
European Patent Publication No. 260 610 B1 by Moeller, A., et al.
Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, K. J.
and Cerami, A. (1994) Annu. Rev. Med. 45:491-503). Accordingly, the
modulated lysine variant species compositions or a low
process-related impurity compositions of the invention may be used
to treat an autoimmune disease, such as rheumatoid arthritis,
juvenile idiopathic arthritis, or psoriatic arthritis, an
intestinal disorder, such as Crohn's disease or ulcerative colitis,
a spondyloarthropathy, such as ankylosing spondylitis, or a skin
disorder, such as psoriasis.
[0334] Disorders in which TNF.alpha. activity is detrimental are
well known in the art and described in detail in U.S. Pat. No.
8,231,876 and U.S. Pat. No. 6,090,382, the entire contents of each
of which are expressly incorporated herein by reference. In one
embodiment, "a disorder in which TNF.alpha. activity is
detrimental" includes sepsis (including septic shock, endotoxic
shock, gram negative sepsis and toxic shock syndrome), autoimmune
diseases (including rheumatoid arthritis, rheumatoid spondylitis,
osteoarthritis and gouty arthritis, allergy, multiple sclerosis,
autoimmune diabetes, autoimmune uveitis, nephrotic syndrome,
multisystem autoimmune diseases, lupus (including systemic lupus,
lupus nephritis and lupus cerebritis), Crohn's disease and
autoimmune hearing loss), active axial spondyloarthritis (active
axSpA) and non-radiographic axial spondyloarthritis (nr-axSpA),
infectious diseases (including malaria, meningitis, acquired immune
deficiency syndrome (AIDS), influenza and cachexia secondary to
infection), allograft rejection and graft versus host disease,
malignancy, pulmonary disorders (including adult respiratory
distress syndrome (ARDS), shock lung, chronic pulmonary
inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis,
silicosis, idiopathic interstitial lung disease and chronic
obstructive airway disorders (COPD), such as asthma), intestinal
disorders (including inflammatory bowel disorders, idiopathic
inflammatory bowel disease, Crohn's disease and Crohn's
disease-related disorders (including fistulas in the bladder,
vagina, and skin; bowel obstructions; abscesses; nutritional
deficiencies; complications from corticosteroid use; inflammation
of the joints; erythem nodosum; pyoderma gangrenosum; lesions of
the eye, Crohn's related arthralgias, fistulizing Crohn's
indeterminant colitis and pouchitis), cardiac disorders (including
ischemia of the heart, heart insufficiency, restenosis, congestive
heart failure, coronary artery disease, angina pectoris, myocardial
infarction, cardiovascular tissue damage caused by cardiac arrest,
cardiovascular tissue damage caused by cardiac bypass, cardiogenic
shock, and hypertension, atherosclerosis, cardiomyopathy, coronary
artery spasm, coronary artery disease, valvular disease,
arrhythmias, and cardiomyopathies), spondyloarthropathies
(including ankylosing spondylitis, psoriatic arthritis/spondylitis,
enteropathic arthritis, reactive arthritis or Reiter's syndrome,
and undifferentiated spondyloarthropathies), metabolic disorders
(including obesity and diabetes, including type 1 diabetes
mellitus, type 2 diabetes mellitus, diabetic neuropathy, peripheral
neuropathy, diabetic retinopathy, diabetic ulcerations, retinopathy
ulcerations and diabetic macrovasculopathy), anemia, pain
(including acute and chronic pains, such as neuropathic pain and
post-operative pain, chronic lower back pain, cluster headaches,
herpes neuralgia, phantom limb pain, central pain, dental pain,
opioid-resistant pain, visceral pain, surgical pain, bone injury
pain, pain during labor and delivery, pain resulting from burns,
including sunburn, post partum pain, migraine, angina pain, and
genitourinary tract-related pain including cystitis), hepatic
disorders (including hepatitis, alcoholic hepatitis, viral
hepatitis, alcoholic cirrhosis, al antitypsin deficiency,
autoimmune cirrhosis, cryptogenic cirrhosis, fulminant hepatitis,
hepatitis B and C, and steatohepatitis, cystic fibrosis, primary
biliary cirrhosis, sclerosing cholangitis and biliary obstruction),
skin and nail disorders (including psoriasis (including chronic
plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular
psoriasis and other psoriasis disorders), pemphigus vulgaris,
scleroderma, atopic dermatitis (eczema), sarcoidosis, erythema
nodosum, hidradenitis suppurative, lichen planus, Sweet's syndrome,
scleroderma and vitiligo), vasculitides (including Behcet's
disease), and other disorders, such as juvenile rheumatoid
arthritis (JRA), endometriosis, prostatitis, choroidal
neovascularization, sciatica, Sjogren's syndrome, uveitis, wet
macular degeneration, osteoporosis and osteoarthritis.
[0335] As used herein, 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.
[0336] As used herein, the term "treatment" or "treat" refers to
both therapeutic treatment and prophylactic or preventative
measures. Those in need of treatment include those already with the
disorder, as well as those in which the disorder is to be
prevented.
[0337] In one embodiment, the invention provides a method of
administering a modulated lysine variant species composition
comprising an anti-TNF.alpha. antibody, or antigen binding portion
thereof, to a subject such that TNF.alpha. activity is inhibited or
a disorder in which TNF.alpha. activity is detrimental is treated.
In one embodiment, the TNF.alpha. is human TNF.alpha. and the
subject is a human subject. In one embodiment, the anti-TNF.alpha.
antibody is adalimumab.
[0338] The modulated lysine variant species compositions can be
administered by a variety of methods known in the art. Exemplary
routes/modes of administration include subcutaneous injection,
intravenous injection or infusion. In certain aspects, a modulated
lysine variant species compositions may be orally administered. As
will be appreciated by the skilled artisan, the route and/or mode
of administration will vary depending upon the desired results.
[0339] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. In certain embodiments it is
especially advantageous to formulate parenteral compositions in
dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the mammalian subjects
to be treated; each unit comprising 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 active compound and the particular
therapeutic or prophylactic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals.
[0340] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a modulated lysine variant
species composition of the invention is 0.01-20 mg/kg, or 1-10
mg/kg, or 0.3-1 mg/kg. With respect to modulated lysine variant
species compositions comprising an anti-TNF.alpha. antibody, or
antigen-binding portion thereof, such as adalimumab, an exemplary
dose is 40 mg every other week. In some embodiments, in particular
for treatment of ulcerative colitis or Crohn's disease, an
exemplary dose includes an initial dose (Day 1) of 160 mg (e.g.,
four 40 mg injections in one day or two 40 mg injections per day
for two consecutive days), a second dose two weeks later of 80 mg,
and a maintenance dose of 40 mg every other week beginning two
weeks later. Alternatively, for psoriasis for example, a dosage can
include an 80 mg initial dose followed by 40 mg every other week
starting one week after the initial dose.
[0341] It is to be noted that dosage values may vary with the type
and 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.
VI. PHARMACEUTICAL FORMULATIONS CONTAINING THE MODULATED LYSINE
VARIANT SPECIES COMPOSITIONS OF THE INVENTION
[0342] The present invention further provides preparations and
formulations comprising the modulated lysine variant species
compositions of the invention. It should be understood that any of
the antibodies and antibody fragments described herein, including
antibodies and antibody fragments having any one or more of the
structural and functional features described in detail throughout
the application, may be formulated or prepared as described below.
When various formulations are described in this section as
including an antibody, it is understood that such an antibody may
be an antibody or an antibody fragment having any one or more of
the characteristics of the antibodies and antibody fragments
described herein. In one embodiment, the antibody is an
anti-TNF.alpha. antibody, or antigen-binding portion thereof.
[0343] In certain embodiments, the modulated lysine variant species
compositions of the invention may be formulated with a
pharmaceutically acceptable carrier as pharmaceutical (therapeutic)
compositions, and may be administered by a variety of methods known
in the art. As will be appreciated by the skilled artisan, the
route and/or mode of administration will vary depending upon the
desired results. The term "pharmaceutically acceptable carrier"
means one or more non-toxic materials that do not interfere with
the effectiveness of the biological activity of the active
ingredients. Such preparations may routinely contain salts,
buffering agents, preservatives, compatible carriers, and
optionally other therapeutic agents. Such pharmaceutically
acceptable preparations may also routinely contain compatible solid
or liquid fillers, diluents or encapsulating substances which are
suitable for administration into a human. The term "carrier"
denotes an organic or inorganic ingredient, natural or synthetic,
with which the active ingredient is combined to facilitate the
application. The components of the pharmaceutical compositions also
are capable of being co-mingled with the antibodies of the present
invention, and with each other, in a manner such that there is no
interaction which would substantially impair the desired
pharmaceutical efficacy.
[0344] The modulated lysine variant species compositions of the
invention are present in a form known in the art and acceptable for
therapeutic uses. In one embodiment, a formulation of the modulated
lysine variant species compositions of the invention is a liquid
formulation. In another embodiment, a formulation of the modulated
lysine variant species compositions of the invention is a
lyophilized formulation. In a further embodiment, a formulation of
the modulated lysine variant species compositions of the invention
is a reconstituted liquid formulation. In one embodiment, a
formulation of the modulated lysine variant species compositions of
the invention is a stable liquid formulation. In one embodiment, a
liquid formulation of the modulated lysine variant species
compositions of the invention is an aqueous formulation. In another
embodiment, the liquid formulation is non-aqueous. In a specific
embodiment, a liquid formulation of the modulated lysine variant
species compositions of the invention is an aqueous formulation
wherein the aqueous carrier is distilled water.
[0345] The formulations of the modulated lysine variant species
compositions of the invention comprise an antibody in a
concentration resulting in a w/v appropriate for a desired dose.
The antibody may be present in the formulation at a concentration
of about 1 mg/ml to about 500 mg/ml, e.g., at a concentration of at
least 1 mg/ml, at least 5 mg/ml, at least 10 mg/ml, at least 15
mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at
least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml, at least 50
mg/ml, at least 55 mg/ml, at least 60 mg/ml, at least 65 mg/ml, at
least 70 mg/ml, at least 75 mg/ml, at least 80 mg/ml, at least 85
mg/ml, at least 90 mg/ml, at least 95 mg/ml, at least 100 mg/ml, at
least 105 mg/ml, at least 110 mg/ml, at least 115 mg/ml, at least
120 mg/ml, at least 125 mg/ml, at least 130 mg/ml, at least 135
mg/ml, at least 140 mg/ml, at least 150 mg/ml, at least 200 mg/ml,
at least 250 mg/ml, or at least 300 mg/ml.
[0346] In a specific embodiment, a formulation of the modulated
lysine variant species compositions of the invention comprises at
least about 100 mg/ml, at least about 125 mg/ml, at least 130
mg/ml, or at least about 150 mg/ml of an antibody of the
invention.
[0347] In one embodiment, the concentration of antibody, which is
included in the formulation of the invention, is between about 1
mg/ml and about 25 mg/ml, between about 1 mg/ml and about 200
mg/ml, between about 25 mg/ml and about 200 mg/ml, between about 50
mg/ml and about 200 mg/ml, between about 75 mg/ml and about 200
mg/ml, between about 100 mg/ml and about 200 mg/ml, between about
125 mg/ml and about 200 mg/ml, between about 150 mg/ml and about
200 mg/ml, between about 25 mg/ml and about 150 mg/ml, between
about 50 mg/ml and about 150 mg/ml, between about 75 mg/ml and
about 150 mg/ml, between about 100 mg/ml and about 150 mg/ml,
between about 125 mg/ml and about 150 mg/ml, between about 25 mg/ml
and about 125 mg/ml, between about 50 mg/ml and about 125 mg/ml,
between about 75 mg/ml and about 125 mg/ml, between about 100 mg/ml
and about 125 mg/ml, between about 25 mg/ml and about 100 mg/ml,
between about 50 mg/ml and about 100 mg/ml, between about 75 mg/ml
and about 100 mg/ml, between about 25 mg/ml and about 75 mg/ml,
between about 50 mg/ml and about 75 mg/ml, or between about 25
mg/ml and about 50 mg/ml.
[0348] In a specific embodiment, a formulation of the modulated
lysine variant species compositions of the invention comprises
between about 90 mg/ml and about 110 mg/ml or between about 100
mg/ml and about 210 mg/ml of an antibody.
[0349] The formulations of the modulated lysine variant species
compositions of the invention comprising an antibody may further
comprise one or more active compounds as necessary for the
particular indication being treated, including those with
complementary activities that do not adversely affect each other.
Such additional active compound/s is/are suitably present in
combination in amounts that are effective for the purpose
intended.
[0350] The formulations of the modulated lysine variant species
compositions of the invention may be prepared for storage by mixing
the antibody having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers,
including, but not limited to buffering agents, saccharides, salts,
surfactants, solubilizers, polyols, diluents, binders, stabilizers,
salts, lipophilic solvents, amino acids, chelators, preservatives,
or the like (Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 12.sup.th edition, L. Brunton, et al. Remington's
Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)), in the
form of lyophilized formulations or aqueous solutions at a desired
final concentration. Acceptable carriers, excipients, or
stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as histidine,
phosphate, citrate, glycine, acetate and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including trehalose, glucose, mannose, or dextrins;
chelating agents such as EDTA; sugars such as sucrose, mannitol,
trehalose or sorbitol; salt-forming counter-ions such as sodium;
metal complexes (e.g., Zn-protein complexes); and/or non-ionic
surfactants such as TWEEN, polysorbate 80, PLURONICS.TM. or
polyethylene glycol (PEG).
[0351] The buffering agent may be histidine, citrate, phosphate,
glycine, or acetate. The saccharide excipient may be trehalose,
sucrose, mannitol, maltose or raffinose. The surfactant may be
polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic F68.
The salt may be NaCl, KCl, MgCl.sub.2, or CaCl.sub.2
[0352] The formulations of the modulated lysine variant species
compositions of the invention may include a buffering or pH
adjusting agent to provide improved pH control. A formulation of
the invention may have a pH of between about 3.0 and about 9.0,
between about 4.0 and about 8.0, between about 5.0 and about 8.0,
between about 5.0 and about 7.0, between about 5.0 and about 6.5,
between about 5.5 and about 8.0, between about 5.5 and about 7.0,
or between about 5.5 and about 6.5. In a further embodiment, a
formulation of the invention has a pH of about 3.0, about 3.5,
about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3,
about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9,
about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5,
about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5,
about 8.0, about 8.5, or about 9.0. In a specific embodiment, a
formulation of the invention has a pH of about 6.0. One of skill in
the art understands that the pH of a formulation generally should
not be equal to the isoelectric point of the particular antibody to
be used in the formulation.
[0353] Typically, the buffering agent is a salt prepared from an
organic or inorganic acid or base. Representative buffering agents
include, but are not limited to, organic acid salts such as salts
of citric acid, ascorbic acid, gluconic acid, carbonic acid,
tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,
tromethamine hydrochloride, or phosphate buffers. In addition,
amino acid components can also function in a buffering capacity.
Representative amino acid components which may be utilized in the
formulations of the invention as buffering agents include, but are
not limited to, glycine and histidine. In certain embodiments, the
buffering agent is chosen from histidine, citrate, phosphate,
glycine, and acetate. In a specific embodiment, the buffering agent
is histidine. In another specific embodiment, the buffering agent
is citrate. In yet another specific embodiment, the buffering agent
is glycine. The purity of the buffering agent should be at least
98%, or at least 99%, or at least 99.5%. As used herein, the term
"purity" in the context of histidine and glycine refers to chemical
purity of histidine or glycine as understood in the art, e.g., as
described in The Merck Index, 13.sup.th ed., O'Neil et al. ed.
(Merck & Co., 2001).
[0354] Buffering agents are typically used at concentrations
between about 1 mM and about 200 mM or any range or value therein,
depending on the desired ionic strength and the buffering capacity
required. The usual concentrations of conventional buffering agents
employed in parenteral formulations can be found in: Pharmaceutical
Dosage Form: Parenteral Medications, Volume 1, 2.sup.nd Edition,
Chapter 5, p. 194, De Luca and Boylan, "Formulation of Small Volume
Parenterals", Table 5: Commonly used additives in Parenteral
Products. In one embodiment, the buffering agent is at a
concentration of about 1 mM, or of about 5 mM, or of about 10 mM,
or of about 15 mM, or of about 20 mM, or of about 25 mM, or of
about 30 mM, or of about 35 mM, or of about 40 mM, or of about 45
mM, or of about 50 mM, or of about 60 mM, or of about 70 mM, or of
about 80 mM, or of about 90 mM, or of about 100 mM. In one
embodiment, the buffering agent is at a concentration of 1 mM, or
of 5 mM, or of 10 mM, or of 15 mM, or of 20 mM, or of 25 mM, or of
30 mM, or of 35 mM, or of 40 mM, or of 45 mM, or of 50 mM, or of 60
mM, or of 70 mM, or of 80 mM, or of 90 mM, or of 100 mM. In a
specific embodiment, the buffering agent is at a concentration of
between about 5 mM and about 50 mM. In another specific embodiment,
the buffering agent is at a concentration of between 5 mM and 20
mM.
