U.S. patent application number 15/601945 was filed with the patent office on 2017-12-14 for cell culture methods to reduce acidic species.
This patent application is currently assigned to AbbVie Inc.. The applicant listed for this patent is AbbVie Inc.. Invention is credited to Christopher CHUMSAE, Diane D. Dong, Kathreen A. Gifford, Wen Chung Lim, Kartik Subramanian, Xiaobei Zeng.
Application Number | 20170355761 15/601945 |
Document ID | / |
Family ID | 48045072 |
Filed Date | 2017-12-14 |
United States Patent
Application |
20170355761 |
Kind Code |
A1 |
Subramanian; Kartik ; et
al. |
December 14, 2017 |
CELL CULTURE METHODS TO REDUCE ACIDIC SPECIES
Abstract
The instant invention relates to the field of protein production
and purification, and in particular to compositions and processes
for controlling the amount of acidic species expressed by host
cells, as well as to compositions and processes for controlling the
amount of acidic species present in purified preparations.
Inventors: |
Subramanian; Kartik;
(Northborough, MA) ; Zeng; Xiaobei; (Carolina,
PR) ; Dong; Diane D.; (Shrewsbury, MA) ; Lim;
Wen Chung; (Worcester, MA) ; Gifford; Kathreen
A.; (Marlborough, MA) ; CHUMSAE; Christopher;
(North Andover, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc. |
North Chicago |
IL |
US |
|
|
Assignee: |
AbbVie Inc.
North Chicago
IL
|
Family ID: |
48045072 |
Appl. No.: |
15/601945 |
Filed: |
May 22, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15008895 |
Jan 28, 2016 |
9683033 |
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15601945 |
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|
14842933 |
Sep 2, 2015 |
9359434 |
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15008895 |
|
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13830583 |
Mar 14, 2013 |
9150645 |
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14842933 |
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61636493 |
Apr 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/165 20130101;
C07K 16/241 20130101; A61K 39/39591 20130101; C07K 1/20 20130101;
C07K 2317/21 20130101; C07K 1/22 20130101; C07K 1/18 20130101; C07K
16/00 20130101; C07K 2317/14 20130101; C12P 21/00 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; C07K 1/18 20060101 C07K001/18; C12P 21/00 20060101
C12P021/00; A61K 39/395 20060101 A61K039/395; C07K 1/22 20060101
C07K001/22; C07K 1/20 20060101 C07K001/20; C07K 1/16 20060101
C07K001/16; C07K 16/00 20060101 C07K016/00 |
Claims
1. A composition comprising adalimumab, wherein the composition
comprises less than 10% total acidic species of adalimumab, wherein
the acidic species of adalimumab do not include process-related
impurities selected from the group consisting of host cells and
lysed host cells, and wherein the acidic species of adalimumab
correspond to the peaks that elute earlier than the main peak in a
WCX-10 HPLC chromatogram of adalimumab.
2. The composition of claim 1, wherein the WCX-10 HPLC chromatogram
is generated using a first mobile phase of 10 mM Sodium Phosphate
dibasic (pH 7.5) and a second mobile phase of 10 mM Sodium
Phosphate dibasic, 500 mM Sodium Chloride (pH 5.5), and wherein the
WCX-10 HPLC chromatogram is generated using detection at 280
nm.
3. The composition of claim 1, wherein the acidic species of
adalimumab comprise a first acidic region (AR1) and a second acidic
region (AR2).
4. The composition of claim 1, wherein the composition comprises 9%
or less acidic species of adalimumab.
5. The composition of claim 1, wherein the composition comprises
6%-8% acidic species of adalimumab.
6. The composition of claim 1, wherein the adalimumab is produced
in a mammalian host cell grown in cell culture.
7. The composition of claim 6, 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.
8. The composition of claim 7, wherein the mammalian host cell is a
CHO cell.
9. The composition of claim 8, wherein the composition is
lyophilized.
10. A pharmaceutical composition suitable for administration to a
subject comprising the composition of claim 8 and a
pharmaceutically acceptable carrier.
11. The pharmaceutical composition of claim 10, wherein adalimumab
is present in the pharmaceutical composition at a concentration of
0.1-250 mg/ml.
12. The pharmaceutical composition of claim 10, wherein the
pharmaceutical composition comprises one or more excipient.
13. The pharmaceutical composition of claim 12, wherein the one or
more excipient is selected from the group consisting of a buffer,
an isotonic agent, a surfactant or a combination thereof.
14. The pharmaceutical composition of claim 13, wherein the
pharmaceutical composition comprises the surfactant polysorbate
80.
15. The pharmaceutical composition of claim 13, wherein the
pharmaceutical composition comprises an amino acid buffer.
16. The pharmaceutical composition of claim 15, wherein the amino
acid is histidine.
17. The pharmaceutical composition of claim 13, wherein the
pharmaceutical composition comprises the isotonic agent
mannitol.
18. A method for treating a subject in need thereof, comprising
administering to the subject the pharmaceutical composition of
claim 10, thereby treating the subject.
19. A composition comprising adalimumab, wherein the composition
comprises less than 10% total acidic species of adalimumab, wherein
the acidic species of adalimumab do not include process-related
impurities selected from the group consisting of host cells and
lysed host cells, wherein the acidic species of adalimumab
correspond to the peaks that elute earlier than the main peak in a
WCX-10 HPLC chromatogram of adalimumab, and wherein the WCX-10 HPLC
chromatogram is generated using a first mobile phase of 10 mM
Sodium Phosphate dibasic (pH 7.5) and a second mobile phase of 10
mM Sodium Phosphate dibasic, 500 mM Sodium Chloride (pH 5.5), and
wherein the WCX-10 HPLC chromatogram is generated using detection
at 280 nm.
20. The composition of claim 19, wherein the acidic species of
adalimumab comprise a first acidic region (AR1) and a second acidic
region (AR2).
21. The composition of claim 19, wherein the composition comprises
9% or less acidic species of adalimumab.
22. The composition of claim 19, wherein the composition comprises
6%-8% acidic species of adalimumab.
23. The composition of claim 19, wherein the adalimumab is produced
in a mammalian host cell grown in cell culture.
24. The composition of claim 23, 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.
25. The composition of claim 24, wherein the mammalian host cell is
a CHO cell.
26. A pharmaceutical composition suitable for administration to a
subject comprising the composition of claim 25 and a
pharmaceutically acceptable carrier.
27. The pharmaceutical composition of claim 26, wherein adalimumab
is present in the pharmaceutical composition at a concentration of
0.1-250 mg/ml.
28. The pharmaceutical composition of claim 26, wherein the
pharmaceutical composition comprises one or more excipient.
29. The pharmaceutical composition of claim 28, wherein the one or
more excipient is selected from the group consisting of a buffer,
an isotonic agent, a surfactant or a combination thereof.
30. A method for treating a subject in need thereof, comprising
administering to the subject the pharmaceutical composition of
claim 26, thereby treating the subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of Ser. No.
15/008,895, filed on Jan. 28, 2016, which is a divisional
application of U.S. patent application Ser. No. 14/842,933, filed
on Sep. 2, 2015, pending, which is a continuation application of
U.S. patent application Ser. No. 13/830,583, now U.S. Pat. No.
9,150,645, filed on Mar. 14, 2013, which claims priority to U.S.
Provisional Application No. 61/636,493, filed on Apr. 20, 2012. The
entire contents of each of the foregoing applications is
incorporated herein by reference.
1. INTRODUCTION
[0002] The instant invention relates to the field of protein
production, and in particular to compositions and processes for
controlling the amount of acidic species generated during
expression of a protein of interest by host cells, as well as the
reduction of acidic species present in the clarified cell culture
broth. In certain aspects of the invention, controlling the amount
of acidic species generated during expression of a protein of
interest is achieved by modifying the culture media of the cells.