[0355] In certain embodiments, the formulation of the modulated
lysine variant species compositions of the invention comprises
histidine as a buffering agent. In one embodiment the histidine is
present in the formulation of the invention at a concentration of
at least about 1 mM, at least about 5 mM, at least about 10 mM, at
least about 20 mM, at least about 30 mM, at least about 40 mM, at
least about 50 mM, at least about 75 mM, at least about 100 mM, at
least about 150 mM, or at least about 200 mM histidine. In another
embodiment, a formulation of the invention comprises between about
1 mM and about 200 mM, between about 1 mM and about 150 mM, between
about 1 mM and about 100 mM, between about 1 mM and about 75 mM,
between about 10 mM and about 200 mM, between about 10 mM and about
150 mM, between about 10 mM and about 100 mM, between about 10 mM
and about 75 mM, between about 10 mM and about 50 mM, between about
10 mM and about 40 mM, between about 10 mM and about 30 mM, between
about 20 mM and about 75 mM, between about 20 mM and about 50 mM,
between about 20 mM and about 40 mM, or between about 20 mM and
about 30 mM histidine. In a further embodiment, the formulation
comprises about 1 mM, about 5 mM, about 10 mM, about 20 mM, about
25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50
mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100
mM, about 150 mM, or about 200 mM histidine. In a specific
embodiment, a formulation may comprise about 10 mM, about 25 mM, or
no histidine.
[0356] The formulations of the modulated lysine variant species
compositions of the invention may comprise a carbohydrate
excipient. Carbohydrate excipients can act, e.g., as viscosity
enhancing agents, stabilizers, bulking agents, solubilizing agents,
and/or the like. Carbohydrate excipients are generally present at
between about 1% to about 99% by weight or volume, e.g., between
about 0.1% to about 20%, between about 0.1% to about 15%, between
about 0.1% to about 5%, between about 1% to about 20%, between
about 5% to about 15%, between about 8% to about 10%, between about
10% and about 15%, between about 15% and about 20%, between 0.1% to
20%, between 5% to 15%, between 8% to 10%, between 10% and 15%,
between 15% and 20%, between about 0.1% to about 5%, between about
5% to about 10%, or between about 15% to about 20%. In still other
specific embodiments, the carbohydrate excipient is present at 1%,
or at 1.5%, or at 2%, or at 2.5%, or at 3%, or at 4%, or at 5%, or
at 10%, or at 15%, or at 20%.
[0357] Carbohydrate excipients suitable for use in the formulations
of the invention include, but are not limited to, monosaccharides
such as fructose, maltose, galactose, glucose, D-mannose, sorbose,
and the like; disaccharides, such as lactose, sucrose, trehalose,
cellobiose, and the like; polysaccharides, such as raffinose,
melezitose, maltodextrins, dextrans, starches, and the like; and
alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol
sorbitol (glucitol) and the like. In one embodiment, the
carbohydrate excipients for use in the present invention are chosen
from, sucrose, trehalose, lactose, mannitol, and raffinose. In a
specific embodiment, the carbohydrate excipient is trehalose. In
another specific embodiment, the carbohydrate excipient is
mannitol. In yet another specific embodiment, the carbohydrate
excipient is sucrose. In still another specific embodiment, the
carbohydrate excipient is raffinose. The purity of the carbohydrate
excipient should be at least 98%, or at least 99%, or at least
99.5%.
[0358] In a specific embodiment, the formulations of the modulated
lysine variant species compositions of the invention may comprise
trehalose. In one embodiment, a formulation of the invention
comprises at least about 1%, at least about 2%, at least about 4%,
at least about 8%, at least about 20%, at least about 30%, or at
least about 40% trehalose. In another embodiment, a formulation of
the invention comprises between about 1% and about 40%, between
about 1% and about 30%, between about 1% and about 20%, between
about 2% and about 40%, between about 2% and about 30%, between
about 2% and about 20%, between about 4% and about 40%, between
about 4% and about 30%, or between about 4% and about 20%
trehalose. In a further embodiment, a formulation of the invention
comprises about 1%, about 2%, about 4%, about 6%, about 8%, about
15%, about 20%, about 30%, or about 40% trehalose. In a specific
embodiment, a formulation of the invention comprises about 4%,
about 6% or about 15% trehalose.
[0359] In certain embodiments, a formulation of the modulated
lysine variant species compositions of the invention comprises an
excipient. In a specific embodiment, a formulation of the invention
comprises at least one excipient chosen from: sugar, salt,
surfactant, amino acid, polyol, chelating agent, emulsifier and
preservative. In one embodiment, a formulation of the invention
comprises a salt, e.g., a salt selected from: NaCl, KCl,
CaCl.sub.2, and MgCl.sub.2. In a specific embodiment, the
formulation comprises NaCl.
[0360] A formulation of the modulated lysine variant species
compositions of the invention may comprise at least about 10 mM, at
least about 25 mM, at least about 50 mM, at least about 75 mM, at
least about 80 mM, at least about 100 mM, at least about 125 mM, at
least about 150 mM, at least about 175 mM, at least about 200 mM,
or at least about 300 mM sodium chloride (NaCl). In a further
embodiment, the formulation may comprise between about 10 mM and
about 300 mM, between about 10 mM and about 200 mM, between about
10 mM and about 175 mM, between about 10 mM and about 150 mM,
between about 25 mM and about 300 mM, between about 25 mM and about
200 mM, between about 25 mM and about 175 mM, between about 25 mM
and about 150 mM, between about 50 mM and about 300 mM, between
about 50 mM and about 200 mM, between about 50 mM and about 175 mM,
between about 50 mM and about 150 mM, between about 75 mM and about
300 mM, between about 75 mM and about 200 mM, between about 75 mM
and about 175 mM, between about 75 mM and about 150 mM, between
about 100 mM and about 300 mM, between about 100 mM and about 200
mM, between about 100 mM and about 175 mM, or between about 100 mM
and about 150 mM sodium chloride. In a further embodiment, the
formulation may comprise about 10 mM, about 25 mM, about 50 mM,
about 75 mM, about 80 mM, about 100 mM, about 125 mM, about 150 mM,
about 175 mM, about 200 mM, or about 300 mM sodium chloride.
[0361] A formulation of the modulated lysine variant species
compositions of the invention may also comprise an amino acid,
e.g., lysine, arginine, glycine, histidine or an amino acid salt.
The formulation may comprise at least about 1 mM, at least about 10
mM, at least about 25 mM, at least about 50 mM, at least about 100
mM, at least about 150 mM, at least about 200 mM, at least about
250 mM, at least about 300 mM, at least about 350 mM, or at least
about 400 mM of an amino acid. In another embodiment, the
formulation may comprise between about 1 mM and about 100 mM,
between about 10 mM and about 150 mM, between about 25 mM and about
250 mM, between about 25 mM and about 300 mM, between about 25 mM
and about 350 mM, between about 25 mM and about 400 mM, between
about 50 mM and about 250 mM, between about 50 mM and about 300 mM,
between about 50 mM and about 350 mM, between about 50 mM and about
400 mM, between about 100 mM and about 250 mM, between about 100 mM
and about 300 mM, between about 100 mM and about 400 mM, between
about 150 mM and about 250 mM, between about 150 mM and about 300
mM, or between about 150 mM and about 400 mM of an amino acid. In a
further embodiment, a formulation of the invention comprises about
1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about 150 mM, about
200 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM
of an amino acid.
[0362] The formulations of the modulated lysine variant species
compositions of the invention may further comprise a surfactant.
The term "surfactant" as used herein refers to organic substances
having amphipathic structures; namely, they are composed of groups
of opposing solubility tendencies, typically an oil-soluble
hydrocarbon chain and a water-soluble ionic group. Surfactants can
be classified, depending on the charge of the surface-active
moiety, into anionic, cationic, and nonionic surfactants.
Surfactants are often used as wetting, emulsifying, solubilizing,
and dispersing agents for various pharmaceutical compositions and
preparations of biological materials. Pharmaceutically acceptable
surfactants like polysorbates (e.g., polysorbates 20 or 80);
polyoxamers (e.g., poloxamer 188); Triton; sodium octyl glycoside;
lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-,
myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or
cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,
linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-betaine (e.g., lauroamidopropyl);
myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUA.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.,
PLURONICS.TM., PF68, etc.), can optionally be added to the
formulations of the invention to reduce aggregation. In one
embodiment, a formulation of the invention comprises Polysorbate
20, Polysorbate 40, Polysorbate 60, or Polysorbate 80. Surfactants
are particularly useful if a pump or plastic container is used to
administer the formulation. The presence of a pharmaceutically
acceptable surfactant mitigates the propensity for the protein to
aggregate. The formulations may comprise a polysorbate which is at
a concentration ranging from between about 0.001% to about 1%, or
about 0.001% to about 0.1%, or about 0.01% to about 0.1%. In other
specific embodiments, the formulations of the invention comprise a
polysorbate which is at a concentration of 0.001%, or 0.002%, or
0.003%, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or
0.009%, or 0.01%, or 0.015%, or 0.02%.
[0363] The formulations of the modulated lysine variant species
compositions of the invention may optionally further comprise other
common excipients and/or additives including, but not limited to,
diluents, binders, stabilizers, lipophilic solvents, preservatives,
adjuvants, or the like. Pharmaceutically acceptable excipients
and/or additives may be used in the formulations of the invention.
Commonly used excipients/additives, such as pharmaceutically
acceptable chelators (for example, but not limited to, EDTA, DTPA
or EGTA) can optionally be added to the formulations of the
invention to reduce aggregation. These additives are particularly
useful if a pump or plastic container is used to administer the
formulation.
[0364] Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,
chlorocresol, benzyl alcohol, phenylmercuric nitrite,
phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride
(for example, but not limited to, hexahydrate), alkylparaben
(methyl, ethyl, propyl, butyl and the like), benzalkonium chloride,
benzethonium chloride, sodium dehydroacetate and thimerosal, or
mixtures thereof can optionally be added to the formulations of the
invention at any suitable concentration such as between about
0.001% to about 5%, or any range or value therein. The
concentration of preservative used in the formulations of the
invention is a concentration sufficient to yield a microbial
effect. Such concentrations are dependent on the preservative
selected and are readily determined by the skilled artisan.
[0365] Other contemplated excipients/additives, which may be
utilized in the formulations of the invention include, for example,
flavoring agents, antimicrobial agents, sweeteners, antioxidants,
antistatic agents, lipids such as phospholipids or fatty acids,
steroids such as cholesterol, protein excipients such as serum
albumin (human serum albumin (HSA), recombinant human albumin
(rHA), gelatin, casein, salt-forming counterions such as sodium and
the like. These and additional known pharmaceutical excipients
and/or additives suitable for use in the formulations of the
invention are known in the art, e.g., as listed in "Remington: The
Science & Practice of Pharmacy", 21.sup.st ed., Lippincott
Williams & Wilkins, (2005), and in the "Physician's Desk
Reference", 60.sup.th ed., Medical Economics, Montvale, N.J.
(2005). Pharmaceutically acceptable carriers can be routinely
selected that are suitable for the mode of administration,
solubility and/or stability of an antibody, as well known those in
the art or as described herein.
[0366] In one embodiment, the modulated lysine variant species
compositions of the invention are formulated with the same or
similar excipients and buffers as are present in the commercial
adalimumab (HUMIRA.RTM.) formulation, as described in the
"Highlights of Prescribing Information" for HUMIRA.RTM.
(adalimumab) Injection (Revised January 2008), the contents of
which are hereby incorporated herein by reference. For example,
each prefilled syringe of HUMIRA.RTM., which is administered
subcutaneously, delivers 0.8 ml (40 mg) of drug product to the
subject. Each 0.8 ml of HUMIRA.RTM. contains 40 mg adalimumab, 4.93
mg sodium chloride, 0.69 mg monobasic sodium phosphate dihydrate,
1.22 mg dibasic sodium phosphate dihydrate, 0.24 mg sodium citrate,
1.04 mg citric acid monohydrate, 9.6 mg mannitol, 0.8 mg
polysorbate 80, and water for Injection, USP. Sodium hydroxide is
added as necessary to adjust pH.
[0367] It will be understood by one skilled in the art that the
formulations of the modulated lysine variant species compositions
of the invention may be isotonic with human blood, wherein the
formulations of the invention have essentially the same osmotic
pressure as human blood. Such isotonic formulations will generally
have an osmotic pressure from about 250 mOSm to about 350 mOSm.
Isotonicity can be measured by, for example, using a vapor pressure
or ice-freezing type osmometer. Tonicity of a formulation is
adjusted by the use of tonicity modifiers. "Tonicity modifiers" are
those pharmaceutically acceptable inert substances that can be
added to the formulation to provide an isotonity of the
formulation. Tonicity modifiers suitable for this invention
include, but are not limited to, saccharides, salts and amino
acids.
[0368] In certain embodiments, the formulations of the modulated
lysine variant species compositions of the invention have an
osmotic pressure from about 100 mOSm to about 1200 mOSm, or from
about 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about
800 mOSm, or from about 200 mOSm to about 600 mOSm, or from about
250 mOSm to about 500 mOSm, or from about 250 mOSm to about 400
mOSm, or from about 250 mOSm to about 350 mOSm.
[0369] The concentration of any one component or any combination of
various components, of the formulations of the modulated lysine
variant species compositions of the invention is adjusted to
achieve the desired tonicity of the final formulation. For example,
the ratio of the carbohydrate excipient to antibody may be adjusted
according to methods known in the art (e.g., U.S. Pat. No.
6,685,940). In certain embodiments, the molar ratio of the
carbohydrate excipient to antibody may be from about 100 moles to
about 1000 moles of carbohydrate excipient to about 1 mole of
antibody, or from about 200 moles to about 6000 moles of
carbohydrate excipient to about 1 mole of antibody, or from about
100 moles to about 510 moles of carbohydrate excipient to about 1
mole of antibody, or from about 100 moles to about 600 moles of
carbohydrate excipient to about 1 mole of antibody.
[0370] The desired isotonicity of the final formulation may also be
achieved by adjusting the salt concentration of the formulations.
Pharmaceutically acceptable salts and those suitable for this
invention as tonicity modifiers include, but are not limited to,
sodium chloride, sodium succinate, sodium sulfate, potassium
chloride, magnesium chloride, magnesium sulfate, and calcium
chloride. In specific embodiments, formulations of the invention
comprise NaCl, MgCl.sub.2, and/or CaCl.sub.2. In one embodiment,
concentration of NaCl is between about 75 mM and about 150 mM. In
another embodiment, concentration of MgCl.sub.2 is between about 1
mM and about 100 mM. Pharmaceutically acceptable amino acids
including those suitable for this invention as tonicity modifiers
include, but are not limited to, proline, alanine, L-arginine,
asparagine, L-aspartic acid, glycine, serine, lysine, and
histidine.
[0371] In one embodiment the formulations of the modulated lysine
variant species compositions of the invention are pyrogen-free
formulations which are substantially free of endotoxins and/or
related pyrogenic substances. Endotoxins include toxins that are
confined inside a microorganism and are released only when the
microorganisms are broken down or die. Pyrogenic substances also
include fever-inducing, thermostable substances (glycoproteins)
from the outer membrane of bacteria and other microorganisms. Both
of these substances can cause fever, hypotension and shock if
administered to humans. Due to the potential harmful effects, even
low amounts of endotoxins must be removed from intravenously
administered pharmaceutical drug solutions. The Food & Drug
Administration ("FDA") has set an upper limit of 5 endotoxin units
(EU) per dose per kilogram body weight in a single one hour period
for intravenous drug applications (The United States Pharmacopeial
Convention, Pharmacopeial Forum 26 (1):223 (2000)). When
therapeutic proteins are administered in amounts of several hundred
or thousand milligrams per kilogram body weight, as can be the case
with antibodies, even trace amounts of harmful and dangerous
endotoxin must be removed. In certain specific embodiments, the
endotoxin and pyrogen levels in the composition are less then 10
EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1
EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
[0372] When used for in vivo administration, the formulations of
the modulated lysine variant species compositions of the invention
should be sterile. The formulations of the invention may be
sterilized by various sterilization methods, including sterile
filtration, radiation, etc. In one embodiment, the antibody
formulation is filter-sterilized with a presterilized 0.22-micron
filter. Sterile compositions for injection can be formulated
according to conventional pharmaceutical practice as described in
"Remington: The Science & Practice of Pharmacy", 21.sup.st ed.,
Lippincott Williams & Wilkins, (2005). Formulations comprising
antibodies, such as those disclosed herein, ordinarily will be
stored in lyophilized form or in solution. It is contemplated that
sterile compositions comprising antibodies are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having an adapter that allows retrieval of the
formulation, such as a stopper pierceable by a hypodermic injection
needle. In one embodiment, a composition of the invention is
provided as a pre-filled syringe.
[0373] In one embodiment, a formulation of the modulated lysine
variant species compositions of the invention is a lyophilized
formulation. The term "lyophilized" or "freeze-dried" includes a
state of a substance that has been subjected to a drying procedure
such as lyophilization, where at least 50% of moisture has been
removed.
[0374] The phrase "bulking agent" includes a compound that is
pharmaceutically acceptable and that adds bulk to a lyo cake.
Bulking agents known to the art include, for example,
carbohydrates, including simple sugars such as dextrose, ribose,
fructose and the like, alcohol sugars such as mannitol, inositol
and sorbitol, disaccharides including trehalose, sucrose and
lactose, naturally occurring polymers such as starch, dextrans,
chitosan, hyaluronate, proteins (e.g., gelatin and serum albumin),
glycogen, and synthetic monomers and polymers.
[0375] A "lyoprotectant" is a molecule which, when combined with a
protein of interest (such as an antibody of the invention),
significantly prevents or reduces chemical and/or physical
instability of the protein upon lyophilization and subsequent
storage. Lyoprotectants include, but are not limited to, sugars and
their corresponding sugar alcohols; an amino acid such as
monosodium glutamate or histidine; a methylamine such as betaine; a
lyotropic salt such as magnesium sulfate; a polyol such as
trihydric or higher molecular weight sugar alcohols, e.g.,
glycerin, dextran, erythritol, glycerol, arabitol, xylitol,
sorbitol, and mannitol; propylene glycol; polyethylene glycol;
PLURONICS.TM.; and combinations thereof. Additional examples of
lyoprotectants include, but are not limited to, glycerin and
gelatin, and the sugars mellibiose, melezitose, raffinose,
mannotriose and stachyose. Examples of reducing sugars include, but
are not limited to, glucose, maltose, lactose, maltulose,
iso-maltulose and lactulose. Examples of non-reducing sugars
include, but are not limited to, non-reducing glycosides of
polyhydroxy compounds selected from sugar alcohols and other
straight chain polyalcohols. Examples of sugar alcohols include,
but are not limited to, monoglycosides, compounds obtained by
reduction of disaccharides such as lactose, maltose, lactulose and
maltulose. The glycosidic side group can be either glucosidic or
galactosidic. Additional examples of sugar alcohols include, but
are not limited to, glucitol, maltitol, lactitol and iso-maltulose.
In specific embodiments, trehalose or sucrose is used as a
lyoprotectant.
[0376] The lyoprotectant is added to the pre-lyophilized
formulation in a "lyoprotecting amount" which means that, following
lyophilization of the protein in the presence of the lyoprotecting
amount of the lyoprotectant, the protein essentially retains its
physical and chemical stability and integrity upon lyophilization
and storage.
[0377] In one embodiment, the molar ratio of a lyoprotectant (e.g.,
trehalose) and antibody molecules of a formulation of the invention
is at least about 10, at least about 50, at least about 100, at
least about 200, or at least about 300. In another embodiment, the
molar ratio of a lyoprotectant (e.g., trehalose) and antibody
molecules of a formulation of the invention is about 1, is about 2,
is about 5, is about 10, about 50, about 100, about 200, or about
300.
[0378] A "reconstituted" formulation is one which has been prepared
by dissolving a lyophilized antibody formulation in a diluent such
that the antibody is dispersed in the reconstituted formulation.
The reconstituted formulation is suitable for administration (e.g.,
parenteral administration) to a patient to be treated with the
antibody and, in certain embodiments of the invention, may be one
which is suitable for intravenous administration.
[0379] The "diluent" of interest herein is one which is
pharmaceutically acceptable (safe and non-toxic for administration
to a human) and is useful for the preparation of a liquid
formulation, such as a formulation reconstituted after
lyophilization. In some embodiments, diluents include, but are not
limited to, sterile water, bacteriostatic water for injection
(BWFI), a pH buffered solution (e.g., phosphate-buffered saline),
sterile saline solution, Ringer's solution or dextrose solution. In
an alternative embodiment, diluents can include aqueous solutions
of salts and/or buffers.
[0380] In certain embodiments, a formulation of the modulated
lysine variant species compositions of the invention is a
lyophilized formulation comprising an antibody of the invention,
wherein at least about 90%, at least about 95%, at least about 97%,
at least about 98%, or at least about 99% of said antibody may be
recovered from a vial upon shaking said vial for 4 hours at a speed
of 400 shakes per minute wherein the vial is filled to half of its
volume with the formulation. In another embodiment, a formulation
of the invention is a lyophilized formulation comprising an
antibody of the invention, wherein at least about 90%, at least
about 95%, at least about 97%, at least about 98%, or at least
about 99% of the antibody may be recovered from a vial upon
subjecting the formulation to three freeze/thaw cycles wherein the
vial is filled to half of its volume with said formulation. In a
further embodiment, a formulation of the invention is a lyophilized
formulation comprising an antibody of the invention, wherein at
least about 90%, at least about 95%, at least about 97%, at least
about 98%, or at least about 99% of the antibody may be recovered
by reconstituting a lyophilized cake generated from said
formulation.
[0381] In one embodiment, a reconstituted liquid formulation may
comprise an antibody at the same concentration as the
pre-lyophilized liquid formulation.
[0382] In another embodiment, a reconstituted liquid formulation
may comprise an antibody at a higher concentration than the
pre-lyophilized liquid formulation, e.g., about 2 fold, about 3
fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about
8 fold, about 9 fold, or about 10 fold higher concentration of an
antibody than the pre-lyophilized liquid formulation.
[0383] In yet another embodiment, a reconstituted liquid
formulation may comprise an antibody of the invention at a lower
concentration than the pre-lyophilized liquid formulation, e.g.,
about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6
fold, about 7 fold, about 8 fold, about 9 fold or about 10 fold
lower concentration of an antibody than the pre-lyophilized liquid
formulation.
[0384] In one embodiment, the pharmaceutical formulations of the
modulated lysine variant species compositions of the invention are
stable formulations, e.g., stable at room temperature.
[0385] The terms "stability" and "stable" as used herein in the
context of a formulation comprising an antibody of the invention
refer to the resistance of the antibody in the formulation to
aggregation, degradation or fragmentation under given manufacture,
preparation, transportation and storage conditions. The "stable"
formulations of the invention retain biological activity under
given manufacture, preparation, transportation and storage
conditions. The stability of the antibody can be assessed by
degrees of aggregation, degradation or fragmentation, as measured
by HPSEC, static light scattering (SLS), Fourier Transform Infrared
Spectroscopy (FTIR), circular dichroism (CD), urea unfolding
techniques, intrinsic tryptophan fluorescence, differential
scanning calorimetry, and/or ANS binding techniques, compared to a
reference formulation. For example, a reference formulation may be
a reference standard frozen at -70.degree. C. consisting of 10
mg/ml of an antibody of the invention in PBS.
[0386] Therapeutic formulations of the modulated lysine variant
species compositions of the invention may be formulated for a
particular dosage. Dosage regimens may be adjusted to provide the
optimum desired response (e.g., a therapeutic response). For
example, a single bolus may be administered, several divided doses
may be administered over time or the dose may 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. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subjects to be treated; each unit contains 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 antibody and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an antibody for the treatment of
sensitivity in individuals.
[0387] Therapeutic compositions of the modulated lysine variant
species compositions of the invention can be formulated for
particular routes of administration, such as oral, nasal,
pulmonary, topical (including buccal and sublingual), rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods known in the art of pharmacy. The amount of active
ingredient which can be combined with a carrier material to produce
a single dosage form will vary depending upon the subject being
treated, and the particular mode of administration. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
composition which produces a therapeutic effect. By way of example,
in certain embodiments, the antibodies (including antibody
fragments) are formulated for intravenous administration. In
certain other embodiments, the antibodies (including antibody
fragments) are formulated for local delivery to the cardiovascular
system, for example, via catheter, stent, wire, intramyocardial
delivery, intrapericardial delivery, or intraendocardial
delivery.
[0388] Formulations of the modulated lysine variant species
compositions of the invention which are suitable for topical or
transdermal administration include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required (U.S. Pat. Nos.
7,378,110; 7,258,873; 7,135,180; 7,923,029; and US Publication No.
20040042972).
[0389] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0390] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the modulated lysine variant species
compositions of the invention may be varied so as to obtain an
amount of the active ingredient which is effective to achieve the
desired therapeutic response for a particular patient, composition,
and mode of administration, without being toxic to the patient. The
selected dosage level will depend upon a variety of pharmacokinetic
factors including the activity of the particular compositions of
the present invention employed, or the ester, salt or amide
thereof, the route of administration, the time of administration,
the rate of excretion of the particular compound being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compositions
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0391] In certain embodiments, antibodies of the invention can be
formulated to ensure proper distribution in vivo. For example, the
blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention can cross the BBB (if desired), they can be formulated,
for example, in liposomes. For methods of manufacturing liposomes,
see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al.,
(1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G.
Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995)
Antimicrob. Agents Chemother. 39:180); surfactant protein A
receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),
different species of which may comprise the formulations of the
invention, as well as components of the invented molecules; p120
(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.
Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion;
I. J. Fidler (1994) Immunomethods 4:273. In one embodiment of the
invention, the therapeutic compounds of the invention are
formulated in liposomes; in another embodiment, the liposomes
include a targeting moiety. In another embodiment, the therapeutic
compounds in the liposomes are delivered by bolus injection to a
site proximal to the desired area. When administered in this
manner, the composition must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and may be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
Additionally or alternatively, the antibodies of the invention may
be delivered locally to the brain to mitigate the risk that the
blood brain barrier slows effective delivery.
[0392] In certain embodiments, the modulated lysine variant species
compositions of the invention may be administered with medical
devices known in the art. For example, in certain embodiments an
antibody or antibody fragment is administered locally via a
catheter, stent, wire, or the like. For example, in one embodiment,
a therapeutic composition of the invention can be administered with
a needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;
5,064,413; 4,941,880; 4,790,824; 4,596,556. Examples of well-known
implants and modules useful in the present invention include: U.S.
Pat. No. 4,487,603, which discloses an implantable micro-infusion
pump for dispensing medication at a controlled rate; U.S. Pat. No.
4,486,194, which discloses a therapeutic device for administering
medicants through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat.
No. 4,475,196, which discloses an osmotic drug delivery system.
Many other such implants, delivery systems, and modules are known
to those skilled in the art.
[0393] The efficient dosages and the dosage regimens for the
modulated lysine variant species compositions of the invention
depend on the disease or condition to be treated and can be
determined by the persons skilled in the art. One of ordinary skill
in the art would be able to determine such amounts based on such
factors as the subject's size, the severity of the subject's
symptoms, and the particular composition or route of administration
selected.
VII. ALTERNATIVE FORMULATIONS CONTAINING THE MODULATED LYSINE
VARIANT SPECIES COMPOSITIONS OF THE INVENTION
Alternative Aqueous Formulations
[0394] The invention also provides a modulated lysine variant
species composition formulated as an aqueous formulation comprising
a protein and water, as described in U.S. Pat. No. 8,420,081 and
WO2012/065072, the contents of which are hereby incorporated by
reference. In these aqueous formulations, the protein is stable
without the need for additional agents. This aqueous formulation
has a number of advantages over conventional formulations in the
art, including stability of the protein in water without the
requirement for additional excipients, increased concentrations of
protein without the need for additional excipients to maintain
solubility of the protein, and low osmolality. These also have
advantageous storage properties, as the proteins in the formulation
remain stable during storage, e.g., stored as a liquid form for
more than 3 months at 7.degree. C. or freeze/thaw conditions, even
at high protein concentrations and repeated freeze/thaw processing
steps. In one embodiment, formulations described herein include
high concentrations of proteins such that the aqueous formulation
does not show significant opalescence, aggregation, or
precipitation.
[0395] In one embodiment, an aqueous modulated lysine variant
species composition comprising a protein, e.g., an antibody, e.g.,
an anti-TNF.alpha. antibody or antigen biding portion thereof, and
water is provided, wherein the formulation has certain
characteristics, such as, but not limited to, low conductivity,
e.g., a conductivity of less than about 2.5 mS/cm, a protein
concentration of at least about 10 .mu.g/ml, an osmolality of no
more than about 30 mOsmol/kg, and/or the protein has a molecular
weight (Mw) greater than about 47 kDa. In one embodiment, the
formulation has improved stability, such as, but not limited to,
stability in a liquid form for an extended time (e.g., at least
about 3 months or at least about 12 months) or stability through at
least one freeze/thaw cycle (if not more freeze/thaw cycles). In
one embodiment, the formulation is stable for at least about 3
months in a form selected from the group consisting of frozen,
lyophilized, or spray-dried.
[0396] In one embodiment, the formulation has a low conductivity,
including, for example, a conductivity of less than about 2.5
mS/cm, a conductivity of less than about 2 mS/cm, a conductivity of
less than about 1.5 mS/cm, a conductivity of less than about 1
mS/cm, or a conductivity of less than about 0.5 mS/cm.
[0397] In another embodiment, modulated lysine variant species
compositions included in the formulation have a given
concentration, including, for example, a concentration of at least
about 1 mg/ml, at least about 10 mg/ml, at least about 50 mg/ml, at
least about 100 mg/ml, at least about 150 mg/ml, at least about 200
mg/ml, or greater than about 200 mg/ml. In another embodiment, the
formulation of the invention has an osmolality of no more than
about 15 mOsmol/kg.
[0398] The aqueous formulations described herein do not rely on
standard excipients, e.g., a tonicity modifier, a stabilizing
agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking
agent, a lyroprotectant, a basic component, and an acidic
component. In other embodiments of the invention, the formulation
contains water, one or more proteins, and no ionic excipients
(e.g., salts, free amino acids).
[0399] In certain embodiments, the aqueous formulation as described
herein comprise a modulated lysine variant species composition
comprising a protein concentration of at least 50 mg/ml and water,
wherein the formulation has an osmolality of no more than 30
mOsmol/kg. Lower limits of osmolality of the aqueous formulation
are also encompassed by the invention. In one embodiment the
osmolality of the aqueous formulation is no more than 15 mOsmol/kg.
The aqueous formulation of the invention may have an osmolality of
less than 30 mOsmol/kg, and also have a high protein concentration,
e.g., the concentration of the protein is at least 100 mg/ml, and
may be as much as 200 mg/ml or greater. Ranges intermediate to the
above recited concentrations and osmolality units are also intended
to be part of this invention. In addition, ranges of values using a
combination of any of the above recited values as upper and/or
lower limits are intended to be included.
[0400] The concentration of the aqueous formulation as described
herein is not limited by the protein size and the formulation may
include any size range of proteins. Included within the scope of
the invention is an aqueous formulation comprising at least 40
mg/ml and as much as 200 mg/ml or more of a protein, for example,
40 mg/ml, 65 mg/ml, 130 mg/ml, or 195 mg/ml, which may range in
size from 5 kDa to 150 kDa or more. In one embodiment, the protein
in the formulation of the invention is at least about 15 kD in
size, at least about 20 kD in size; at least about 47 kD in size;
at least about 60 kD in size; at least about 80 kD in size; at
least about 100 kD in size; at least about 120 kD in size; at least
about 140 kD in size; at least about 160 kD in size; or greater
than about 160 kD in size. Ranges intermediate to the above recited
sizes are also intended to be part of this invention. In addition,
ranges of values using a combination of any of the above recited
values as upper and/or lower limits are intended to be
included.
[0401] The aqueous formulation as described herein may be
characterized by the hydrodynamic diameter (D.sub.h) of the
proteins in solution. The hydrodynamic diameter of the protein in
solution may be measured using dynamic light scattering (DLS),
which is an established analytical method for determining the
D.sub.h of proteins. Typical values for monoclonal antibodies,
e.g., IgG, are about 10 nm. Low-ionic formulations may be
characterized in that the D.sub.h of the proteins are notably lower
than protein formulations comprising ionic excipients. It has been
discovered that the D.sub.h values of antibodies in aqueous
formulations made using the disfiltration/ultrafilteration (DF/UF)
process, as described in U.S. Pat. No. 8,420,081, using pure water
as an exchange medium, are notably lower than the D.sub.h of
antibodies in conventional formulations independent of protein
concentration. In one embodiment, antibodies in the aqueous
formulation as described herein have a D.sub.h of less than 4 nm,
or less than 3 nm.
[0402] In one embodiment, the D.sub.h of the protein in the aqueous
formulation is smaller relative to the D.sub.h of the same protein
in a buffered solution, irrespective of protein concentration.