In certain aspects of the invention, controlling the amount of
acidic species generated during expression of a protein of interest
is achieved by modifying the culture process parameters. In certain
aspects of the invention, controlling the amount of acidic species
of a protein of interest is achieved by modifying a cell culture
clarified harvest comprising the protein of interest.
2. BACKGROUND OF THE INVENTION
[0003] The production of proteins for biopharmaceutical
applications typically involves the use of cell cultures that are
known to produce proteins exhibiting varying levels of
product-related substance heterogeneity. Such heterogeneity
includes, but is not limited to, the presence of acidic species.
For example, in monoclonal antibody (mAb) preparations, such acidic
species heterogeneities can be detected by various methods, such as
WCX-10 HPLC (a weak cation exchange chromatography) or IEF
(isoelectric focusing). In certain embodiments, the acidic species
identified using such techniques comprise a range of
product-related impurities such as antibody product fragments
(e.g., Fc and Fab fragments), and/or post-translation modifications
of the antibody product, such as, deamidated and/or glycoslyated
antibodies. However, because of their similar chemical
characteristics to the antibody product molecules, reduction of
acidic species is a challenge in monoclonal antibody purification.
Control of acidic species heterogeneity is particularly
advantageous in the context of cell culture processes used for
commercially produced recombinant bio-therapeutics as such
heterogeneity has the potential to impact stability.
3. SUMMARY OF THE INVENTION
[0004] The present invention is directed to compositions and
methods that control (modulate or limit) acidic species
heterogeneity in a population of proteins. The presence of such
acidic species corresponds to heterogeneity of the distribution of
charged impurities, e.g., a mixture of protein fragments (e.g., Fc
and Fab fragments of antibodies), and/or post-translation
modifications of the proteins, such as, deamidated and/or
glycoslyated proteins, in the population of proteins, and such
heterogeneity particularly of interest when it arises in the
context of recombinant protein production.
[0005] In certain embodiments, the acidic species heterogeneity
arises from differences in the amount and/or type of acidic species
in a population of proteins.
[0006] In certain embodiments, the acidic species heterogeneity is
present in a population of proteins produced by cell culture. In
certain embodiments, control is exerted over the amount of acidic
species of protein produced by cell culture. In certain
embodiments, the control is exerted over the amount of acidic
species formed while the protein is present in a cell culture
broth, while the culture is actively maintained or while the cells
are removed. In certain embodiments, the protein is an
antibody.
[0007] In certain embodiments, control over the amount of acidic
species produced by cell culture is exerted by employing certain
media components during production of a protein, for example, an
antibody, of interest. In certain embodiments, control over the
amount of acidic species of protein produced by cell culture is
exerted by supplementing the media of cells expressing the protein
of interest with one or more amino acids. In certain embodiments,
the one or more amino acids are arginine, lysine, ornithine,
histidine or combinations thereof.
[0008] In certain embodiments, control over the amount of acidic
species of protein produced by cell culture is exerted by
supplementing the media of cells expressing the protein of interest
with calcium, for example, by supplementing the media with calcium
chloride dihydrate.
[0009] In certain embodiments, control over the amount of acidic
species of protein produced by cell culture is exerted by
supplementing the media of cells expressing the protein of interest
with vitamin niacinamide.
[0010] In certain embodiments, control over the amount of acidic
species of protein produced by cell culture is exerted by
supplementing the media of cells expressing the protein of interest
with suitable combinations of arginine, lysine, calcium chloride
and niacinamide.
[0011] In certain embodiments, control over the amount of acidic
species produced by cell culture is exerted by ensuring that the
production of a protein, for example, an antibody, of interest
occurs under specific conditions, including specific pH.
[0012] In certain embodiments, control over the amount of acidic
species of protein produced by cell culture is exerted by
supplementing the media of cells expressing the protein of interest
with arginine and lysine and by controlling the pH of the cell
culture. In certain embodiments, the pH of the cell culture is
adjusted to a pH of about 6.9. In certain embodiments, the pH of
the cell culture is adjusted to a lower pH of about 6.8.
[0013] In certain embodiments, control over the amount of acidic
species of protein produced by cell culture is exerted by
supplementing the media of cells expressing the protein of interest
with arginine and lysine and by choice of cell culture harvest
criteria. In certain embodiments, the harvest criterion is a
particular culture day. In certain embodiments, the harvest
criterion is based on harvest viability.
[0014] In certain embodiments, control over the amount of acidic
species produced by cell culture is exerted by supplementing a cell
culture clarified harvest comprising a protein or antibody of
interest with one or more amino acids. In certain embodiments, the
one or more amino acids is arginine, histidine, or combinations
thereof.
[0015] In certain embodiments, control over the amount of acidic
species produced by cell culture is exerted by adjusting the pH of
a cell culture clarified harvest comprising a protein or antibody
of interest. In certain embodiments, the pH of the cell culture
clarified harvest is adjusted to a pH of about 5. In certain
embodiments, the pH of the cell culture clarified harvest is
adjusted to a pH of about 6.
[0016] In certain embodiments, control over the amount of acidic
species produced by cell culture is exerted by the use of a
continuous or perfusion technology. In certain embodiments, this
may be attained through choice of medium exchange rate. In certain,
non-limiting, embodiments, maintenance of the medium exchange rates
(working volumes/day) of a cell culture run between 0 and 20, or
between 0.5 and 12 or between 1 and 8 or between 1.5 and 6 can be
used to achieve the desired reduction in acidic species. In certain
embodiments, the choice of cell culture methodology that allows for
control of acidic species heterogeneity can also include, for
example, but not by way of limitation, employment of an
intermittent harvest strategy or through use of cell retention
device technology.
[0017] In certain embodiments, the methods of culturing cells
expressing a protein of interest, such as an antibody or
antigen-binding portion thereof, reduces the amount of acidic
species present in the resulting composition. In certain
embodiments, the resulting composition is substantially free of
acidic species. In one aspect, the sample comprises a cell culture
harvest wherein the cell culture is employed to produce specific
proteins of the present invention. In a particular aspect, the
sample matrix is prepared from a cell line used to produce
anti-TNF-.alpha. antibodies.
[0018] The purity 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, HCP ELISA, Protein A ELISA,
and western blot analysis.
[0019] In yet another embodiment, the invention is directed to one
or more pharmaceutical compositions comprising an isolated protein,
such as an antibody or antigen-binding portion thereof, and an
acceptable carrier. In another aspect, the compositions further
comprise one or more pharmaceutical agents.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 depicts the effect of total arginine concentration in
adalimumab producing cell line 2, media 1 on viable cell density
(n=2).
[0021] FIG. 2 depicts the effect of total arginine concentration in
adalimumab producing cell line 2, media 1 on viability (n=2).
[0022] FIG. 3 depicts the effect of total arginine concentration in
adalimumab producing cell line 2, media 1 on harvest titer
(n=2).
[0023] FIG. 4 depicts the effect of total arginine concentration in
adalimumab producing cell line 2, media 1 on day 10 WCX 10 profile
total acidic regions (n=2).
[0024] FIG. 5 depicts the effect of total arginine concentration in
adalimumab producing cell line 2, media 1 on day 12 WCX 10 profile
total acidic regions (n=2).
[0025] FIG. 6 depicts the effect of total arginine concentration in
adalimumab producing cell line 3, media 1 on viable cell density
(n=2).
[0026] FIG. 7 depicts the effect of total arginine concentration in
adalimumab producing cell line 3, media 1 on viability (n=2).
[0027] FIG. 8 depicts the effect of total arginine concentration in
adalimumab producing cell line 3, media 1 on harvest titer
(n=2).
[0028] FIG. 9 depicts the effect of total arginine concentration in
adalimumab producing cell line 3, media 1 on WCX 10 profile total
acidic regions (n=2).
[0029] FIG. 10 depicts the effect of total arginine concentration
in adalimumab producing cell line 1, media 1 on WCX 10 profile
total acidic regions (n=2).
[0030] FIG. 11 depicts the effect of arginine addition to
adalimumab producing cell line 1, media 2 on day 11 on WCX-10
profile total acidic regions (n=2).