Thus, in certain embodiments, protein in an aqueous formulation
made in accordance with the methods described herein, will have a
D.sub.h which is at least 25% less than the D.sub.h of the protein
in a buffered solution at the same given concentration. Examples of
buffered solutions include, but are not limited to phosphate
buffered saline (PBS). In certain embodiments, proteins in the
aqueous formulation of the invention have a D.sub.h that is at
least 50% less than the D.sub.h of the protein in PBS in at the
given concentration; at least 60% less than the D.sub.h of the
protein in PBS at the given concentration; at least 70% less than
the D.sub.h of the protein in PBS at the given concentration; or
more than 70% less than the D.sub.h of the protein in PBS at the
given concentration. Ranges intermediate to the above recited
percentages are also intended to be part of this invention, e.g.,
55%, 56%, 57%, 64%, 68%, and so forth. In addition, ranges of
values using a combination of any of the above recited values as
upper and/or lower limits are intended to be included, e.g., 50% to
80%.
[0403] In one aspect, the aqueous formulation includes the protein
at a dosage of about 0.01 mg/kg-10 mg/kg. In another aspect, the
dosages of the protein include approximately 1 mg/kg administered
every other week, or approximately 0.3 mg/kg administered weekly. A
skilled practitioner can ascertain the proper dosage and regime for
administering to a subject.
Alternative Solid Unit Formulations
[0404] The invention also provides a modulated lysine variant
species composition of the invention formulated as a stable solid
composition of a protein (preferably a therapeutic protein) and a
stabilizer, referred to herein as solid units, as described in
Attorney Docket No. 117813-31001, U.S. Provisional Patent
Application 61/893,123, the contents of which are hereby
incorporated by reference herein.
[0405] Specifically, it has been discovered that despite having a
high proportion of sugar relative to the protein, the solid units
of the invention maintain structural rigidity and resist changes in
shape and/or volume when stored under ambient conditions, room
temperature and humidity, for extended periods of time. The solid
units of the invention remain free-flowing and are able to maintain
long-term physical and chemical stability of the protein without
significant degradation and/or aggregate formation. The solid units
of the invention have many advantages over the art, including that
they can be formulated for oral delivery and are easily
reconstituted in a diluent, such as water. Because the solid units
are readily dissolved, they may be used in dual chamber delivery
devices and may be prepared directly in a device for patient
use.
[0406] As used herein, the term "solid unit," refers to a
composition which is suitable for pharmaceutical administration and
comprises a protein, an antibody or peptide, and a stabilizer,
e.g., a sugar. The solid unit has a structural rigidity and
resistance to changes in shape and/or volume. In a preferred
embodiment, the solid unit is obtained by lyophilizing a
pharmaceutical formulation of a therapeutic protein. The solid unit
may be any shape, e.g., geometric shape, including, but not limited
to, a sphere, a cube, a pyramid, a hemisphere, a cylinder, a
teardrop, and so forth, including irregularly shaped units. In one
embodiment, the solid unit has a volume ranging from about 1 ml to
about 20 ml. In one embodiment, the solid unit is not obtained
using spray drying techniques, e.g., the solid unit is not a powder
or granule.
[0407] As used herein, the phrase "a plurality of solid units"
refers to a collection or population of solid units, wherein the
collection comprises two or more solid units having a substantially
uniform shape, e.g., sphere, and/or volume distribution. In one
embodiment, the plurality of solid units is free-flowing.
VIII. KITS AND ARTICLES OF MANUFACTURE COMPRISING THE MODULATED
LYSINE VARIANT SPECIES COMPOSITIONS OF THE INVENTION
[0408] Also within the scope of the present invention are kits
comprising the modulated lysine variant species compositions of the
invention and instructions for use. The term "kit" as used herein
refers to a packaged product comprising components with which to
administer the antibody, or antigen-binding portion thereof, of the
invention for treatment of a disease or disorder. The kit can
comprise a box or container that holds the components of the kit.
The box or container is affixed with a label or a Food and Drug
Administration approved protocol. In one embodiment, the box or
container holds components of the invention which are contained
within plastic, polyethylene, polypropylene, ethylene, or propylene
vessels. The vessels can be capped-tubes or bottles. The kit can
also include instructions for administering an antibody of the
invention.
[0409] The kit can further contain one more additional reagents,
such as an immunosuppressive reagent, a cytotoxic agent or a
radiotoxic agent or one or more additional antibodies of the
invention (e.g., an antibody having a complementary activity which
binds to an epitope in the TNF.alpha. antigen distinct from a first
anti-TNF.alpha. antibody). Kits typically include a label
indicating the intended use of the contents of the kit. The term
label includes any writing, or recorded material supplied on or
with the kit, or which otherwise accompanies the kit.
[0410] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with a liquid formulation
or lyophilized formulation of an antibody or antibody fragment
thereof of the invention. In one embodiment, a container filled
with a liquid formulation of the invention is a pre-filled syringe.
In a specific embodiment, the formulations of the invention are
formulated in single dose vials as a sterile liquid. For example,
the formulations may be supplied in 3 cc USP Type I borosilicate
amber vials (West Pharmaceutical Services--Part No. 6800-0675) with
a target volume of 1.2 ml. Optionally associated with such
container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0411] In one embodiment, a container filled with a liquid
formulation of the invention is a pre-filled syringe. Any
pre-filled syringe known to one of skill in the art may be used in
combination with a liquid formulation of the invention. Pre-filled
syringes that may be used are described in, for example, but not
limited to, PCT Publications WO05032627, WO08094984, WO9945985,
WO03077976, U.S. Pat. No. 6,792,743, U.S. Pat. No. 5,607,400, U.S.
Pat. No. 5,893,842, U.S. Pat. No. 7,081,107, U.S. Pat. No.
7,041,087, U.S. Pat. No. 5,989,227, U.S. Pat. No. 6,807,797, U.S.
Pat. No. 6,142,976, U.S. Pat. No. 5,899,889, U.S. Pat. No.
7,699,811, U.S. Pat. No. 7,540,382, U.S. Pat. No. 7,998,120, U.S.
Pat. No. 7,645,267, and US Patent Publication No. US20050075611.
Pre-filled syringes may be made of various materials. In one
embodiment a pre-filled syringe is a glass syringe. In another
embodiment a pre-filled syringe is a plastic syringe. One of skill
in the art understands that the nature and/or quality of the
materials used for manufacturing the syringe may influence the
stability of a protein formulation stored in the syringe. For
example, it is understood that silicon based lubricants deposited
on the inside surface of the syringe chamber may affect particle
formation in the protein formulation. In one embodiment, a
pre-filled syringe comprises a silicone based lubricant. In one
embodiment, a pre-filled syringe comprises baked on silicone. In
another embodiment, a pre-filled syringe is free from silicone
based lubricants. One of skill in the art also understands that
small amounts of contaminating elements leaching into the
formulation from the syringe barrel, syringe tip cap, plunger or
stopper may also influence stability of the formulation. For
example, it is understood that tungsten introduced during the
manufacturing process may adversely affect formulation stability.
In one embodiment, a pre-filled syringe may comprise tungsten at a
level above 500 ppb. In another embodiment, a pre-filled syringe is
a low tungsten syringe. In another embodiment, a pre-filled syringe
may comprise tungsten at a level between about 500 ppb and about 10
ppb, between about 400 ppb and about 10 ppb, between about 300 ppb
and about 10 ppb, between about 200 ppb and about 10 ppb, between
about 100 ppb and about 10 ppb, between about 50 ppb and about 10
ppb, between about 25 ppb and about 10 ppb.
[0412] In certain embodiments, kits comprising antibodies of the
invention are also provided that are useful for various purposes,
e.g., research and diagnostic including for purification or
immunoprecipitation of protein of interest from cells, detection of
the protein of interest in vitro or in vivo. For isolation and
purification of a protein of interest, the kit may contain an
antibody coupled to beads (e.g., sepharose beads). Kits may be
provided which contain the antibodies for detection and
quantitation of a protein of interest in vitro, e.g., in an ELISA
or a Western blot. As with the article of manufacture, the kit
comprises a container and a label or package insert on or
associated with the container. The container holds a composition
comprising at least one antibody of the invention. Additional
containers may be included that contain, e.g., diluents and
buffers, control antibodies. The label or package insert may
provide a description of the composition as well as instructions
for the intended in vitro or diagnostic use.
[0413] The present invention also encompasses a finished packaged
and labeled pharmaceutical product. This article of manufacture
includes the appropriate unit dosage form in an appropriate vessel
or container such as a glass vial, pre-filled syringe or other
container that is hermetically sealed. In one embodiment, the unit
dosage form is provided as a sterile particulate free solution
comprising an antibody that is suitable for parenteral
administration. In another embodiment, the unit dosage form is
provided as a sterile lyophilized powder comprising an antibody
that is suitable for reconstitution.
[0414] In one embodiment, the unit dosage form is suitable for
intravenous, intramuscular, intranasal, oral, topical or
subcutaneous delivery. Thus, the invention encompasses sterile
solutions suitable for each delivery route. The invention further
encompasses sterile lyophilized powders that are suitable for
reconstitution.
[0415] As with any pharmaceutical product, the packaging material
and container are designed to protect the stability of the product
during storage and shipment. Further, the products of the invention
include instructions for use or other informational material that
advise the physician, technician or patient on how to appropriately
prevent or treat the disease or disorder in question, as well as
how and how frequently to administer the pharmaceutical. In other
words, the article of manufacture includes instruction means
indicating or suggesting a dosing regimen including, but not
limited to, actual doses, monitoring procedures, and other
monitoring information.
[0416] Specifically, the invention provides an article of
manufacture comprising packaging material, such as a box, bottle,
tube, vial, container, pre-filled syringe, sprayer, insufflator,
intravenous (i.v.) bag, envelope and the like; and at least one
unit dosage form of a pharmaceutical agent contained within said
packaging material, wherein said pharmaceutical agent comprises a
liquid formulation containing an antibody. The packaging material
includes instruction means which indicate how that said antibody
can be used to prevent, treat and/or manage one or more symptoms
associated with a disease or disorder.
[0417] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way.
IX. EXAMPLES
Example 1: Methods for Modulating the Lysine Variant Distribution
in Cell Culture by the Modulation of Zinc Concentration in Culture
Medium
[0418] This Example provides methods to modulate the lysine variant
distribution of monoclonal antibodies by modulating the levels of
zinc in the medium.
[0419] Materials and Methods
[0420] Cell Lines and Adaptation Cultures
[0421] Two adalimumab producing cell lines were employed in the
studies discussed herein (cell line 1 and cell line 2). Upon thaw,
cells were typically cultured in a combination of 250 ml and 500 ml
Corning vented non-baffled shake flasks on a shaker platform at 110
RPM for cell line 1 and 180 rpm for cell line 2 in a 35.degree. C.,
5% CO.sub.2 incubator. Subsequent to the initial cell growth in the
standard IVGN CD basal growth media, cells were adapted for two
passages in separate flasks in basal media containing different
concentrations of zinc. Only the cultures that demonstrated good
cell growth in the adaptation phase were carried forward to the
production stage.
Cell Culture Media
[0422] The initial growth media was prepared from proprietary basal
CD media GIA1 (Invitrogen, media 1). For the adaptation and
production culture stages (in different concentrations of zinc),
media was prepared starting from either proprietary basal CD media
GIA1 (media 1) or CD media without zinc (Basal 2). The control
cultures were carried through the adaptation and production stage
in Basal 1 media. The test conditions were carried through both the
adaptation and production stages in Basal 2 media supplemented with
different concentrations of zinc. The trace element compounds
supplemented to media are listed in Table 1. The detailed
descriptions of the culture media for the different conditions for
both cell lines are listed in Table 2. All media was filtered
through Corning 1 L filter systems (0.22 .mu.m PES) and stored at
4.degree. C. until usage.
TABLE-US-00001 TABLE 1 List of trace element compounds supplemented
to culture media Compound Catalog No./Source Zinc Chloride Fluka,
96468 Zinc Sulfate Heptahydrate Sigma, Z0251
TABLE-US-00002 TABLE 2 Detailed description of culture media for
different experimental conditions Estimated final Cell
concentration line Condition of zinc (.mu.M) 1 1 10 2 3.4 2 1 10 2
6.7 3 3.4
Production Cultures
[0423] Production cultures were initiated in duplicates in 500 ml
Corning vented non-baffled shake flasks (200 ml working volume).
The shake flasks were kept in incubators maintained at 35.degree.
C. and 5% CO.sub.2 on shaker platforms that were either set at 110
rpm for cell line 1 or 180 rpm for cell line 2. In all experiments,
the cells were transferred from the adaptation stage to the
production stage at a split ratio of 1:5.
[0424] The harvest procedure of the shake flasks and reactors
involved centrifugation of the culture sample at 3,000 RPM for 30
minutes and storage of supernatant in PETG bottles at -80.degree.
C. before submission for Protein A purification and WCX-10
analysis.
[0425] WCX-10 Assay
[0426] For quantification of charge variants of antibodies, cation
exchange chromatography was performed on a Dionex ProPac WCX-10,
Analytical column 4 mm.times.250 mm (Dionex, CA). A Shimadzu LC10A
HPLC system was used as the HPLC. The mobile phases used were 10 mM
sodium phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM sodium
phosphate dibasic, 500 mM sodium phosphate pH 5.5 (Mobile phase B).
A binary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min;
0% A, 100% B: 22-28 min; 94% A, 6% B: 28-34 min) was used with
detection at 280 nm. Quantitation was based on the relative area
percent of detected peaks (FIG. 1).
[0427] Results and Discussion
[0428] Effect of Varying Zinc Concentration in Chemically Defined
Media with Cell Line 1
[0429] In this Example, the effect of varying total zinc
concentration (control (10 .mu.M), 6.7 .mu.M, 3.4 .mu.M) in cell
culture media on culture performance and product quality was
evaluated using cell line 1. The ratios of the concentration of the
two zinc salts (zinc chloride and zinc sulfate) were kept constant
between the test conditions. As described in the materials and
methods, above, each of the production stage cultures were
initiated from respective adaptation cultures with corresponding
levels of total zinc.
[0430] A difference in cell growth and viability profiles was
observed between the test conditions and the control (FIG. 2, FIG.
3). While the peak viable cell density (VCD) in the control
condition was about 11.times.10.sup.6 cells/ml, the peak VCD for
the 3.4 .mu.M zinc condition was about 8.times.10.sup.6 cells/ml.
Corresponding to difference in peak VCD, the harvest titer was also
slightly reduced in the 3.4 .mu.M zinc condition (1.0 g/L) compared
to the control (1.3 g/L) (FIG. 4). The cultures were harvested on
day 10 at viability of 50% or lower for each condition and the
harvest was taken through Protein A purification before WCX-10
analysis. From the WCX-10 analysis, the lysine variant distribution
was characterized and the relative proportion of Lys 0, Lys 1, and
Lys 2 variants were quantified as a fraction of the total lysine
sum. A zinc dose dependent decrease in the relative fraction of
relative Lys 0 was observed from 92.8% in the control condition to
69.0% in the 3.4 .mu.M zinc condition. A corresponding relative
increase in both Lys 1/Lys 2 variants was also observed (FIG.
5).
[0431] Thus, lowering the zinc concentration provides an effective
method to increase the relative proportion of the product antibody
with a C-terminal lysine on one or both the heavy chains (Lys 1/Lys
2).
[0432] Effect of Varying Zinc Concentration in Chemically Defined
Media with Cell Line 2
[0433] In this Example, the effect of varying total zinc
concentration (control (10 .mu.M), 3.4 .mu.M) in basal cell culture
media on cell culture performance and product quality was evaluated
using cell line 2. The ratios of the concentration of the two zinc
salts (zinc chloride, zinc sulfate) were kept constant between the
test conditions. As described in the materials and methods, above,
each of the production stage cultures were initiated from
respective adaptation cultures with corresponding levels of total
zinc.
[0434] A significant difference in cell growth and viability
profile was observed between the two test conditions (FIG. 6, FIG.
7). While the peak viable cell density (VCD) in the control
condition was about 22.times.10.sup.6 cells/ml, the peak VCD for
the 3.4 .mu.M zinc condition was about 11.times.10.sup.6 cells/ml.
Corresponding to difference in peak VCD, the harvest titer was also
significantly reduced in the 3.4 .mu.M zinc condition compared to
the control (FIG. 8). The cultures were harvested on day 10 at the
target viability of 50% for each condition and the harvest was
taken through Protein A purification before WCX-10 analysis. From
the WCX-10 analysis, the lysine variant distribution was
characterized and the relative proportion of Lys 0, Lys 1, and Lys
2 variants were quantified as a fraction of the total lysine sum.
There was a decrease in the relative fraction of Lys 0 in the
control condition (92.7%) versus the relative Lys 0 in the 3.4
.mu.M zinc condition (67.9%). The relative fractions of Lys 1/Lys 2
variants were also correspondingly higher (FIG. 9).
[0435] Thus, an increase in the relative proportion of the product
antibody with C-terminal lysine on one or both the heavy chains
(Lys 1/Lys 2) corresponding to reduction in levels of zinc in basal
media, was also observed in this Example.
Example 2: Methods for Modulating the Lysine Variant Distribution
in Cell Culture by the Addition of Amino Acids
[0436] This Example provides methods to modulate the lysine variant
distribution of monoclonal antibodies by supplementing amino acids
(arginine, lysine, and histidine added individually and ornithine
in combination with arginine, lysine and/or histidine) to the cell
culture medium.
[0437] Materials and Methods
[0438] Cell Source and Adaptation Cultures
[0439] Three adalimumab producing cell lines (cell line 1, cell
line 2, and cell line 3), one mAb1 producing cell line and one mAb2
producing cell line were employed in the experiments described in
this Example.
[0440] For adalimumab producing cell lines, cells were cultured in
their respective growth media (chemically defined media (media 1)
or a hydrolysate based media (media 2 or media 3)) in a combination
of vented non-baffled shake flasks (Corning) on a shaker platform
at 110 RPM (cell line 1), 180 RPM (cell line 2), 140 RPM (cell line
3) and 10 L or 20 L wave bags (GE). For experiments with cells in
the hydrolysate based media (media 3), cells were thawed in media 1
and then adapted to media 3 over a few passages. Cultures were
propagated in a 35.degree. C., 5% CO.sub.2 incubator for cell line
1 and 2 and in a 36.degree. C., 5% CO.sub.2 incubator for cell line
3 in order to obtain the required number of cells to be able to
initiate production stage cultures.
[0441] For the mAb1 producing cell line, cells were cultured in
chemically defined growth media (media 1) in a combination of
vented non-baffled shake flasks (Corning) on a shaker platform at
130 RPM and 20 L wave bags (GE). Cultures were propagated in a
36.degree. C., 5% CO.sub.2 incubator to obtain the required number
of cells to be able to initiate production stage cultures.
[0442] For the mAb2 producing cell line, cells were cultured in
chemically defined growth media (media 1) in a combination of
vented non-baffled shake flasks (Corning) on a shaker platform at
140 RPM and 20 L wave bags (GE). Cultures were propagated in a
35.degree. C., 5% CO.sub.2 incubator to obtain the required number
of cells to be able to initiate production stage cultures.
[0443] Cell Culture Media
[0444] Growth and production media were prepared from either a
chemically defined media formulation (media 1) or hydrolysate-based
medium formulations (media 2 and media 3). For preparation of the
media 1, the media (IVGN GIA-1, proprietary formulation) was
supplemented with L-glutamine, sodium bicarbonate, sodium chloride,
and methotrexate solution. Production media consisted of all the
components in the growth medium, excluding methotrexate. For cell
line 1, mAb1, and mAb2, both growth and production medium were also
supplemented with insulin.
[0445] For the hydrolysate-based formulation (media 2), the growth
media was composed of PFCHO (proprietary CD formulation from SAFC),
dextrose, L-glutamine, L-asparagine, HEPES, Poloxamer 188, ferric
citrate, recombinant human insulin, Yeastolate (BD), Phytone
Peptone (BD), mono- and di-basic sodium phosphate, sodium
bicarbonate, sodium phosphate and methotrexate. Production media
consisted of all the components listed in the growth medium,
excluding methotrexate.
[0446] For the hydrolysate-based formulation (media 3), the growth
media was composed of OptiCHO (Invitrogen), L-glutamine, Yeastolate
(BD), Phytone Peptone (BD) and methotrexate. Production media
consisted of all the components listed in the growth medium,
excluding methotrexate.
[0447] Amino acids used for the experiments were reconstituted in
Milli-Q water to make a 100 g/L stock solution, which was
subsequently supplemented to both growth and production basal
media. After addition of amino acids, media was brought to a pH
similar to non-supplemented (control) media using 5N hydrochloric
acid/5N NaOH, and it was brought to an osmolality similar to
unsupplemented (control) media by adjusting the concentration of
sodium chloride. All media was filtered through Corning 1 L filter
systems (0.22 .mu.m PES) and stored at 4.degree. C. until used.
[0448] The amino acids supplemented to media are listed in Table
3.
TABLE-US-00003 TABLE 3 List of Amino Acids Supplemented to Culture
Media and the Relevant Concentration Ranges Tested Amino Acid
Catalog No./Source Arginine Sigma, A8094 Lysine Calbiochem, 4400
Histidine Sigma, H5659 Ornithine Sigma, 06503
[0449] Production Cultures
[0450] Production cultures were initiated either in 500 ml shake
flasks (Corning) or in 3 L Bioreactors (Applikon). For shake flask
experiments, duplicate 500 ml Corning vented non-baffled shake
flasks (200 ml working volume) were used for each condition. The
shake flasks were kept in incubators either maintained at
35.degree. C. or 36.degree. C. and 5% CO.sub.2 on shaker platforms
that were either set at 110 rpm for adalimumab producing cell line
1, 180 rpm for adalimumab producing cell line 2, 140 rpm for
adalimumab producing cell line 3, 130 rpm for mAb1 producing cell
line, or 140 rpm for mAb2 producing cell line. For the bioreactor
experiments, 3 L bioreactors (1.5 L working volume) were run at
37.degree. C.-33.degree. C. (temperature shift), 30% dissolved
oxygen (DO), 200 rpm, pH profile from 7.1 to 6.9 in three days and
pH 6.9 thereafter. In all experiments, the cells were transferred
from the seed train to the production stage at a split ratio of
about 1:5.
[0451] Cultures were run in either batch or fed-batch mode. In the
batch mode, cells were cultured in the respective production
medium. A 1.25% (v/v) of 40% glucose stock solution was fed when
the media glucose concentration reduced to less than 3 g/L. In the
fed-batch mode, cultures were run with either the IVGN feed as per
the following feed schedule--(4% (v/v)--day 3, 6%--day 5, 8%--day
7, 10%--day 9, 10%--day 11) or 10.times. Ex-Cell PFCHO feed (SAFC,
67411)--3% (v/v) on day 3. In fed-batch cultures with IVGN feed,
cultures were also fed with 1.25% (v/v) of 40% glucose stock
solution when the glucose concentration fell below 1.5 g/L on IVGN
feed days and when the concentration fell below 2.5 g/L on other
days. In fed-batch cultures with 10.times.PFCHO feed, 1.25% (v/v)
of 40% glucose stock solution was fed when the media glucose
concentration was below 3 g/L.
[0452] Retention samples for titer analysis of 2.times.1.5 ml, were
collected daily for the bioreactor experiments beginning on Day 8,
and frozen at -80.degree. C. The samples taken from each were later
subjected to titer analysis.
[0453] The harvest procedure of the shake flasks and reactors
involved centrifugation of the culture sample at 3,000 RPM for 30
min and storage of supernatant in PETG bottles at -80.degree. C.
before submission for Protein A purification and WCX-10
analysis.
[0454] WCX-10 Assay
[0455] The acidic species and other charge variants present in cell
culture harvest samples were quantified. Cation exchange
chromatography was performed on a Dionex ProPac WCX-10, Analytical
column (Dionex, CA). A Shimadzu LC10A HPLC system was used as the
HPLC.
[0456] For the adalimumab and mAb1 samples, the mobile phases used
were 10 mM sodium phosphate dibasic pH 7.5 (Mobile phase A) and 10
mM sodium phosphate dibasic, 500 mM sodium phosphate pH 5.5 (Mobile
phase B). A binary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B:
20-22 min; 0% A, 100% B: 22-28 min; 94% A, 6% B: 28-34 min) was
used with detection at 280 nm.
[0457] For mAb2 samples, the mobile phases used were 20 mM
(4-Morpholino)ethanesulfonic Acid Monohydrate (MES) pH 6.5 (Mobile
phase A) and 20 mM MES, 500 mM sodium phosphate pH 6.5 (Mobile
phase B). An optimized gradient (minute/% B): 0/3, 1/3, 46/21,
47/100, 52/100, 53/3, 58/3 was used with detection at 280 nm.
[0458] Quantitation is based on the relative area percent of
detected peaks. The peaks that elute at relative residence time
earlier than the main peak (Lys 0) corresponding to the drug
product are together represented as the acidic peaks. The peaks
that eluted at a relative residence time later than the main peak
in the basic region correspond to Lys 1 and Lys 2,
respectively.
[0459] Results and Discussion
[0460] Effect of Supplementation of Arginine to Culture Media
[0461] Two experiments where two different adalimumab producing
cell lines (cell line 1 and cell line 2) were cultured in a
chemically defined media (media 1) are described below.
[0462] Cell line 2 was cultured in media 1 with different total
concentrations of arginine (1 (control), 1.25, 1.5, 2, 3, 5, 9
g/L). The cultures were performed in shake flasks in batch format
with only glucose feed as described in the materials and methods.
The cells grew to maximum viable cell densities (VCD) in the range
of 18-22.times.10.sup.6 cells/ml for the different conditions
tested. The growth and viability profiles were comparable between
the different conditions, although a slight decrease in viable cell
density profile was observed in samples with the 9 g/L arginine
condition (FIG. 10, FIG. 11). The harvest titers were comparable
between the conditions (FIG. 12). On Day 10 and Day 12 of culture,
duplicate shake flasks for each of the conditions were harvested
and then subsequently analyzed using WCX-10 after Protein A
purification and the percentages of total peak(s) area
corresponding to Lys 0, Lys 1 and Lys 2 were quantified (FIG. 13,
FIG. 14). The percentage of Lys 0 relative to lysine sum (sum of
areas corresponding to peaks Lys 0, Lys 1 and Lys 2) in the control
sample was as 91.9% on day 10. In the sample with the highest
tested concentration of arginine in this experiment (9 g/L), the
relative percentage of Lys 0 was reduced to 77.2%. A dose dependent
decrease in relative Lys 0 was observed in conditions with arginine
concentrations beyond 2 g/L (FIG. 13). The decrease in relative Lys
0 corresponded with the increase in relative levels of both Lys 1
and Lys 2. A dose dependent decrease in relative levels of Lys 0,
and a corresponding dose dependent increase in Lys 1 and Lys 2 were
observed in conditions with increased arginine. A similar trend in
reduction of relative levels of Lys 0 with arginine increase was
also observed in the day 12 harvest samples (FIG. 14).
[0463] Cell line 3 was cultured in media 1 with different
concentrations of arginine (1 (control), 3, 5, 7, 9 g/L). The
cultures were performed in shake flasks in batch format with only
glucose feed as described in the materials and methods above. The
cells grew to maximum viable cell densities (VCD) in the range of
7-10.times.10.sup.6 cells/ml for the different conditions tested.
The growth and viability profiles were comparable between the
different test conditions, although a slight decrease in viable
cell density and viability profiles was observed in samples with
the 9 g/L arginine condition (FIG. 15, FIG. 16). The product titer
was also comparable between the conditions (FIG. 17). On Day 10 of
culture, duplicate shake flasks for each of the conditions were
harvested and then subsequently analyzed using WCX-10 post Protein
A purification and the percentages of total peak(s) area
corresponding to the Lys 0, Lys 1 and Lys 2 species were quantified
(FIG. 18). The percentage of Lys 0 relative to lysine sum in the
control sample was 82.9% on day 10. In the sample with the highest
concentration of arginine in this experiment (9 g/L), the
percentage of relative Lys 0 was reduced to 73.4%. The decrease in
Lys 0 corresponded with the increase in relative levels of both Lys
1 and Lys 2. This relative modulation of Lys 0, Lys 1 and Lys 2 was
directly related to the concentration of arginine in the media.
[0464] Thus, although the lysine variant distributions were
substantially different between the control conditions in the
studies presented above, significant modulation in the relative
levels of the lysine variants (decrease in Lys 0 and increase in
Lys 1/Lys 2) with increased arginine concentration was observed in
both cases.
[0465] Additional experiments were performed with multiple cell
lines in chemically defined or hydrolysate based media to
demonstrate the wide range of applicability of this method. The
experimental setup for each of these experiments was similar to
that described above. The summaries of results of the different
experiments performed for adalimumab are set forth in FIG. 19, FIG.
20 and FIG. 21. A reduction in relative Lys 0, and increase in
relative Lys 1 and Lys 2 species with increased arginine
concentration was observed in each case.
[0466] In addition to adalimumab, the utility of this method for
lysine variant modulation was also demonstrated for processes
involving two other mAbs. The experimental setup for each of these
experiments was similar to that described in the materials and
methods above. The reduction of acidic species with arginine
increase for experiments corresponding to each mAb is summarized in
FIG. 22 and FIG. 23. A reduction in relative Lys 0, and increase in
relative Lys 1 and Lys 2 species with increase in arginine
concentration was observed in both cases.
[0467] Effect of Supplementation of Lysine to Culture Media
[0468] The following is a description of two experiments where two
different adalimumab producing cell lines (cell line 1 and cell
line 2) were cultured in a chemically defined media (media 1).
[0469] Cell line 2 was cultured in media 1 with different
concentrations of lysine (1 (control), 5, 7, 9, 11 g/L). The
cultures were performed in shake flasks in batch format with only
glucose feed as described in the materials and methods above. The
cells grew to maximum viable cell densities (VCD) in the range of
17-23.times.10.sup.6 cells/ml for the different conditions tested.
A slight dose dependent decrease in viable cell density profile was
observed in all test conditions, with no significant effect on
viability profiles (FIG. 24 and FIG. 25). On Days 10 and 11 of
culture samples were collected for titer analysis. The harvest
titers for all conditions were comparable (FIG. 26). On Day 11 of
culture, duplicate shake flasks for each of the conditions were
harvested and then subsequently analyzed using WCX-10 post Protein
A purification and the percentages of total peak(s) area
corresponding to the Lys 0, Lys 1 and Lys 2 peaks were quantified
(FIG. 27). The percentage of Lys 0 relative to lysine sum (sum of
the peak areas corresponding to Lys 0, Lys 1 and Lys 2) in the
control sample was 92.5%. In the sample with the highest
concentration of lysine in this experiment (11 g/L), the percentage
of relative levels of Lys 0 was reduced to 67.7%. The decrease in
Lys 0 corresponded with the increase in relative levels of both Lys
1 and Lys 2. A dose dependent decrease in relative Lys 0, and a
dose dependent increase in relative Lys 1 and Lys 2 regions was
observed in test conditions with increased lysine
concentration.
[0470] Cell line 3 was cultured in media 1 with different
concentrations of lysine (1 (control), 3, 5, 7, 9, 11 g/L). The
cultures were performed in shake flasks in batch format with only
glucose feed as described in the materials and methods above. The
cells grew to maximum viable cell densities (VCD) in the range of
9.5-11.5.times.10.sup.6 cells/ml for the different conditions
tested. The growth and viability profiles were comparable between
the different conditions, although a slight decrease in viable cell
density and viability profiles was observed in samples with lysine
concentration greater than 1 g/L, (FIG. 28, FIG. 29). On Days 10,
11 and 12 of culture samples were collected for titer analysis. The
harvest titers for all conditions were comparable (FIG. 30). On Day
12 of culture, duplicate shake flasks for each of the conditions
were harvested and then subsequently analyzed using WCX-10 post
Protein A purification and the percentages of total peak(s) area
corresponding to the Lys 0, Lys 1 and Lys 2 peaks were quantified
(FIG. 31). The percentage of Lys 0 relative to lysine sum in the
control sample was 94.2%. In the sample with the highest
concentration of lysine in this experiment (11 g/L), the percentage
of relative level of Lys 0 was reduced to 76.0%. The decrease in
Lys 0 corresponded with the increase in relative levels of both Lys
1 and Lys 2. A dose dependent decrease in relative levels of Lys 0,
and a corresponding increase in relative levels of Lys 1 and Lys 2
was observed in test conditions with increased lysine
concentration.
[0471] Additional experiments were performed with multiple cell
lines in chemically defined or hydrolysate based media to
demonstrate the wide range of applicability of this method. The
experimental setup for each of these experiments was similar to
that described in the materials and methods above. The summaries of
results of the different experiments performed for adalimumab are
set forth in FIG. 32, FIG. 33 and FIG. 34. A reduction in relative
levels of Lys 0, and a corresponding increase in relative levels of
Lys 1 and Lys 2 with increased lysine was also observed in each
case.
[0472] In addition to adalimumab, the utility of this method for
acidic species reduction was also demonstrated for processes
involving two other mABs. The experimental setup for each of these
experiments was similar to that described in the materials and
methods above. The modulation of lysine variants with arginine
addition for experiments corresponding to each mAb is summarized in
FIG. 35 and FIG. 36. A reduction in relative levels of Lys 0, and
increase in relative levels of Lys 1 and Lys 2 species with
increased lysine was observed in each case.
[0473] Effect of Supplementation of Histidine to Culture Media
[0474] The following describes two experiments where two different
adalimumab producing cell lines (cell line 1 and cell line 2), were
cultured in a chemically defined media (media 1).
[0475] Cell line 2 was cultured in media 1 with different
concentrations of histidine (0 (control), 4, 6, 8, 10 g/L). The
cultures were performed in shake flasks in batch format with only
glucose feed as described in the materials and methods above. The
cells grew to maximum viable cell densities (VCD) in the range of
12-22.times.10.sup.6 cells/ml for the different conditions tested.