[0031] FIG. 12 depicts the effect of arginine addition to
adalimumab producing cell line 2, media 3 on WCX-10 profile total
acidic regions (n=2).
[0032] FIG. 13 depicts the effect of total arginine concentration
in mAB1 producing cell line on WCX-10 profile total acidic regions
(n=1).
[0033] FIG. 14 depicts the effect of total arginine concentration
in mAB2 producing cell line on WCX-10 profile total acidic regions
(n=2)
[0034] FIG. 15 depicts the effect of carboxypeptidase digestion of
product from adalimumab producing cell line 3, media 1 experiment
on WCX-10 profile total acidic regions (n=1).
[0035] FIG. 16 depicts the effect of carboxypeptidase digestions of
product from mAB2 producing cell line on WCX-10 profile total
acidic regions (n=2).
[0036] FIG. 17 depicts the effect of total lysine concentration in
adalimumab producing cell line 2, media 1 on viable cell density
(n=2).
[0037] FIG. 18 depicts the effect of total lysine concentration in
adalimumab producing cell line 2, media 1 on viability (n=2).
[0038] FIG. 19 depicts the effect of total lysine concentration in
adalimumab producing cell line 2, media 1 on harvest titer
(n=2).
[0039] FIG. 20 depicts the effect of total lysine concentration in
adalimumab producing cell line 2, media 1 on WCX 10 profile total
acidic regions (n=2).
[0040] FIG. 21 depicts the effect of total lysine concentration in
adalimumab producing cell line 3, media 1 on viable cell density
(n=2).
[0041] FIG. 22 depicts the effect of total lysine concentration in
adalimumab producing cell line 3, media 1 on viability (n=2).
[0042] FIG. 23 depicts the effect of total lysine concentration in
adalimumab producing cell line 3, media 1 on harvest titer
(n=2).
[0043] FIG. 24 depicts the effect of total lysine concentration in
adalimumab producing cell line 3, media 1 on WCX 10 profile total
acidic regions (n=2).
[0044] FIG. 25 depicts the effect of total lysine concentration in
adalimumab producing cell line 1, media 1 on WCX 10 profile total
acidic regions (n=2).
[0045] FIG. 26 depicts the effect of lysine addition to adalimumab
producing cell line 1, media 2 on WCX-10 profile total acidic
regions (n=2).
[0046] FIG. 27 depicts the effect of lysine addition to adalimumab
producing cell line 2, media 3 on WCX-10 profile total acidic
regions (n=2).
[0047] FIG. 28 depicts the effect of total lysine concentration in
mAB1 producing cell line on WCX-10 profile total acidic regions
(n=1).
[0048] FIG. 29 depicts the effect of total lysine concentration in
mAB2 producing cell line on WCX-10 profile total acidic regions
(n=2).
[0049] FIG. 30 depicts the effect of carboxypeptidase digestion of
product from cell line 3, media 1 experiment on WCX-10 profile
total acidic regions (n=1).
[0050] FIG. 31 depicts the effect of carboxypeptidase digestions of
product from mAB2 producing cell line on WCX-10 profile total
acidic regions (n=2).
[0051] FIG. 32 depicts the effect of total histidine concentration
in adalimumab producing cell line 2, media 1 on viable cell density
(n=2).
[0052] FIG. 33 depicts the effect of total histidine concentration
in adalimumab producing cell line 2, media 1 on viability
(n=2).
[0053] FIG. 34 depicts the effect of total histidine concentration
in adalimumab producing cell line 2, media 1 on harvest titer
(n=2).
[0054] FIG. 35 depicts the effect of total histidine concentration
in adalimumab producing cell line 2, media 1 on WCX 10 profile
total acidic regions (n=2).
[0055] FIG. 36 depicts the effect of total histidine concentration
in adalimumab producing cell line 3, media 1 on viable cell density
(n=2).
[0056] FIG. 37 depicts the effect of total histidine concentration
in adalimumab producing cell line 3, media 1 on viability
(n=2).
[0057] FIG. 38 depicts the effect of total histidine concentration
in adalimumab producing cell line 3, media 1 on harvest titer
(n=2).
[0058] FIG. 39 depicts the effect of total histidine concentration
in adalimumab producing cell line 3, media 1 on WCX 10 profile
total acidic regions (n=2).
[0059] FIG. 40 depicts the effect of total histidine concentration
in adalimumab producing cell line 1, media 1 on WCX 10 profile
total acidic regions (n=2).
[0060] FIG. 41 depicts the effect of histidine addition to
adalimumab producing cell line 1, media 2 on WCX-10 profile total
acidic regions (n=2).
[0061] FIG. 42 depicts the effect of histidine addition to
adalimumab producing cell line 2, media 3 on WCX-10 profile total
acidic regions (n=2).
[0062] FIG. 43 depicts the effect of total histidine concentration
in mAB1 producing cell line on WCX-10 profile total acidic regions
(n=1).
[0063] FIG. 44 depicts the effect of total histidine concentration
in mAB2 producing cell line on WCX-10 profile total acidic regions
(n=2).
[0064] FIG. 45 depicts the effect of carboxypeptidase digestion of
product from cell line 3, media 1 experiment on WCX-10 profile
total acidic regions (n=1).
[0065] FIG. 46 depicts the effect of carboxypeptidase digestions of
product from mAB2 producing cell line on WCX-10 profile total
acidic regions (n=2).
[0066] FIG. 47 depicts the effect of total ornithine concentration
in adalimumab producing cell line 2, media 1 on viable cell density
(n=2).
[0067] FIG. 48 depicts the effect of total ornithine concentration
in adalimumab producing cell line 2, media 1 on viability
(n=2).
[0068] FIG. 49 depicts the effect of total ornithine concentration
in adalimumab producing cell line 2, media 1 on harvest titer
(n=2).
[0069] FIG. 50 depicts the effect of total ornithine concentration
in adalimumab producing cell line 2, media 1 on WCX 10 profile
total acidic regions.
[0070] FIG. 51 depicts the effect of total ornithine concentration
in adalimumab producing cell line 3, media 1 on viable cell density
(n=2).
[0071] FIG. 52 depicts the effect of total ornithine concentration
in adalimumab producing cell line 3, media 1 on viability
(n=2).
[0072] FIG. 53 depicts the effect of total ornithine concentration
in adalimumab producing cell line 3, media 1 on harvest titer
(n=2).
[0073] FIG. 54 depicts the effect of total ornithine concentration
in adalimumab producing cell line 3, media 1 on WCX 10 profile
total acidic regions (n=2).
[0074] FIG. 55 depicts the effect of total ornithine concentration
in adalimumab producing cell line 1, media 1 on WCX 10 profile
total acidic regions (n=2).
[0075] FIG. 56 depicts the effect of ornithine addition to
adalimumab producing cell line 1, media 2 on WCX-10 profile total
acidic regions (n=2).
[0076] FIG. 57 depicts the effect of ornithine addition to
adalimumab producing cell line 2, media 3 on WCX-10 profile total
acidic regions (n=2).
[0077] FIG. 58 depicts the effect of total ornithine concentration
in mAB1 producing cell line on WCX-10 profile total acidic regions
(n=1).
[0078] FIG. 59 depicts the effect of total ornithine concentration
in mAB2 producing cell line on WCX-10 profile total acidic regions
(n=2).
[0079] FIG. 60 depicts the effect of carboxypeptidase digestion of
product from cell line 3, media 1 experiment on WCX-10 profile
total acidic regions (n=1).
[0080] FIG. 61 depicts the effect of carboxypeptidase digestions of
product from mAB2 producing cell line on WCX-10 profile total
acidic regions (n=2).
[0081] FIG. 62 depicts the effect of multiple amino acid additions
to adalimumab producing cell line 2, media 1 on WCX 10 profile
total acidic regions (n=2).
[0082] FIG. 63 depicts the effect of increased arginine and lysine
concentration in adalimumab producing cell line 1, media 1 on
viable cell density (n=3).