A dose dependent decrease in viable cell density profile was
observed in all conditions, with the 10 g/L histidine condition
having significant reduction in growth (FIG. 37). A corresponding
significant impact on the viability profile was also observed (FIG.
38). There was a small dose dependent decrease in titers for all
conditions with histidine supplementation (FIG. 39). On Day 11 for
control sample and Day 12 for the remaining conditions, duplicate
shake flasks were harvested and then subsequently analyzed using
WCX-10 post Protein A purification and the percentages of total
peak(s) area corresponding to the lysine species were quantified
(FIG. 40). The percentage of Lys 0 relative to lysine sum in the
control sample was 92.5%. In the sample with the highest
concentration of histidine in this experiment (10 g/L), the
percentage of relative Lys 0 was reduced to 80.6%. The decrease in
Lys 0 corresponded with the increase in relative levels of both Lys
1 and Lys 2.
[0476] Cell line 3 was cultured in media 1 with different
concentrations of histidine (0 (control), 2, 4, 6, 8 g/L). The
cultures were performed in shake flasks in batch format with only
glucose feed as described in the materials and methods above. The
cells grew to maximum viable cell densities (VCD) in the range of
6-10.times.10.sup.6 cells/ml for the different conditions tested. A
dose dependent decrease in viable cell density profile was observed
in all samples supplemented with histidine (FIG. 41). In comparison
to the impact on VCD profile, the viability profiles were more
comparable between the conditions (FIG. 42). The harvest titers for
all conditions were comparable (FIG. 43). On Day 12 of culture,
duplicate shake flasks for each of the conditions were harvested
and then subsequently analyzed using WCX-10 post Protein A
purification and the percentages of total peak(s) area
corresponding to Lys 0, Lys 1 and Lys 2 species were quantified
(FIG. 44). The percentage of Lys 0 relative to lysine sum in the
control sample was 94.2%. In the sample with the highest
concentration of histidine in this experiment (8 g/L), the
percentage of relative Lys 0 was reduced to 81.5%. The decrease in
Lys 0 corresponded with the increase in relative levels of both Lys
1 and Lys 2. A dose dependent decrease in relative Lys 0, and a
dose dependent increase in relative Lys 1 and Lys 2 regions was
observed in test conditions with increased histidine
concentration.
[0477] Additional experiments were performed with multiple cell
lines in chemically defined or hydrolysate based media to
demonstrate the wide range of applicability of this method. The
experimental setup for each of these experiments was similar to
that described above. The results of the different experiments
performed for adalimumab are summarized in FIG. 45, FIG. 46 and
FIG. 47. A reduction in relative Lys 0, and increase in relative
Lys 1 and Lys 2 species with increased histidine was observed in
each case.
[0478] In addition to adalimumab, the utility of this method for
modulation of lysine variants was evaluated for processes involving
two other mAbs. The experimental setup for each of these
experiments was similar to that described in the materials and
methods. The results for experiments corresponding to each mAb are
summarized in FIG. 48 and FIG. 49. For mAb1, a dose dependent
reduction in relative levels of Lys 0 was evident with increased
histidine. However, for mAb2, the relative change was minimal
within the histidine concentration range tested.
[0479] Effect of Amino Acid Modulation on Culture Media in 3 L
Bioreactors
[0480] In this study, 3 L bioreactors were set up to confirm the
effect of amino acid addition on lysine variant distribution in
more controlled conditions (pH and dissolved oxygen (DO)) at a
larger scale. Eight Bioreactors (1.5 L working volume) were set up
with cell line 2 in IVGN production media. The process included a
temperature shift from 37.degree. C. to 33.degree. C. when the cell
density criterion of 6.times.10.sup.6 cells/ml was met. The pH was
controlled via CO.sub.2 gas flow/0.5 N Sodium Hydroxide base at a
starting pH of 7.1 that was subsequently allowed to ramp down to
6.9 over the initial three days of the process. The DO was
controlled at 30% and the agitation rate was maintained at 200 rpm.
The cultures were fed with Ex-Cell PFCHO (SAFC, 67411) (3% (v/v))
on Day 3 of culture and with 18.8 g of 40% (w/v) glucose solution
on days when glucose in the reactor was measured to be below 3 g/L.
The test conditions included the amino acid concentration in media
to be as follows: control (1 g/L arginine and 1 g/L lysine), 3 g/L
arginine, 3 g/L arginine/2 g/L lysine and 3 g/L arginine/3 g/L
lysine. Reactors were run in duplicates for each condition.
[0481] The culture performance was comparable between the different
conditions with similar growth and viability profiles (FIG. 50,
51). The cultures were harvested on Day 11 with the harvest
viability between 40-50% in all the different conditions. Culture
harvests were processed through Protein A purification and WCX-10
analysis for quantification of the lysine variants. The lysine
variant distribution in the control samples were 86.7% (Lys 0),
11.1% (Lys 1) and 2.2% (Lys 2). The Lys 0 was reduced to 72.1% in
the condition with the highest concentration of amino acids (3 g/L
arginine/3 g/L lysine sample) (FIG. 53). The decrease in Lys 0
corresponded with the increase in relative levels of both Lys 1 and
Lys 2. Thus, increase of amino acids arginine and lysine can
modulate lysine distribution even in 3 L bioreactors under
controlled conditions of DO and pH.
[0482] Effect of Ornithine Modulation on Culture Media
[0483] In this Example, the effect of increased ornithine
concentration was tested both individually as well as in
combination with other amino acids arginine and lysine. The study
was performed with adalimumab producing cell line 2 in media 1. The
experiment was carried out in 500 ml shake flasks (200 ml working
volume) and was run on shaker platforms set at 180 rpm in
incubators set to be controlled at 35.0.degree. C. and 5% CO.sub.2.
The conditions tested included a control (only 1 g/L arginine and 1
g/L lysine), and test conditions including condition 2 (1 g/L
ornithine, 1 g/L arginine, 1 g/L lysine), test condition 3 (4 g/L
ornithine, 1 g/L arginine and 1 g/L lysine), test conditions 4 (1
g/L ornithine, 5 g/L arginine, 1 g/L lysine), test condition 5 (0
g/L ornithine, 5 g/L arginine, 2 g/L lysine), and test condition 6
(1 g/L ornithine, 5 g/L arginine, 2 g/L lysine). The cell culture
performed comparably between the control and the test conditions
with similar growth and viability profiles (FIG. 54, FIG. 55).
Samples were collected for all conditions on day 10 for titer,
which were comparable (FIG. 56). The cultures were harvested at day
10, processed through Protein A purification and WCX-10 analysis,
and the relative fractions of lysine variants were estimated. There
was no significant change in the lysine variant distribution in the
conditions where only the ornithine concentration was increased (at
1 g/L or 4 g/L) compared to the control. However, the combination
of ornithine increase with arginine or lysine increase reduced the
relative level of Lys 0 in comparison with the condition with just
arginine and lysine increase with a relative level of Lys 0 (FIG.
57). Thus, the increase of ornithine may exhibit synergistic
effects in modulating lysine variant distribution when added in
combination with arginine and lysine.
[0484] Effect of Increase in Concentration of a Combination of
Arginine, Lysine, Histidine, and Ornithine to Culture Media
[0485] In this experiment, the combined use of the four amino acids
arginine, lysine, histidine and ornithine for modulation of the
lysine variants is demonstrated. The experiment described here was
performed using adalimumab producing cell line 2 in chemically
defined media (media 1). The concentration range for arginine and
lysine in this experiment was 1-3 g/L while the concentration range
for histidine and ornithine in this experiment was between 0-2 g/L.
In comparison to the lower concentrations, or conditions where
amino acids were supplemented individually, a further reduction in
Lys 0 relative to lysine sum was observed in conditions where
combinations of amino acids were increased in the media (FIG. 58).
A progressive decrease was observed in relative Lys 0 when more
amino acids were increased in combination. The percentage of
relative Lys 0 was reduced from 94.9% in the control sample to
73.9% in the sample with all four amino acid concentrations
increased.
[0486] Effect of Increase in Concentration of a Combination of
Arginine, Lysine, Histidine, and Zinc to Culture Media
[0487] In this experiment, the combined use of zinc and the three
amino acids arginine, lysine, and histidine for lysine species
modulation is demonstrated. The experiment described here was
performed using adalimumab producing cell line 1 in chemically
defined media (media 1). The concentration range for arginine and
lysine in this experiment was between 1-3 g/L. The concentration
range for histidine was between 0-1 g/L. The concentration range
for zinc in this experiment was 30 .mu.M-60 .mu.M. Using the data
from the experiment, a model predicting the effects of addition of
these supplements to media for relative Lys 0 reduction (R.sup.2:
0.98, P=0.09) is described in FIG. 59. The model predicted a
contribution from each of the amino acids towards relative Lys 0
reduction. The model also predicted an increase in relative Lys 0
with an increase in zinc, which further supports the claim that
reduction of zinc in culture causes a reduction in relative Lys 0.
It may be also possible to utilize this model to predict the choice
of concentrations of these different components to the media, in
order to achieve a target reduction in relative Lys 0.
[0488] Effect of Supplementation of Single Peptides, Dipeptides and
Tripeptides to Culture Media
[0489] In this experiment, the use of the three amino acid monomers
arginine, lysine, and histidine, use of the dipeptides lys-lys and
arg-lys, and use of the tripeptides lys-lys-lys, his-arg-lys, and
arg-his-lys for lysine species modulation is demonstrated. The
experiment described here was performed using adalimumab producing
cell line 1 in chemically defined media (media 1). The
concentration range for each peptide set tested in this experiment
was between 0-4 g/L. In comparison to the control, a reduction in
Lys 0 relative to lysine sum was also observed in conditions where
dipeptides and tripeptides were supplemented to the media (FIG.
60). A decrease was observed in relative Lys 0 when polypeptides
were supplemented. The percentage of relative Lys 0 was reduced
from 88.0% in the control sample to 71.9% in a sample supplemented
with tripeptide arg-his-lys (4 g/L), and to 74.0% in a sample
supplemented with his-arg-lys (2 g/L).
[0490] The experiments outlined above demonstrate the different
methods that can be used either alone or in suitable combinations
to modulate the lysine variant distribution profile of a protein of
interest. Specifically, increasing the concentration in culture
media of the amino acids lysine, arginine, histidine, or
combinations thereof along with ornithine and limiting the
concentration of zinc in media, resulted in the relative modulation
of the lysine variants with a decrease in the relative levels of
Lys 0 and a corresponding increase in both Lys 1 and Lys 2.
Example 3: Methods for Modulating the Lysine Variant Distribution
in Cell Culture by Adjusting Process Parameters
[0491] Materials and Methods
[0492] Cell Source and Adaptation Cultures
[0493] Three adalimumab producing CHO cell lines (call line 1, cell
line 2, and cell line 3) were employed in the studies in this
Example.
[0494] For adalimumab producing cell lines, cells were cultured in
their respective growth media (chemically defined media (media 1)
or a hydrolysate based media (media 2 or media 3)) in a combination
of vented non-baffled shake flasks (Corning) on a shaker platform
at 110 RPM (cell line 1), 180 RPM (cell line 2), 140 RPM (cell line
3) and 10 L or 20 L wave bags (GE). For experiments with cells in
the hydrolysate based media (media 3), cells were thawed in media 1
and then adapted to media 3 over a few passages. Cultures were
propagated in a 35.degree. C., 5% CO.sub.2 incubator for cell line
1 and 2 and in a 36.degree. C., 5% CO.sub.2 incubator for cell line
3 in order to obtain the required number of cells to be able to
initiate production stage cultures.
[0495] In some cases, the culture might be transferred into a seed
reactor with pH 7.1, 35.degree. C. and 30% dissolved oxygen (DO).
In some cases, the culture was adapted to either media 1 or media 2
by propagated in a 10 L or 20 L wavebag for 7-13 days with one or
two passages before initiating production stage cultures.
[0496] Cell Culture Media
[0497] Media 1, the chemical defined growth or production media,
was prepared from basal IVGN CD media GIA1. For preparation of the
IVGN CD media formulation, the proprietary media was supplemented
with L-glutamine, sodium bicarbonate, sodium chloride, and
methotrexate solution. Production media consisted of all the
components in the growth medium, excluding methotrexate. For
cultures with adalimumab producing cell line 1, the medium was also
supplemented with insulin. In some cases, 10 mM or 5 mM of
Galactose (Sigma, G5388) and 0.4 .mu.M or 10 .mu.M of Manganese
(Sigma, M1787) were supplemented into production medium for
cultures with adalimumab producing cell line 3 and adalimumab
producing cell line 1, respectively. Osmolality was adjusted by the
addition of sodium chloride. All media was filtered through filter
systems (0.22 .mu.m PES) and stored at 4.degree. C. until
usage.
[0498] For the hydrolysate-based formulation (media 2), the growth
media was composed of PFCHO (proprietary CD formulation from SAFC),
dextrose, L-glutamine, L-asparagine, HEPES, Poloxamer 188, ferric
citrate, recombinant human insulin, yeastolate (BD), phytone
peptone (BD), mono- and di-basic sodium phosphate, sodium
bicarbonate, sodium chloride and methotrexate. Production media
consisted of all the components listed in the growth medium,
excluding methotrexate.
[0499] Production Cultures
[0500] Production cultures were initiated in 3 L Bioreactors
(Applikon). The bioreactors (1.5-2.0 L working volume) were run at
the following conditions (except for the different experimental
conditions): 35.degree. C., 30% DO (dissolved oxygen), 200 rpm, pH
profile from 7.1 to 6.9 in three days and pH 6.9 thereafter. In all
experiments, the cells were transferred from the wavebag to the
production stage at a split ratio of 1:5.6. When the media glucose
concentration reduced to less than 3 g/L, approximately 1.25% (v/v)
of 40% glucose stock solution was fed.
[0501] The harvest procedure of reactors involved centrifugation of
the culture sample at 3,000 RPM for 30 min and storage of
supernatant in PETG bottles at -80.degree. C. before submission for
Protein A purification and WCX-10 analysis.
[0502] WCX-10 Assay
[0503] The acidic species and other charge variants present in cell
culture harvest samples were quantified. Cation exchange
chromatography was performed on a Dionex ProPac WCX-10, Analytical
column (Dionex, CA).
[0504] For adalimumab samples, the mobile phases used were 10 mM
sodium phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM sodium
phosphate dibasic, 500 mM sodium chloride pH 5.5 (Mobile phase B).
A binary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min;
0% A, 100% B: 22-28 min; 94% A, 6% B: 28-34 min) was used with
detection at 280 nm.
[0505] Results and Discussion
[0506] Effect of Process pH in Media 1 with Cell Line 1
[0507] Five different pH conditions were assessed in this study:
7.1, 7.0, 6.9, 6.8 and 6.7. The cultures were started at pH set
point of 7.1; then were ramped down to the target pH set points
within 4 days. All cultures reached similar maximum viable cell
densities on day 8, except for the culture at pH 6.7 condition, for
which the maximum cell density was much lower than the other
cultures (FIG. 61). In addition, the viability of the culture at pH
7.1 and pH 7.0 dropped much earlier than the other cultures (FIG.
62). The viability of cultures at pH 7.1 and pH 7.0 were 38% and
54% on day 10, respectively; while the viability of the cultures at
lower pH (including pH 6.9, 6.8 and 6.7) was above 70% on the same
day. Samples were taken on the day of the cultures and measured for
titer. The titer of each tested condition increased corresponding
to the decrease in pH, from 1.2 g/L in the pH 7.1 condition to 1.8
g/L in the pH 6.8 condition; however, product titer was not
continued to increase at pH 6.7 (1.6 g/L) (FIG. 63). The cultures
were harvested at .ltoreq.50% viability. The harvest was purified
via Protein A chromatography, then analyzed using WCX-10. From the
WCX-10 analysis, the lysine variant distribution was characterized
and the relative proportion of Lys 0, Lys 1 and Lys 2 variants were
quantified as a fraction of the total lysine sum. The relative
fraction of Lys 0 increased with decrease in pH from 82.0% in the
pH 7.1 condition to 87.9% in the pH 6.7 condition, with
corresponding 5.0% decrease in relative levels of Lys 1 and 0.9%
decrease in Lys 2 (FIG. 64).
[0508] Effect of Process pH in Media 2 with Cell Line 1
[0509] Three different pH conditions were assessed in this study:
7.0, 6.9, and 6.8. The cultures were started at pH of 7.1; then
were ramped down to the target pH set points within 3 days of
culture. The viable cell density and viability were comparable
across the different pH set points until day 8. After day 8, the
viable cell density and viability were slightly higher
corresponding to lower pH set points (FIG. 65, FIG. 66). The
cultures were harvested at .about.50% viability. The product titer
was slightly higher at pH 6.8 comparing to pH 6.9 and 7.0 (FIG.