[0083] FIG. 64 depicts the effect of increased arginine and lysine
concentration in adalimumab producing cell line 3, media 1 on
viability (n=3).
[0084] FIG. 65 depicts the effect of increased arginine and lysine
concentration in adalimumab producing cell line 3, media 1 on
culture titer (n=3).
[0085] FIG. 66 depicts the effect of increased arginine and lysine
concentration in adalimumab producing cell line 1, media 1 on WCX
10 profile total acidic regions (n=2).
[0086] FIG. 67 depicts the effect of arginine, lysine and pH
modulation to adalimumab producing cell line 1, media 1 on viable
cell density (n=2).
[0087] FIG. 68 depicts the effect of arginine, lysine and pH
modulation to adalimumab producing cell line 3, media 1 on
viability (n=2).
[0088] FIG. 69 depicts the effect of arginine, lysine and pH
modulation to adalimumab producing cell line 3, media 1 on culture
titer (n=2).
[0089] FIG. 70 depicts the effect of arginine, lysine and pH
modulation to adalimumab producing cell line 1, media 1 on WCX 10
profile total acidic regions (n=2).
[0090] FIG. 71 depicts the effect of total calcium concentration in
adalimumab producing cell line 2, media 1 on viable cell density
(n=2).
[0091] FIG. 72 depicts the effect of total calcium concentration in
adalimumab producing cell line 2, media 1 on viability (n=2).
[0092] FIG. 73 depicts the effect of total calcium concentration in
adalimumab producing cell line 2, media 1 on harvest titer
(n=2).
[0093] FIG. 74 depicts the effect of total calcium concentration in
adalimumab producing cell line 2, media 1 on WCX 10 profile total
acidic regions (n=2).
[0094] FIG. 75 depicts the effect of total calcium concentration in
adalimumab producing cell line 3, media 1 on viable cell density
(n=2).
[0095] FIG. 76 depicts the effect of total calcium concentration in
adalimumab producing cell line 3, media 1 on viability (n=2).
[0096] FIG. 77 depicts the effect of total calcium concentration in
adalimumab producing cell line 3, media 1 on harvest titer
(n=2)
[0097] FIG. 78 depicts the effect of total calcium concentration in
adalimumab producing cell line 3, media 1 on WCX 10 profile total
acidic regions (n=2).
[0098] FIG. 79 depicts the effect of total calcium concentration in
adalimumab producing cell line 1, media 1 on WCX 10 profile total
acidic regions (n=2).
[0099] FIG. 80 depicts the effect of calcium addition to adalimumab
producing cell line 1, media 2 on WCX-10 profile total acidic
regions (n=2).
[0100] FIG. 81 depicts the effect of calcium addition to adalimumab
producing cell line 2, media 3 on WCX-10 profile total acidic
regions (n=2).
[0101] FIG. 82 depicts the effect of total calcium concentration in
mAB1 producing cell line on WCX-10 profile total acidic regions
(n=2).
[0102] FIG. 83 depicts the effect of total calcium concentration in
mAB2 producing cell line on WCX-10 profile total acidic regions
(n=2).
[0103] FIG. 84 depicts the effect of multiple amino acid additions
to cell line 1, media 1 on WCX 10 profile total acidic regions a)
overall prediction plot, b) prediction plots for each additive
(n=2).
[0104] FIG. 85 depicts the effect of niacinamide addition to
adalimumab producing cell line 1, media 1 on viable cell density
(n=2).
[0105] FIG. 86 depicts the effect of niacinamide addition to
adalimumab producing cell line 1, media 1 on viability (n=2).
[0106] FIG. 87 depicts the effect of niacinamide addition to
adalimumab producing cell line 1, media 1 on harvest titer
(n=2).
[0107] FIG. 88 depicts the effect of niacinamide addition to
adalimumab producing cell line 1, media 1 on Day 11 WCX 10 profile
total acidic regions (n=2).
[0108] FIG. 89 depicts the effect of niacinamide addition to
adalimumab producing cell line 1, media 1 on Day 12 WCX-10 profile
total acidic regions (n=2).
[0109] FIG. 90 depicts the effect of niacinamide addition to mAB2
producing cell line, media 1 on viable cell density (n=2).
[0110] FIG. 91 depicts the effect of niacinamide addition to mAB2
producing cell line, media 1 on viability (n=2).
[0111] FIG. 92 depicts the effect of niacinamide addition to mAB2
producing cell line, media 1 on harvest titer (n=2).
[0112] FIG. 93 depicts the effect of niacinamide addition to mAB2
producing cell line, media 1 on WCX 10 profile total acidic regions
(n=2).
[0113] FIG. 94 depicts the effect of pH modulation of adalimumab
producing cell line 1, media 1 on viable cell density (n=2).
[0114] FIG. 95 depicts the effect of pH modulation adalimumab
producing cell line 1, media 1 on viability (n=2).
[0115] FIG. 96 depicts the effect of pH modulation of adalimumab
producing cell line 1, media 1 on harvest titer (n=2).
[0116] FIG. 97 depicts the effect of pH modulation of adalimumab
producing cell line 1, media 1 on WCX 10 profile total acidic
regions (n=2).
[0117] FIG. 98 depicts the effect of pH modulation of adalimumab
producing cell line 1, media 2 on viable cell density (n=2).
[0118] FIG. 99 depicts the effect of pH modulation addition of
adalimumab producing adalimumab producing cell line 1, media 2 on
viability (n=2).
[0119] FIG. 100 depicts the effect of pH modulation of adalimumab
producing cell line 1, media 2 on harvest titer (n=2).
[0120] FIG. 101 depicts the effect of pH modulation of adalimumab
producing cell line 1, media 2 on WCX 10 profile total acidic
regions (n=2).
[0121] FIG. 102 depicts the effect of pH modulation of adalimumab
producing cell line 3, media 1 on viable cell density (n=2).
[0122] FIG. 103 depicts the effect of pH modulation adalimumab
producing cell line 3, media 1 on viability (n=2).
[0123] FIG. 104 depicts the effect of pH modulation of adalimumab
producing cell line 3, media 1 on harvest titer (n=2).
[0124] FIG. 105 depicts the effect of pH modulation of adalimumab
producing cell line 3, media 1 on WCX 10 profile total acidic
regions (n=2).
[0125] FIG. 106 depicts an acidification sample preparation
scheme.
[0126] FIG. 107 depicts an arginine sample preparation scheme.
[0127] FIG. 108 depicts a histidine sample preparation scheme.
[0128] FIG. 109 depicts a lysine sample preparation scheme.
[0129] FIG. 110 depicts a methionine sample preparation scheme.
[0130] FIG. 111 depicts an amino acid sample preparation
scheme.
[0131] FIG. 112 depicts a CDM clarified harvest sample preparation
scheme.
[0132] FIG. 113 depicts an acid-type pH study sample preparation
scheme.
[0133] FIG. 114 depicts the effect of low pH treatment with
subsequent neutralization on initial acidic variant content.
[0134] FIG. 115 depicts the effect of low pH treatment with
subsequent neutralization on acidic variant formation rate.
[0135] FIG. 116 depicts the effect of sample preparation method on
initial acidic variant content.
[0136] FIG. 117 depicts the effect of sample preparation method on
initial acidic variant content.
[0137] FIG. 118 depicts the dose dependent effect of arginine on
reduction of acidic variant formation rate.
[0138] FIG. 119 depicts the effect of histidine concentration on
initial acidic variant content.
[0139] FIG. 120 depicts the effect of histidine concentration on
acidic variant formation rate.
[0140] FIG. 121 depicts the effect of lysine on initial acid
variant content.
[0141] FIG. 122 depicts the effect of lysine on acidic variant
formation rate.
[0142] FIG. 123 depicts the effect of methionine on initial acid
variant content.
[0143] FIG. 124 depicts the effect of methionine on acidic variant
formation rate.
[0144] FIG. 125 depicts the effect of amino acids on initial acid
variant content.
[0145] FIG. 126 depicts the effect of amino acids on acidic variant
formation rate.
[0146] FIG. 127 depicts the effect of alternative additives on
initial acid variant content.
[0147] FIG. 128 depicts the effect of alternative additives on
acidic variant formation rate.
[0148] FIG. 129 depicts the effect of low pH/arginine treatment on
D2E7 CDM initial acid variant content.
[0149] FIG. 130 depicts the effect of low pH/arginine treatment on
D2E7 CDM acidic variant formation rate.
[0150] FIG. 131 depicts the effect of low pH/arginine treatment on
mAb B hydrolysate initial acid variant content.
[0151] FIG. 132 depicts the effect of low pH/arginine treatment on
mAb B hydrolysate acidic variant formation rate.
[0152] FIG. 133 depicts the effect of low pH/arginine treatment on
mAb C hydrolysate initial acid variant content.
[0153] FIG. 134 depicts the effect of low pH/arginine treatment on
mAb C hydrolysate acidic variant formation rate.
[0154] FIG. 135 depicts the effect of acid type/pH on acid variant
content.
[0155] FIG. 136 depicts the effect of acid concentration on acid
variant content.
[0156] FIG. 137 depicts the effect of acid concentration on acid
variant content.
[0157] FIG. 138 depicts the effect of neutralization on acid
variant content.
[0158] FIG. 139 depicts the effect of neutralization on acid
variant content.
[0159] FIG. 140 depicts LC/MS peptide mapping analysis of exemplary
antibodies expressed in the context of the cell culture conditions
of the instant invention, including preparation of specific mass
traces for both modified and non-modified peptides in order to
accurately quantify the total amount of MGO modification. Mass
spectra are also analyzed for the specific region of the
chromatogram to confirm the peptide identity.
[0160] FIG. 141 depicts a chromatogram wherein the total acidic
species associated with the expression of Adalimiumab is divided
into a first acidic species region (AR1) and a second acidic
species region (AR2).
[0161] FIG. 142 depicts the AR Growth at 25.degree. C. of low and
high AR containing samples.
5. DETAILED DESCRIPTION OF THE INVENTION
[0162] The instant invention relates to the field of protein
production. In particular, the instant invention relates to
compositions and processes for controlling the amount of acidic
species expressed by host cells when used to produce a protein of
interest. Certain embodiments of the invention relate to culturing
said cells to express said proteins under conditions that limit the
amount of acidic species that are expressed by the cells. In
certain embodiments, the methods described herein employ culturing
said cells in media supplemented with one or more amino acids
and/or calcium (e.g., as calcium chloride dihydrate) and/or
niacinamide. In certain embodiments, the methods described herein
employ culturing said cells in a culture with appropriate control
of process parameters, such as pH. In certain embodiments, methods
described herein employ culturing cells at a lower process pH. In
certain embodiments of the instant invention, control of acidic
species heterogeneity can be attained by the choice of cell culture
methodology. In certain embodiments, use of a continuous or
perfusion technology may be utilized to achieve the desired control
over acidic species heterogeneity. In certain embodiments, this may
be attained through choice of medium exchange rate. In certain
embodiments, the present invention is directed toward
pharmaceutical compositions comprising one or more proteins, such
as, but not limited to an antibody or antigen-binding portion
thereof, purified by a method described herein.
[0163] For clarity and not by way of limitation, this detailed
description is divided into the following sub-portions:
[0164] (i) Definitions;
[0165] (ii) Antibody Generation;
[0166] (iii) Protein Production;
[0167] (iv) Protein Purification;
[0168] (v) Pharmaceutical Compositions
5.1 Definitions
[0169] In order that the present invention may be more readily
understood, certain terms are first defined.
[0170] As used herein, the terms "acidic species" and "acidic
species heterogeneity" refer to a characteristic of a population of
proteins wherein the population includes a distribution of
product-related impurities identifiable by the presence of charge
heterogeneities. For example, in monoclonal antibody (mAb)
preparations, such acidic species heterogeneities can be detected
by various methods, such as, for example, WCX-10 HPLC (a weak
cation exchange chromatography), or IEF (isoelectric focusing). In
certain embodiments, the acidic species identified using such
techniques comprise a mixture of product-related impurities
containing antibody product fragments (e.g., Fc and Fab fragments),
chemical modifications (e.g., methylglyoxal modified species (as
described in U.S. patent application Ser. No. 14/078,181), glycated
species) and/or post-translation modifications of the antibody
product, such as, deamidated and/or glycoslyated antibodies.
[0171] In certain embodiments, the acidic species heterogeneity
comprises a difference in the type of acidic species present in the
population of proteins. For example, the population of proteins may
comprise more than one acidic species variant. For example, but not
by way of limitation, the total acidic species can be divided based
on chromatographic residence time. FIG. 141 depicts a non-limiting
example of such a division wherein the total acidic species
associated with the expression of Adalimiumab is divided into a
first acidic species region (AR1) and a second acidic species
region (AR2). The compositions of particular acidic species regions
may differ depending on the particular antibody of interest, as
well as the particular cell culture, purification, and/or
chromatographic conditions employed.
[0172] In certain embodiments, the heterogeneity of the
distribution of acidic species comprises a difference in the amount
of acidic species in the population of proteins. For example, the
population of proteins may comprise more than one acidic species
variant, and each of the variants may be present in different
amounts.
[0173] The term "antibody" includes an immunoglobulin molecule
comprised of four polypeptide chains, two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as HCVR or VH) and a heavy chain constant region (CH). 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 (CDRs), 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.
[0174] The term "antigen-binding portion" of an antibody (or
"antibody portion") includes fragments of an antibody that retain
the ability to specifically bind to an antigen (e.g., in the case
of Adalimumab, hTNF.alpha.). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
comprising the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment,
a bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; (iii) a Fd fragment
comprising the VH and CH1 domains; (iv) a Fv fragment comprising
the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546, the entire
teaching of which is incorporated herein by reference), which
comprises a VH domain; and (vi) an isolated complementarity
determining region (CDR). 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, the entire teachings of which are incorporated herein
by reference). 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, the entire teachings of which are
incorporated herein by reference). Still further, an antibody or
antigen-binding portion thereof may be part of a larger
immunoadhesion molecule, formed by covalent or non-covalent
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, the entire teaching of which is
incorporated herein by reference) 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, the entire teaching of which is
incorporated herein by reference). Antibody portions, such as Fab
and F(ab')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. In one
aspect, the antigen binding portions are complete domains or pairs
of complete domains.
[0175] 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.
[0176] The term "human antibody" includes antibodies having
variable and constant regions corresponding to human germline
immunoglobulin sequences as described by Kabat et al. (See Kabat,
et al. (1991) Sequences of proteins of Immunological Interest,
Fifth Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242). 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),
e.g., in the CDRs and in particular CDR3. The mutations can be
introduced using the "selective mutagenesis approach." The human
antibody can have at least one position replaced with an amino acid
residue, e.g., an activity enhancing amino acid residue which is
not encoded by the human germline immunoglobulin sequence. The
human antibody can have up to twenty positions replaced with amino
acid residues which are not part of the human germline
immunoglobulin sequence. In other embodiments, up to ten, up to
five, up to three or up to two positions are replaced. In one
embodiment, these replacements are within the CDR regions. 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.
[0177] The phrase "recombinant human antibody" includes human
antibodies that are prepared, expressed, created or isolated by
recombinant means, such as antibodies expressed using a recombinant
expression vector transfected into a host cell, antibodies isolated
from a recombinant, combinatorial human antibody library,
antibodies isolated from an animal (e.g., a mouse) that is
transgenic for human immunoglobulin genes (see, e.g., Taylor, L.
D., et al. (1992) Nucl. Acids Res. 20:6287-6295, the entire
teaching of which is incorporated herein by reference) 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 (see, 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). 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. In certain embodiments, however, such recombinant
antibodies are the result of selective mutagenesis approach or
back-mutation or both.