67). The resulting peak areas from WCX-10 analysis were quantified
(FIG. 68). The relative fraction of Lys 0 increased with decrease
in pH from 76.8% in the pH 7.0 condition to 80.5 in the pH 6.8
condition, with corresponding 2.8% decrease in relative levels of
Lys 1 and 0.9% decrease in Lys 2.
[0510] Effect of Process pH in Media 1 with Cell Line 3
[0511] Five different pH conditions were assessed in this study:
7.1 7.0, 6.9, 6.8, and 6.7. The cultures were started at pH set
point of 7.1; then were ramped down to the target pH set points
within 4 days of culture. The pH set points showed significant
effect on the cell growth and viability with this cell line and
media. Cell density was lower at higher pH and viability also
dropped earlier at higher pH (FIG. 69, FIG. 70). The cells were
harvested at an approximate viability of 50%. The titer was
slightly increased as the pH was reduced, reached the highest titer
at pH 6.8 condition (FIG. 71). The resulting peak areas from WCX-10
analysis were quantified (FIG. 72). The relative fraction of Lys 0
increased with decrease in pH from 88.0% in the pH 7.1 condition to
94.1% in the pH 6.7 condition, with a corresponding 4.8% decrease
in relative levels of Lys 1 and 1.3% decrease in Lys 2.
[0512] Effect of Process Dissolved Oxygen (DO) in Media 2 with Cell
Line 1
[0513] Three different dissolved oxygen (DO) concentrations were
assessed in this study: 20%, 30% and 50% at a culture temperature
of 35.degree. C. The cell density and viability were very
comparable at different DO conditions (FIGS. 73 and 74). The
cultures were harvested at the target viability of 50% for each
condition. The harvest titer was higher at 50% DO concentration
compared to 20% DO concentration (FIG. 75). The harvest was also
purified via Protein A chromatography before WCX-10 analysis. From
the WCX-10 analysis, the lysine variant distribution was
characterized and the relative proportion of Lys 0, Lys 1 and Lys 2
variants were quantified as a fraction of the total lysine sum
(FIG. 76). The relative fraction of Lys 0 decreased with an
increase in DO concentration from 83.6% in the 20% DO concentration
to 77.2% in the 50% DO concentration, with a corresponding 4.7%
increase in relative levels of Lys 1 and 1.8% increase in Lys
2.
[0514] In the next study, three different DO concentrations were
assessed: 20%, 30% and 60% at a culture temperature of 33.degree.
C. The cell density, viability and product titer were very
comparable at different DO conditions (FIGS. 77, 78 and 79). The
cultures were harvested at the target viability of 50% for each
condition. The harvest was also purified via Protein A
chromatography before WCX-10 analysis. From the WCX-10 analysis,
the lysine variant distribution was characterized and the relative
proportion of Lys 0, Lys 1 and Lys 2 variants were quantified as a
fraction of the total lysine sum (FIG. 80). The relative fraction
of Lys 0 decreased with an increase in DO concentration from 73.8%
in the 20% DO concentration to 66.3% in the 60% DO concentration,
with a corresponding 4.5% increase in relative levels of Lys 1 and
3.0% increase in Lys 2.
[0515] Effect of Process Dissolved Oxygen (DO) in Media 1 with Cell
Line 1
[0516] In this study, three different dissolved oxygen (DO)
concentrations were assessed: 20%, 30% and 50%. The cultures were
set at a temperature of 35.degree. C. The cell density and
viability were very comparable at different DO conditions (FIGS.
81, 82). The cultures were harvested at the target viability of 40%
for each condition. The harvest titer was higher at 30% and 50% DO
comparing to 20% DO (FIG. 83). The harvest was also purified via
Protein A chromatography before WCX-10 analysis. From the WCX-10
analysis, the lysine variant distribution was characterized and the
relative proportion of Lys 0, Lys 1 and Lys 2 variants were
quantified as a fraction of the total lysine sum (FIG. 84). The
relative fraction of Lys 0 decreased with an increase in DO
concentration from 84.8% in the 20% DO concentration to 81.0% in
the 50% DO concentration, with a corresponding 3.2% increase in
relative levels of Lys 1 and 0.7% increase in Lys 2.
[0517] Effect of Process Temperature in Media 1 with Cell Line
1
[0518] Three different temperature conditions were assessed:
33.degree. C., 35.degree. C. and 37.degree. C. The cultures were
harvested at the target viability of 50% for each condition. At a
lower temperature, the culture duration was longer with higher
viability through the culture (FIG. 85, FIG. 86). Samples were
collected for titer analysis on harvest days. The titer for all
conditions was comparable (FIG. 87). The harvest was purified via
Protein A chromatography before WCX-10 analysis. From the WCX-10
analysis, the lysine variant distribution was characterized and the
relative proportion of Lys 0, Lys 1 and Lys 2 variants were
quantified as a fraction of the total lysine sum (FIG. 88). The
relative fraction of Lys 0 increased with increase in temperature
from 80.3% in the 33.degree. C. concentration to 86.6% in the
37.degree. C. concentration, with corresponding 5.3% decrease in
relative levels of Lys 1 and 1.0% decrease in Lys 2. Thus, lowering
the process temperature provides an effective method to increase
the relative proportion of the product antibody with C-terminal
lysine on one or both the heavy chains.
[0519] Effect of Process Temperature in Media 1 with Cell Line
2
[0520] Three different temperature conditions were assessed:
33.degree. C., 35.degree. C., and 37.degree. C. The cultures were
harvested at the target viability of 50% for each condition. The
viability cell density (VCD) and viability profiles were similar
for the 37.degree. C. and 35.degree. C. conditions, but the
33.degree. C. condition took longer to drop to 50% (FIG. 89, FIG.
90). The product titers were lower at 31.degree. C. compared to the
titers at 33.degree. C. and 35.degree. C. (FIG. 91). The harvest
was purified via Protein A chromatography before WCX-10 analysis.
From the WCX-10 analysis, the lysine variant distribution was
characterized and the relative proportion of Lys 0, Lys 1 and Lys 2
variants were quantified as a fraction of the total lysine sum
(FIG. 92). The relative fraction of Lys 0 increased with increase
in temperature from 88.6% in the 33.degree. C. condition to 93.1%
in the 37.degree. C. condition, with corresponding 3.8% decrease in
relative levels of Lys 1 and 0.7% decrease in Lys 2. Thus, the
results here are consistent with that observed for cell line 1.
[0521] Effect of Process Dissolved Oxygen and Temperature in Media
1 with Cell Line 3
[0522] The study was performed at four different temperature levels
(33.degree. C., 34.degree. C., 35.degree. C. and 36.degree. C.)
with two different DO concentrations (20% DO and 50% DO). In
general, the cell growth at different dissolved oxygen
concentrations was similar except at 35.degree. C., in which the
cell density was lower at 50% DO concentration (FIG. 93). In
addition, the cultures reached higher maximum cell density at
higher temperatures (at 36.degree. C. and 35.degree. C.), while
viability dropped earlier and faster (FIG. 94). The cultures were
harvested at approximately 50% viability. The product titers were
comparable at all conditions (FIG. 95). The harvest was purified
via Protein A chromatography before WCX-10 analysis. From the
WCX-10 analysis, the lysine variant distribution was characterized
and the relative proportion of Lys 0, Lys 1 and Lys 2 variants were
quantified as a fraction of the total lysine sum (FIG. 96).
[0523] The relative distribution of lysine variants was influenced
by the choice of % DO concentration and temperature. The effect of
lowering temperature was more significant than increasing % DO
concentration in this case. At 20% DO, the relative fraction of Lys
0 increased with increase in temperature from 80.5% in the
33.degree. C. condition to 91.4% in the 36.degree. C. condition,
with corresponding 8.5% decrease in relative levels of Lys 1 and
2.4% decrease in Lys 2. At 50% DO, the relative fraction of Lys 0
increased with increase in temperature from 80.1% in the 33.degree.
C. condition to 90.8% in the 36.degree. C. condition, with
corresponding 8.2% decrease in relative levels of Lys 1 and 2.5%
decrease in Lys 2. On the other hand, at different temperatures,
the relative fraction of Lys 0 decreased with an increase in DO
concentration, with the maximum difference observed at 35.degree.
C. from 90.1% in the 20% DO concentration to 88.7% in the 50% DO
concentration, with a corresponding 1.2% increase in relative
levels of Lys 1 and 0.2% increase in Lys 2. Therefore the results
here are consistent with that observed with other cell lines.
[0524] The experiments described above demonstrate the different
methods that can be used either alone or in suitable combination to
control the lysine variant distribution profile of a protein of
interest, e.g., the antibody adalimumab. These experiments also
indicate that altering cell culture process parameters on-line can
be used to modulate the lysine variant distribution. Increasing
dissolved oxygen concentration, increasing pH set points or
reducing temperature set points results in a relative shift in
lysine variant distribution from Lys 0 to Lys 1 Lys 2.
Example 4: Effect of Phosphate Salt Concentration on the Lysine
Variant Distribution of Product Antibody
[0525] In this Example, the effect of varying the concentration of
phosphate salt individually and in combination with other amino
acids (lysine or arginine) and trace elements (zinc ion) in
chemically defined media on the Lys variant distribution of the
antibody adalimumab was studied. Subsections 4.1 and 4.2, below,
show that increasing the concentration of phosphate in media leads
to a relative shift in lysine variant distribution from Lys 0 to
Lys 1/Lys 2 in two adalimumab producing CHO cell lines (cell line 1
and cell line 2). Subsection 4.3 demonstrates that increasing the
concentration of phosphate salt in combination with the addition of
lysine or arginine provides modulation of lysine variants in an
additive manner in cell line 3.
Materials and Methods
Cell Lines and Adaptation Cultures
[0526] Two adalimumab producing CHO cell lines were employed in the
studies covered here (cell line 1 and cell line 2).
Cell Culture Media
[0527] The initial growth media used in the seed train phase was
prepared from proprietary growth CD media GIA1 (Invitrogen). In the
experiments testing phosphate salt individually, the production
media was prepared staring from proprietary basal CD media GIA1
(Basal) and supplemented with various level of
NaH.sub.2PO.sub.4--H.sub.2O. A description of the culture
conditions for both cell lines is listed in Table 4, below. In the
combination experiment, the production media were prepared staring
from proprietary basal CD media GIA1 (Basal) and supplemented with
various level of NaH.sub.2PO.sub.4--H.sub.2O, L-lysine-HCl,
L-arginine and zinc salt (containing ZnSO.sub.4 and ZnCl.sub.2)
based on central composite design with two center points. A
description of the culture conditions for cell line 1 is listed in
Table 5. The L-lysine, L-arginine, phosphate salt and zinc ion
concentration in the CD media GIA1 were approximately 6.0 mM, 4.0
mM, 5.8 mM and 10 .mu.M, respectively.
TABLE-US-00004 TABLE 4 Cell culture media and cell line conditions
used for Examples 4.1 and 4.2 Production media
NaH.sub.2PO.sub.4.cndot.H.sub.2O Experiment Cell line Condition
Basal media Supplementation Example 4.1 Cell line Control CD media
None 1 batch 6 mM GIA1 6 mM 12 mM 12 mM 18 mM 18 mM 24 mM 24 mM
Example 4.2 Cell line Control CD media None 2 batch 6 mM GIA1 6 mM
12 mM 12 mM 18 mM 18 mM 24 mM 24 mM
TABLE-US-00005 TABLE 5 Cell culture media conditions used for
Example 4.3 conducted on cell line 1. Production media
Supplementation Lysine.cndot.HCl Arginine
NaH.sub.2PO.sub.4.cndot.H.sub.2O Zn * Experiment Condition Basal
Media (mM) (mM) (mM) (.mu.M) Example 4.3 1 CD 5.5 5.7 6.0 30 2
media 10.9 0.0 0.0 0 3 GIA1 5.5 11.5 6.0 15 4 5.5 5.7 6.0 15 5 5.5
0.0 6.0 15 6 5.5 5.7 12.0 15 7 5.5 5.7 6.0 15 8 10.9 11.5 12.0 0 9
0.0 0.0 0.0 0 10 10.9 5.7 6.0 15 11 0.0 0.0 0.0 30 12 10.9 0.0 12.0
30 13 10.9 11.5 12.0 30 14 0.0 11.5 12.0 0 15 5.5 5.7 0.0 15 16
10.9 11.5 0.0 30 17 0.0 11.5 0.0 30 18 10.9 11.5 0.0 0 19 0.0 5.7
6.0 15 20 5.5 5.7 6.0 0 21 10.9 0.0 0.0 30 22 0.0 11.5 0.0 0 23
10.9 0.0 12.0 0 23 0.0 0.0 12.0 0 25 0.0 0.0 12.0 30 26 0.0 11.5
12.0 30
[0528] * Zn was supplemented using combination of ZnCl.sub.2 and
ZnSO.sub.4 with fixed molar ratio of 0.56:1.
Cell Culture Conditions
[0529] Upon thaw, cells were typically cultured in a combination of
250 ml and 500 ml Corning vented non-baffled shake flasks on a
shaker platform at 110 RPM for cell line 1 and 180 rpm for cell
line 2 in a 35.degree. C., 5% CO.sub.2 incubator. Growth media was
used to carry both cell lines in seed train stages.
[0530] In the experiments that tested phosphate individually
(Example 4.1 and Example 4.2, below), production cultures were
initiated in duplicates according to Table 4. In Example 4.2, which
tested phosphate in combination with lysine, arginine and zinc ion,
a production culture was started according to Table 5 based on
centre composite design, which is an experimental design that is
useful in response surface methodology, for building a second order
(quadratic) model for the response variable without needing to use
a complete three-level factorial experiment. All the cultures were
kept in 500 ml Corning vented non-baffled shake flasks (200 ml
working volume) with temperature and CO.sub.2 maintained at
35.degree. C. and 5%. The shaker platforms were set at 110 rpm for
cell line 1 and 180 rpm for cell line 2. In all experiment
conditions, the cells were transferred from the seed train stage to
the production stage at a split ratio of 1:5. 40% glucose solution
was feed as necessary to maintain the culture at adequate levels.
The cultures were harvested on day 11 (generally maintaining
harvest viability at approximately 50%).
[0531] The harvest procedure of the shake flasks involved
centrifugation of the culture sample at 3,000 RPM for 30 min and
storage of supernatant in PETG bottles at -80.degree. C. before
submission for Protein A purification and WCX-10 analysis.
[0532] WCX-10 Assay
[0533] For quantification of charge variants of antibodies, cation
exchange chromatography was performed on a Dionex ProPac WCX-10,
Analytical column 4 mm.times.250 mm (Dionex, CA). A Shimadzu LC10A
HPLC system was used as the HPLC. The mobile phases used were 10 mM
sodium phosphate dibasic pH 7.5 (Mobile phase A) and 10 mM sodium
phosphate dibasic, 500 mM sodium phosphate pH 5.5 (Mobile phase B).
A binary gradient (94% A, 6% B: 0-20 min; 84% A, 16% B: 20-22 min;
0% A, 100% B: 22-28 min; 94% A, 6% B: 28-34 min) was used with
detection at 280 nm.
[0534] Quantitation was based on the relative area percent of
detected peaks.
Example 4.1: Effect of Phosphate Supplementation in Chemically
Defined Media with Cell Line 1
[0535] In this Example, the effect of varying phosphate
concentration in production cell culture media on cell culture
performance and product quality was evaluated using cell line 1.
Slightly increased peak viable cell density (VCD) was observed at 6
mM phosphate supplementation level compared with control (FIG.
97A). Nevertheless, slightly to moderate growth inhibition was
observed as the phosphate supplementation amount was increased from
12 mM to 24 mM (FIG. 97A). All the cultures were harvested on day
11, and slightly higher viabilities were observed in phosphate
supplemented conditions compared with control (FIG. 97B).
Corresponding to the growth inhibition, the harvest titer was also
reduced in the phosphate supplemented conditions (1.1 to 1.3 g/L)
compared with control (1.4 g/L) (FIG. 97C). All the harvested
cultures were purified via Protein A chromatography before WCX-10
analysis. From the WCX-10 analysis, the lysine variant distribution
was characterized and the relative proportion of Lys 0, Lys 1 and
Lys 2 variants were quantified as a fraction of the total lysine
sum. Dose dependent modification of lysine variant distribution was
shown with the increase of the phosphate concentration. As the
phosphate concentration in the production media increased from 5.8
mM (control condition) to 29.8 mM (24 mM phosphate supplementation
condition), the relative fraction of Lys 0 was reduced from 91.3 to
85.7%, Lys 1 was increased from 7.0 to 11.5% and Lys 2 was
increased from 1.7 to 2.8% (FIG. 97D).
[0536] Thus, the higher phosphate concentrations provide an
effective method to increase the relative proportion of the product
antibody with C-terminal lysine on both the heavy chains (Lys 1/Lys
2).
Example 4.2: Effect of Varying Phosphate Concentration in
Chemically Defined Media with Cell Line 2
[0537] In this Example, the effect of varying phosphate
concentration in production cell culture media on cell culture
performance and product quality was evaluated using cell line 2.