[0178] An "isolated antibody" includes an antibody that is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated antibody that specifically binds
hTNF.alpha. is substantially free of antibodies that specifically
bind antigens other than hTNF.alpha.). An isolated antibody that
specifically binds hTNF.alpha. may bind TNF.alpha. molecules from
other species. Moreover, an isolated antibody may be substantially
free of other cellular material and/or chemicals. A suitable
anti-TNF.alpha. antibody is Adalimumab (Abbott Laboratories).
[0179] As used herein, the term "adalimumab", also known by its
trade name Humira.RTM. (AbbVie) refers to a human IgG antibody that
binds the human form of tumor necrosis factor 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). Weak cation-exchange
chromatography (WCX) analysis of the antibody has shown that it has
three main charged-variants (i.e. Lys 0, Lys 1, and Lys 2). These
variants, or charged isomers, are the result of incomplete
posttranslational cleavage of the C-terminal lysine residues. In
addition to the lysine variants, the WCX-10 analysis measures the
presence acidic species. These acidic regions (i.e., acidic
species) are classified as product-related impurities that are
relatively acidic when compared to the lysine variants and elute
before the Lys 0 peak in the chromatogram (FIG. 1).
[0180] The term "activity" includes activities such as the binding
specificity/affinity of an antibody for an antigen, and includes
activities such as the binding specificity/affinity of an
anti-TNF.alpha. antibody for its antigen, e.g., an anti-TNF.alpha.
antibody that binds to a TNF.alpha. antigen and/or the neutralizing
potency of an antibody, e.g., an anti-TNF.alpha. antibody whose
binding to hTNF.alpha. inhibits the biological activity of
hTNF.alpha..
[0181] The phrase "nucleic acid molecule" includes DNA molecules
and RNA molecules. A nucleic acid molecule may be single-stranded
or double-stranded, but in one aspect is double-stranded DNA.
[0182] The phrase "isolated nucleic acid molecule," as used herein
in reference to nucleic acids encoding antibodies or antibody
portions (e.g., VH, VL, CDR3), e.g. those that bind hTNF.alpha.,
and includes a nucleic acid molecule in which the nucleotide
sequences encoding the antibody or antibody portion are free of
other nucleotide sequences encoding antibodies or antibody portions
that bind antigens other than hTNF.alpha., which other sequences
may naturally flank the nucleic acid in human genomic DNA. Thus,
e.g., an isolated nucleic acid of the invention encoding a VH
region of an anti-TNF.alpha. antibody contains no other sequences
encoding other VH regions that bind antigens other than, for
example, hTNF.alpha.. The phrase "isolated nucleic acid molecule"
is also intended to include sequences encoding bivalent, bispecific
antibodies, such as diabodies in which VH and VL regions contain no
other sequences other than the sequences of the diabody.
[0183] The phrase "recombinant host cell" (or simply "host cell")
includes a cell into which a recombinant expression vector 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.
[0184] As used herein, the term "recombinant protein" refers to a
protein produced as the result of the transcription and translation
of a gene carried on a recombinant expression vector that has been
introduced into a host cell. In certain embodiments the recombinant
protein is an antibody, preferably a chimeric, humanized, or fully
human antibody. In certain embodiments the recombinant protein is
an antibody of an isotype selected from group consisting of: IgG
(e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In
certain embodiments the antibody molecule is a full-length antibody
(e.g., an IgG1 or IgG4 immunoglobulin) or alternatively the
antibody can be a fragment (e.g., an Fc fragment or a Fab
fragment).
[0185] As used herein, the term "cell culture" refers to methods
and techniques employed to generate and maintain 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 high level
expression of the relevant nucleotide coding sequences, and the
collection and purification of the desired recombinant protein.
Mammalian cells are preferred for expression and production of the
recombinant protein of the present invention, however other
eukaryotic cell types can also be employed in the context of the
instant invention. See, e.g., Winnacker, From Genes to Clones, VCH
Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells for
expressing recombinant proteins according to 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. 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.
[0186] 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, by 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, which can then be subjected to one or more
additional purification techniques, including but not limited to
affinity chromatography, including protein A affinity
chromatography, ion exchange chromatography, such as anion or
cation exchange chromatography, and hydrophobic interaction
chromatography.
[0187] As used herein the term "on-line" refers to processes that
are accomplished in the context of an on-going cell culture run.
For example, the administration of a particular nutrient or changes
in temperature, pH, or dissolved oxygen level occur on-line when
such administrations or changes are implemented in an existing cell
culture run. Similarly, measurements of certain data are considered
on-line if that data is being collected in the context of a
particular cell culture run. For example, on-line gas analysis
refers to the measurement of gases introduced into or released from
a particular cell culture run. In contrast, the term "off-line", as
used herein, refers to actions taken outside the context of a
particular cell culture run. For example, the production of cell
culture media comprising specific concentrations of particular
components is an example of an off-line activity.
[0188] The term "modifying", as used herein, is intended to refer
to changing one or more amino acids in the antibodies or
antigen-binding portions 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 known techniques, such
as PCR mutagenesis.
[0189] The term "about", as used herein, is intended to refer to
ranges of approximately 10-20% greater than or less than the
referenced value. In certain circumstances, one of skill in the art
will recognize that, due to the nature of the referenced value, the
term "about" can mean more or less than a 10-20% deviation from
that value.
[0190] The term "control", as used herein, is intended to refer to
both limitation as well as to modulation. For example, in certain
embodiments, the instant invention provides methods for controlling
diversity that decrease the diversity of certain characteristics of
protein populations, including, but not limited to, the presence of
acidic species. Such decreases in diversity can occur by: (1)
promotion of a desired characteristic; (2) inhibition of an
unwanted characteristic; or (3) a combination of the foregoing. As
used herein, the term "control" also embraces contexts where
heterogeneity is modulated, i.e., shifted, from one diverse
population to a second population of equal, or even greater
diversity, where the second population exhibits a distinct profile
of the characteristic of interest.
5.2 Antibody Generation
[0191] The term "antibody" as used in this section refers to an
intact antibody or an antigen binding fragment thereof.
[0192] The antibodies of the present disclosure 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. Although somatic cell hybridization procedures are preferred,
in principle, other techniques for producing monoclonal antibody
can be employed e.g., viral or oncogenic transformation of B
lymphocytes.
[0193] One preferred 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.
[0194] An antibody preferably can be a human, a chimeric, or a
humanized antibody. Chimeric or humanized antibodies of the present
disclosure 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.).
[0195] In one non-limiting embodiment, the antibodies of this
disclosure 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).
[0196] 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.
[0197] Recombinant human antibodies 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.
[0198] 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.
[0199] Human monoclonal antibodies of this disclosure 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.
[0200] In certain embodiments, the methods of the invention include
anti-TNF.alpha. antibodies and antibody portions,
anti-TNF.alpha.-related antibodies and antibody portions, and human
antibodies and antibody portions 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 invention provides treatment with 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 purified according to the
invention competitively inhibits binding of Adalimumab to
TNF.alpha. under physiological conditions.
[0201] Antibodies or fragments thereof, 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 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.
5.3 Protein Production
[0202] To express a protein 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
in the case of antibodies, 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. 6,914,128, 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 comprising 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 no limited to,
antibody constant region sequences. For example, one approach to
converting the anti-TNF.alpha. antibody or anti-TNF.alpha.
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).
[0203] 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.
[0204] 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).
[0205] An antibody, or antibody portion, 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.
[0206] 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).
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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. No. 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, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0213] 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 antibody, 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.
[0214] 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.
[0215] 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.
[0216] 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 acidic species, and
are therefore amenable to the instant invention's methods for
control of acidic species heterogeneity. For example, weak
cation-exchange chromatography (WCX) analysis of adalimumab has
shown the presence of acidic regions. These acidic species are
classified as product-related impurities that are relatively acidic
when compared to the adalimumab protein population. The presence of
these acidic species provides an exemplary system to identify those
cell culture conditions that allow for control over acidic species
heterogeneity.
[0217] 5.3.1 Adjusting Amino Acid Concentration to Control Acidic
Species
[0218] The variation in raw materials used in cell culture,
particularly in the context of media preparation, can vary product
quality significantly.
[0219] In certain embodiments of the instant invention, control of
acidic species heterogeneity can be attained by adjustment of the
media composition of the cell culture run. In certain embodiments,
such adjustment will be to increase the amount of one or more amino
acids in the media, while in other embodiments the necessary
adjustment to achieve the desired control over acidic species
heterogeneity will involve a decrease in the amount of one or more
amino acids in the media. 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.
[0220] In certain embodiments, a cell culture media will include
one or more of the amino acids, or other compositions, described
herein as facilitating a reduction in acidic species. In certain
embodiments the amount of the amino acid, or other composition,
that is necessary to be supplemented may be adjusted to account for
the amount present in the media prior to supplementation.
[0221] 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 0.25
g/L, or 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.
[0222] 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 acidic species heterogeneity 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.
[0223] In certain embodiments, the one or more amino acids used to
supplement the cell culture media is a basic amino acid. In certain
embodiments the one or more amino acids is arginine, lysine,
histidine, ornithine, or certain combinations of arginine or lysine
with ornithine or of all four amino acids. 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. In certain embodiments, the amount of amino
acid supplemented to the cell culture to achieve concentrations of
about 0 to about 9 g/l for arginine, about 0 to about 11 g/l for
lysine, about 0 to about 11 g/l histidine, and about 0 to about 11
g/l ornithine. Although wider ranges are also within the scope of
the instant invention, including, but not limited to: about 0 to
about 30 g/l for arginine, about 0 to about 30 g/l for lysine,
about 0 to about 30 g/l histidine, and about 0 to about 30 g/l
ornithine.
[0224] For example, and not by way of limitation, as detailed in
Example 6.1, below, when the production medium employed in the
example was supplemented with arginine to achieve a total
concentration of 9 g/L arginine, the total amount of acidic species
of adalimumab present in a cell culture sample after purification
was reduced from 19.7% of a control sample to 12.2% of the sample
purified from the cells cultured with the arginine supplemented
media. Similarly, when the production medium employed in the
example was supplemented with lysine, or histidine, or ornithine to
achieve total concentrations of 11 g/L lysine, 10 g/L ornithine or
10 g/L histidine, respectively, the total amount of acidic species
of adalimumab present in a cell culture sample after purification
was reduced by 11.5%, 10.4% and 10.9%, respectively, compared to a
control sample.
[0225] In certain embodiments, control over the amount of acidic
species of protein produced by cell culture is exerted by
supplementing the media of cells expressing the protein of interest
medium supplements described herein 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 feed amounts may be
calculated to achieve a certain concentration based on offline or
online measurements. The supplements may be added as multimers,
e.g., arg-arg, his-his, arg-his-orn, etc., and/or as chemical
variants, e.g., 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. The addition of one or more supplement may be based on
measured amount of acidic species. The resulting media can be used
in various 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. In
addition, the harvest criterion for these cultures may be chosen,
for example based on choice of harvest viability or culture
duration, to further optimize a certain targeted acidic species
profile.
[0226] 5.3.1 Adjusting CaCl.sub.2 and/or Niacinamide Concentration
to Control Acidic Species
[0227] In certain embodiments, the cell culture media is
supplemented with calcium (e.g., as calcium chloride dihydrate),
wherein the calcium is supplemented to achieve a calcium
concentration of between about 0.05 and 2.5 mM, or between about
0.05 and 1 mM, or between about 0.1 and 0.8 mM, or between about
0.15 and 0.7 mM, or between about 0.2 and 0.6 mM, or between about
0.25 and 0.5 mM, or between about 0.3 and 0.4 mM.
[0228] In certain embodiments, the cell culture media is
supplemented with calcium (e.g., as calcium chloride dihydrate)
wherein the calcium is supplemented in an amount effective to
reduce the amount of acidic species heterogeneity 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.
[0229] For example, and not by way of limitation, as detailed in
Example 6.3, below, when the production medium employed in the
example was supplemented with calcium (e.g., as calcium chloride
dihydrate) at a concentration of 1.05 mM, the total amount of
acidic species of adalimumab present in a cell culture sample after
purification was reduced from 23.2% of a control sample to 16.5% of
the sample purified from the cells cultured with the calcium
supplemented media.
[0230] In certain embodiments, the cell culture can be supplemented
with a combination of calcium, e.g., CaCl.sub.2, and one or more a
basic amino acids. In certain embodiments the one or more basic
amino acids is arginine, lysine, histidine, ornithine, or
combinations of arginine or lysine with ornithine or of all four
amino acids. 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. In certain embodiments, the amount of basic amino acid
concentrations in combination with calcium in the cell culture is
between about 0 to about 9 g/l for arginine, about 0 to about 11
g/l for lysine, about 0 to about 11 g/l histidine, and about 0 to
about 11 g/l ornithine. Although wider ranges are also within the
scope of the instant invention, including, but not limited to:
about 0 to about 30 g/l for arginine, about 0 to about 30 g/l for
lysine, about 0 to about 30 g/l histidine, and about 0 to about 30
g/l ornithine.
[0231] In certain embodiments, the cell culture media is
supplemented with niacinamide, wherein the niacinamide is
supplemented to achieve a niacinamide concentration of between
about 0.2 and 3.0 mM, or between about 0.4 and 3.0 mM, or between
about 0.8 and 3.0 mM.
[0232] In certain embodiments, the cell culture media is
supplemented with niacinamide wherein the niacinamide is
supplemented in an amount effective to reduce the amount of acidic
species heterogeneity 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.
[0233] For example, and not by way of limitation, as detailed in
Example 6.3, below, when the production medium employed in the
example was supplemented with niacinamide at a concentration of 1.6
mM, the total amount of acidic species of adalimumab present in a
cell culture sample after purification was reduced from 19.9% of a
control sample to 15.9% of the sample purified from the cells
cultured with the niacinamide supplemented media. In a separate
example, where the media was supplemented with 3 mM niacinamide,
the total amount of acidic species of adalimumab present in a cell
culture sample after purification was reduced from 27.0% of a
control sample to 19.8% of the sample purified from the cells
cultured with the niacinamide supplemented media.
[0234] In certain embodiments, the cell culture can be supplemented
with a combination of niacinamide, calcium, e.g., CaCl.sub.2,
and/or one or more a basic amino acids. In certain embodiments the
one or more basic amino acids is arginine, lysine, histidine,
ornithine, or combinations of arginine or lysine with ornithine or
of all four amino acids. 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. In certain embodiments, the amount of basic amino acid
concentrations (after supplementation) in combination with calcium
in the cell culture is between about 0 to about 9 g/l for arginine,
about 0 to about 11 g/l for lysine, about 0 to about 11 g/l
histidine, and about 0 to about 11 g/l ornithine. Although wider
ranges are also within the scope of the instant invention,
including, but not limited to: about 0 to about 30 g/l for
arginine, about 0 to about 30 g/l for lysine, about 0 to about 30
g/l histidine, and about 0 to about 30 g/l ornithine.
[0235] In certain embodiments, control over the amount of acidic
species of protein produced by cell culture is exerted by
supplementing the media of cells expressing the protein of interest
medium supplements described herein 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 feed amounts may be
calculated to achieve a certain concentration based on offline or
online measurements. The addition of the supplement may be based on
measured amount of acidic species. Other salts of particular
supplements, e.g., calcium, may also be used, for example Calcium
Nitrate. The resulting media can be used in various 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.
[0236] In certain embodiments, control over amount and/or rate of
formation of acidic species is achieved by supplementing a
clarified harvest. For example, but not by way of limitation, such
clarified harvests can be supplemented as described above (e.g.,
with calcium, niacinamide, and/or basic amino acids) to achieve a
reduction the amount of acidic species and/or a reduction in the
rate such acidic species form.