Compared with cell line 1, the reduction of peak viable cell
density (VCD) and the increase of harvest viability effects
associated with high concentration level phosphate supplementation
(18 mM and 24 mM) were also observed on cell line 2 (FIGS. 98A and
98B). The harvest titer reduction resulting from phosphate
supplementation also remained significant. The harvest titers were
in the range of 1.5-2.0 g/L with 6 mM to 24 mM phosphate
supplementation, compared with 2.1 g/L titer for the control
condition (FIG. 98C). All the cultures were harvested on day 11 and
purified via Protein A chromatography before WCX-10 analysis. From
the WCX-10 analysis, the lysine variant distribution was
characterized and the relative proportion of Lys 0, Lys 1 and Lys 2
variants were quantified as a fraction of the total lysine sum.
Dose dependent modification of lysine variant distribution was also
shown with the increase of the phosphate concentration in cell line
2. As the phosphate concentration in the production media increased
from 5.8 mM (control condition) to 29.8 mM (24 mM phosphate
supplementation condition), the relative fraction of Lys 0 was
reduced from 91.7 to 84.8%, Lys 1 was increased from 6.6 to 12.3%
and Lys 2 was increased from 1.7 to 2.9% (FIG. 98D).
[0538] Therefore, the increase in the relative proportion of the
product antibody with C-terminal lysine on one or both the heavy
chains (Lys 1/Lys 2) corresponding to an increase in levels of
phosphate in basal media, was also observed in this Example.
Example 4.3: Effect of Varying Phosphate, Lysine, Arginine and Zinc
Concentrations in Chemically Defined Media with Cell Line 1
[0539] In this Example, the effect of varying phosphate was
evaluated in combination with altering lysine, arginine and zinc
concentrations in production cell culture media on cell culture
performance and product quality. The study was conducted using cell
line 1 based on centre composite design. Data were analyzed using
JMP software (a computer program for statistics developed by the
JMP business unit of SAS Institute). The effects of varying lysine,
arginine, zinc and phosphate concentration on peak viable cell
density (VCD), harvest viability, harvest titer were evaluated and
ARE shown in FIGS. 99A-99C. Within the studied range, no
significant peak VCD, harvest viability and harvest tier impact was
observed as the concentration of lysine or arginine was varied.
However, increasing the zinc concentration corresponded to the
significantly increased peak VCD and harvest titer; while
increasing the phosphate concentration resulted in significantly
reduced harvest viability and titer.
[0540] All the cultures were harvested on day 11 and purified via
Protein A chromatography before WCX-10 analysis. From the WCX-10
analysis, the lysine variant distribution was characterized and the
relative proportion of Lys 0, Lys 1 and Lys 2 variants were
quantified as a fraction of the total lysine sum. The effects of
varying lysine, arginine, zinc and phosphate concentration on
lysine variant distribution are shown in FIGS. 99D-99F.
Supplementation of lysine, arginine, and phosphate all lead to
significantly decreased Lys 0 and increased Lys 1 and Lys 2. The
effect of varying zinc concentration on lysine variant was minimal
within the evaluated range. No interaction effects between the
additives had statistical significance indicating that the
modulation of lysine variants was additive and not synergistic.
[0541] Thus, manipulating phosphate concentration in the production
media provides not only one additional way to modulate the lysine
variant distribution but also provides additive modulation of
lysine variants in combination with the addition of amino
acids.
[0542] The Examples above (Examples 4.1, 4.2, and 4.3) demonstrate
that altering the concentration of phosphate in chemically defined
media can lead to modulation of the lysine variant distribution. In
Examples 4.1 and 4.2, it is demonstrated that increasing the
concentration of phosphate in media leads to a relative shift in
lysine variant distribution from Lys 0 to Lys 1/Lys 2. In Example
4.3 it is demonstrated that the effect of phosphate salt in
combination with lysine or arginine provides additive modulation of
lysine variants.
Example 5: Increased Biological Activity of Modified Lysine
Compositions
[0543] This Example describes the increased efficacy of an
exemplary modulated lysine variant species composition comprising
adalimumab in vivo. The modulated lysine variant species
composition used in this Example was produced by collecting
fractions from a preparative scale HPLC column of WCX-10, using
purified adalimumab. Specifically, the Lys 1 and Lys2 peaks from
the WCX column were collected and then subsequently combined and
further concentrated to prepare the modulated lysine variant
species composition, referred to in this Example as Lys-1/2.
[0544] Animal Model for Arthritis
[0545] In order to study the efficacy of this modulated lysine
variant species composition, experiments were carried out in vivo
using human TNF-Tg197 mice. The TNF-Tg197 mouse model is a well
recognized mouse model of arthritis used to test anti-human
TNF.alpha. treatment modalities. The TNF-Tg197 mouse model is
described in Keffer, J. et al., (1991) EMBO J 10:4025-4031, the
contents of which are incorporated herein by reference. The
transgenic mice carrying human TNF gene were developed to study the
effects of excess TNF.alpha. production in vivo.
[0546] Tg197 mice develop swelling in the ankle joints of both hind
paws and impaired movement, which is very similar to human
rheumatoid arthritis. Clinical signs of disease in Tg197 mice start
at 4 weeks of age and include slower weight gain, joint distortion
and swelling, joint deformation and ankylosis and impaired
movement. Histopathological analysis reveals hyperplasia of
synovial membrane, leukocyte infiltration at around 3 weeks of age,
and then pannus formation, articular cartilage destruction and
massive production of fibrous tissue at advanced stage of disease
at 9-11 weeks of age. This model has been used in the development
of anti-TNF.alpha. biologics, including adalimumab.
[0547] Methods
[0548] Groups of mice (6 males and 6 females), were administered
one of the following adalimumab formulations: low AR composition
(group 5; which contains 3.1% acidic species (AR), wherein the
composition comprises 0.1% acidic region 1 (AR1) acidic variants
and 3.0% acidic region 2 (AR2) acidic variants); low host cell
protein (HCP) composition (group 7); AR1 composition (containing
only AR1 acidic variants) (group 8); and a Lys 1 and Lys 2
composition (referred to in this Example as the modulated lysine
variant species composition or Lys-1/2, and which contains only Lys
1 and Lys 2 variants) (group 9). These compositions (fractions) are
shown in the chromatograph in FIG. 100. Another group of mice was
administered a control composition, also referred to as the
"control AR composition," or "normal" composition, which contains
adalimumab with unmodified AR levels and unmodified Lys variants. A
placebo group, comprising 6 mice, was also included.
[0549] Each composition, including the control AR composition, was
administered to the mice in each group beginning with a tolerizing
dose of adalimumab at age 1 week, and followed by additional weekly
dosages of 1 mg/kg for 10 weeks. From weeks 2.5 through weeks 13.5,
weekly measurements of weight and arthritic scores were taken and
weekly serum collection was made. In addition, at the end of the
study, tissue samples from perfused mice were obtained and
analyzed. The following tissues were harvested for testing drug
levels, anti-drug antibodies (ADA), and complexed and free TNF
levels: front paws, inguinal, popliteal and mesenteric lymph nodes,
spleen, tail (for skin sample), knees. The femur and spine tissues
were harvested for micro-CT scanning.
[0550] Results
[0551] As shown in FIG. 101A, the animals receiving the modulated
lysine variant species composition (Lys-1/2) had arthritic scores
similar to the control AR composition. Furthermore, as shown in
FIG. 101B, the mice administered the modulated lysine variant
species composition exhibited an average weight gain that was
comparable to the control AR composition, indicating safety of the
modulated lysine variant species composition, and a lack of adverse
effects that impact weight gain and growth of the mice.
[0552] As shown in FIG. 102, during the 12-13 week treatment period
of the mice, the modulated lysine variant species composition
(Lys-1/2) provided good protection against developing arthritic
scores, as it was more effective than the control, or "normal," AR
composition. The AR1 composition offered the least protection
against development of arthritic scores, and it was less protective
than the control AR composition.
[0553] Serum levels of ADA and drug levels were measured from 3 to
14 weeks of age. As shown in FIG. 103B, the modulated lysine
variant species composition (Lys-1/2) exhibited low average levels
of ADA across the time frame measured. In addition, the modulated
lysine variant species composition (Lys-1/2) composition exhibited
drug levels comparable to the control AR composition (FIG. 103A),
indicating that a lack of presence of the drug in the serum was not
responsible for the low levels of serum ADA.
[0554] As set forth in FIG. 104, cumulative serum concentration
values (PK) during the ten week treatment period was high in the
treated mice for the modulated lysine variant species composition
(Lys-1/2). As also shown in FIG. 104, the highest ADA titers were
observed for animals administered the AR1 composition and the
lowest for animals administered the modulated lysine variant
species composition and the low AR composition.
[0555] Furthermore, complexed TNF levels show that cumulative serum
concentration values during the ten week treatment period were
highest for the animals administered the control (normal) AR
composition and lowest for the AR1 fraction (FIG. 105). Cumulative
serum concentration values for the modulated lysine variant species
composition (Lys-1/2) were lower than the control AR composition,
but higher than the AR1 fraction.
[0556] A histopathology evaluation of the joints of the mice
indicated that good protection was afforded by the modulated lysine
variant species composition (Lys-1/2), indicating that the
modulated lysine variant species composition protects against the
formation of arthritis in the joint in vivo. As shown in FIG. 106,
the modulated lysine variant species composition (Lys-1/2)
protected against chondrocyte death, synovial proliferation,
proteoglycan loss, cartilage destruction, and bone erosion more
effectively than the control AR formulation.
[0557] FIG. 107 illustrates the average drug levels for various
tissues (paw, lymph node, spleen, skin, knee and serum) for the low
AR composition, the control AR composition, the AR1 composition,
and the Lys-1/2 composition. As shown therein, animals administered
the modulated lysine variant species composition (Lys-1/2) has drug
levels comparable to the other compositions tested, including the
AR control.
[0558] FIG. 108 illustrates average ADA levels in the same tissues
for the same compositions (the low AR composition, the control AR
composition, the AR1 composition, and the Lys-1/2 composition). As
shown there, the modulated lysine variant species composition, the
highest ADA concentrations are present in the paws (which
corresponds to the location of the highest levels of inflammation
in the animals), and the serum.
[0559] FIGS. 109 and 110 show the results of a micro CT analysis of
spines and femurs obtained from the transgenic mice at the end of
the study that were administered low AR composition, control AR
composition, AR1 composition, modulated lysine variant species
composition, as well as naive, (control) and placebo. Samples were
analyzed for L5 vertebra bone volume, L5 vertebra trabecular
number, L5 vertebra trabecular thickness, and L5 vertebra
trabecular space. As shown in the Figures, the modulated lysine
variant species composition (Lys-1/2) resulted in greater bone
volume, trabecular number, trabecular thickness and trabecular
space, as compared to the vehicle alone.
[0560] FIG. 111 shows additional results of a micro CT analysis of
spines and femurs obtained from the transgenic mice. Samples were
analyzed for trabecula bone volume at the femoral metaphysis,
trabecular number at the femoral metaphysis, trabecular thickness
at the femoral metaphysis, and trabecular separation at the femoral
metaphysis. As shown in FIG. 111, the modulated lysine variant
species composition (Lys-1/2) resulted in similar trabecula bone
volume at the femoral metaphysis, trabecular number at the femoral
metaphysis, and trabecular thickness at the femoral metaphysis, as
compared to the control (normal) AR composition.
[0561] Furthermore, FIGS. 112A-F and 113A-F show actual micro CT
images of the spine and femur, respectively, from each of six
groups of mice administered the following compositions: naive,
vehicle (control), low AR composition (group 5), low host cell
protein (HCP) composition (group 7), AR1 composition (containing
only AR1 acidic variants) (group 8), and Lys 1 and Lys 2
composition (containing only Lys 1 and Lys 2 variants and referred
to as the modulated lysine variant species composition) (group 9).
As seen in both the spine and the femur, the Lys 1/2 composition
(group 9), provided protection from bone erosion, as compared to
the vehicle, as there is less bone erosion visible in the "group 9"
image as compared to the vehicle.
[0562] The results of these experiments demonstrate that a weekly
dose of 1 mg/kg adalimumab in TNF-Tg197 mice provides protection
from arthritis development as measured by arthritic scores and
histopathology scores (radiologic damage involving cartilage and
bone as well as local inflammation) in the TNF-Tg197 mouse model.
Thus, the control AR group with unmodified Lys variant levels and
normal AR levels was efficacious.
[0563] Furthermore, formulations containing the modulated lysine
variant species composition (Lys-1/2) and the low AR composition
provided greatest protection, as compared to the control AR group,
from development of arthritic and histopathology scores and showed
increased efficacy as compared to the control AR group in all
parameters tested including cell infiltration, synovial
proliferation, proteoglycan loss, cartilage destruction, and bone
erosion.
[0564] Accordingly, the modulated lysine variant species
composition (Lys-1/2) and the low AR compositions have increased
efficacy in the treatment and prevention of arthritis as compared
to the control adalimumab formulation.
[0565] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0566] The contents of all cited references, including literature
references, issued patents, and published patent applications, as
cited throughout this application are hereby expressly incorporated
herein by reference. It should further be understood that the
contents of all the figures and tables attached hereto are
expressly incorporated herein by reference. The entire contents of
the following applications are also expressly incorporated herein
by reference: U.S. Provisional Patent Application 61/893,123,
entitled "STABLE SOLID PROTEIN COMPOSITIONS AND METHODS OF MAKING
SAME", Attorney Docket Number 117813-31001, filed on Oct. 18, 2013;
U.S. Provisional Application Ser. No. 61/892,833, entitled "LOW
ACIDIC SPECIES COMPOSITIONS AND METHODS FOR PRODUCING THE SAME
USING DISPLACEMENT CHROMATOGRAPHY", Attorney Docket Number
117813-73602, filed on Oct. 18, 2013; U.S. Provisional Patent
Application 61/892,710, entitled "MUTATED ANTI-TNF.alpha.
ANTIBODIES AND METHODS OF THEIR USE", Attorney Docket Number
117813-73802, filed on Oct. 18, 2013; U.S. Provisional Patent
Application 61/893,068, entitled "LOW ACIDIC SPECIES COMPOSITIONS
AND METHODS FOR PRODUCING THE SAME", Attorney Docket Number
117813-73901, filed on Oct. 18, 2013; and U.S. Provisional Patent
Application 61/893,131, entitled "PURIFICATION OF PROTEINS USING
HYDROPHOBIC INTERACTION CHROMATOGRAPHY", Attorney Docket Number
117813-74301, filed on Oct. 18, 2013.
Sequence CWU 1
1
121107PRTArtificial Sequenceadalimumab light chain variable region
1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 2121PRTArtificial
Sequenceadalimumab heavy chain variable region 2Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val
50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser
Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 39PRTArtificial Sequenceadalimumab light chain variable
region CDR3 3Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa1 5
412PRTArtificial Sequenceadalimumab heavy chain variable region
CDR3 4Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa1 5 10
57PRTArtificial Sequenceadalimumab light chain variable region CDR2
5Ala Ala Ser Thr Leu Gln Ser1 5 617PRTArtificial Sequenceadalimumab
heavy chain variable region CDR2 6Ala Ile Thr Trp Asn Ser Gly His
Ile Asp Tyr Ala Asp Ser Val Glu1 5 10 15 Gly711PRTArtificial
Sequenceadalimumab light chain variable region CDR1 7Arg Ala Ser
Gln Gly Ile Arg Asn Tyr Leu Ala1 5 10 85PRTArtificial
Sequenceadalimumab heavy chain variable region CDR1 8Asp Tyr Ala
Met His1 5 9321DNAArtificial Sequenceadalimumab light chain
variable region 9gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtagggga cagagtcacc 60atcacttgtc gggcaagtca gggcatcaga aattacttag
cctggtatca gcaaaaacca 120gggaaagccc ctaagctcct gatctatgct
gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctacagcct 240gaagatgttg
caacttatta ctgtcaaagg tataaccgtg caccgtatac ttttggccag
300gggaccaagg tggaaatcaa a 32110363DNAArtificial Sequenceadalimumab
heavy chain variable region 10gaggtgcagc tggtggagtc tgggggaggc
ttggtacagc ccggcaggtc cctgagactc 60tcctgtgcgg cctctggatt cacctttgat
gattatgcca tgcactgggt ccggcaagct 120ccagggaagg gcctggaatg
ggtctcagct atcacttgga atagtggtca catagactat 180gcggactctg
tggagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat
240ctgcaaatga acagtctgag agctgaggat acggccgtat attactgtgc
gaaagtctcg 300taccttagca ccgcgtcctc ccttgactat tggggccaag
gtaccctggt caccgtctcg 360agt 36311214PRTArtificial
Sequenceadalimumab light chain 11Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala
Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205 Phe Asn Arg Gly Glu Cys 210 12451PRTArtificial
Sequenceadalimumab heavy chain 12Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala
Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu
Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310
315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435
440 445 Pro Gly Lys 450
* * * * *