[0237] 5.3.3 Adjusting Process Parameters to Control Acidic
Species
[0238] In certain embodiments of the instant invention, control of
acidic species heterogeneity can be attained by adjustment of pH of
the cell culture run. In certain embodiments, such adjustment will
be to decrease in the pH of the cell culture. Such decreases in the
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
amount.
[0239] In certain embodiments, pH is either increased or decreased
in order to increase or decrease the amount of acidic species
and/or the rate at which such acidic species form. 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 decrease the acidic species during
cell culture and the rate of acidic species formation in the
context of a clarified harvest.
[0240] In certain embodiments, the pH is maintained in such a
manner as to reduce the amount of acidic species 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.
[0241] In certain embodiments, control over the amount of acidic
species of protein produced by cell culture can be exerted by
maintaining the pH of the cell culture expressing the protein of
interest as described herein along with choice of suitable
temperature or temperature shift strategies, for example, but not
limited to, lower process temperature of operation, temperature
shift to a lower temperature or a temperature shift at an earlier
culture time point. These culture conditions can be used in various
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. These may
also be used in combination with supplementation of culture media
with amino acids, niacinamide, and/or calcium salt, as described
above.
[0242] 5.3.4 Continuous/Perfusion Cell Culture Technology to
Control Acidic Species
[0243] In certain embodiments of the instant invention, control of
acidic species heterogeneity can be attained by the choice of cell
culture methodology. In certain embodiments, use of a continuous or
perfusion technology may be utilized to achieve the desired control
over acidic species heterogeneity. In certain embodiments, this may
be attained through choice of medium exchange rate (where the
exchange rate is the rate of exchange of medium in/out of a
reactor). Such increases or decreases in medium exchange rates may
be of 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.
[0244] In certain, non-limiting, embodiments, maintenance of the
medium exchange rates (working volumes/day) of a cell culture run
between 0 and 20, or between 0.5 and 12 or between 1 and 8 or
between 1.5 and 6 can be used to achieve the desired reduction in
acidic species.
[0245] For example, and not by way of limitation, as detailed in
Example 6.4, below, when the medium exchange rate was chosen to be
1.5, the acidic species was 8.1%. With further increase in exchange
rates to 6, a further reduction in acidic species to 6% was
obtained.
[0246] In certain embodiments, the choice of cell culture
methodology that allows for control of acidic species heterogeneity
can also include, for example, but not by way of limitation,
employment of an intermittent harvest strategy or through use of
cell retention device technology.
5.4 Protein Purification
[0247] 5.4.1 Protein Purification Generally
[0248] In certain embodiments, the methods 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.
[0249] 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 can be
performed 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. The separation steps
separate mixtures of proteins on the basis of their charge, degree
of hydrophobicity, or size. In one aspect of the invention,
separation is performed using chromatography, including cationic,
anionic, and hydrophobic interaction. Several different
chromatography resins are 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 down a column, achieving a physical separation that
increases as they pass further down the column, or to adhere
selectively to the separation medium, being then differentially
eluted by different solvents. In some cases, the antibody is
separated from impurities when the impurities specifically adhere
to the column and the antibody does not, i.e., the antibody is
present in the flow through.
[0250] As noted above, accurate tailoring of a purification scheme
relies on consideration of the protein to be purified. In certain
embodiments, the separation steps of 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
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, in certain
embodiments, the use of Protein A affinity chromatography would
prove useful, for example in the context of the purification of
IgG3 antibodies, as 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.
[0251] 5.4.2 Primary Recovery and Virus Inactivation
[0252] 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 teaching
of which is incorporated herein by reference.
[0253] 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.
[0254] 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.
[0255] 5.4.3 Affinity Chromatography
[0256] In certain embodiments, it will be advantageous to subject a
sample produced by the techniques 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.
[0257] 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..
[0258] 5.4.4 Ion Exchange Chromatography
[0259] In certain embodiments, it will be advantageous to subject a
sample produced by the techniques 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.
[0260] 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.
[0261] 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 Flow
are 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.
[0262] 5.4.5 Ultrafiltration/Diafiltration
[0263] In certain embodiments, it will be advantageous to subject a
sample produced by the techniques 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, Munir Cheryan
(Technomic Publishing, 1986; ISBN No. 87762-456-9). A preferred
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.
[0264] 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.
[0265] 5.4.6 Hydrophobic Interaction Chromatography
[0266] In certain embodiments, it will be advantageous to subject a
sample produced by the techniques 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.
[0267] 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.
[0268] 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).
[0269] 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
PO.sup.---; SO.sub.4.sup.--; CH.sub.3CO.sub.3.sup.-; Cl.sup.-;
Br.sup.-; NO.sub.3.sup.-; ClO.sub.4.sup.-; I.sup.-; SCN.sup.-.
[0270] 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.
[0271] 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).
[0272] 5.4.7 Multimodal Chromatography
[0273] In certain embodiments, it will be advantageous to subject a
sample produced by the techniques 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.
[0274] 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.
[0275] 5.5 Pharmaceutical Compositions
[0276] The proteins, for example, antibodies and antibody-portions,
produced using the cell culture techniques of the instant invention
can be incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises a protein of the invention and a
pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it is desirable to include
isotonic agents, e.g., sugars, polyalcohols such as mannitol,
sorbitol, or sodium chloride in the composition. Pharmaceutically
acceptable carriers may further comprise minor amounts of auxiliary
substances such as wetting or emulsifying agents, preservatives or
buffers, which enhance the shelf life or effectiveness of the
antibody or antibody portion.
[0277] The protein compositions of the invention can be
incorporated into a pharmaceutical composition suitable for
parenteral administration. The protein can be prepared as an
injectable solution containing, e.g., 0.1-250 mg/mL antibody. The
injectable solution can be composed of either a liquid or
lyophilized dosage form in a flint or amber vial, ampule or
pre-filled syringe. The buffer can be L-histidine approximately
1-50 mM, (optimally 5-10 mM), at pH 5.0 to 7.0 (optimally pH 6.0).
Other suitable buffers include but are not limited to sodium
succinate, sodium citrate, sodium phosphate or potassium phosphate.
Sodium chloride can be used to modify the toxicity of the solution
at a concentration of 0-300 mM (optimally 150 mM for a liquid
dosage form). Cryoprotectants can be included for a lyophilized
dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other
suitable cryoprotectants include trehalose and lactose. Bulking
agents can be included for a lyophilized dosage form, principally
1-10% mannitol (optimally 24%). Stabilizers can be used in both
liquid and lyophilized dosage forms, principally 1-50 mM
L-methionine (optimally 5-10 mM). Other suitable bulking agents
include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ
surfactants.
[0278] In one aspect, the pharmaceutical composition 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.
[0279] The compositions of this invention may be in a variety of
forms. These include, e.g., liquid, semi-solid and solid dosage
forms, such as liquid solutions (e.g., injectable and infusible
solutions), dispersions or suspensions, tablets, pills, powders,
liposomes and suppositories. The form depends on, e.g., the
intended mode of administration and therapeutic application.
Typical compositions are in the form of injectable or infusible
solutions, such as compositions similar to those used for passive
immunization of humans with other antibodies. One mode of
administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular). In one aspect, the protein is
administered by intravenous infusion or injection. In another
aspect, the protein is administered by intramuscular or
subcutaneous injection.
[0280] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., protein, antibody or
antibody portion) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile, lyophilized powders for the preparation of sterile
injectable solutions, the methods of preparation are vacuum drying
and spray-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, e.g., by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, e.g.,
monostearate salts and gelatin.
[0281] The protein of the present invention can be administered by
a variety of methods known in the art, one route/mode of
administration is subcutaneous injection, intravenous injection or
infusion. As will be appreciated by the skilled artisan, the route
and/or mode of administration will vary depending upon the desired
results. In certain embodiments, the active compound may be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978, the entire teaching of which
is incorporated herein by reference.
[0282] In certain aspects, a protein of the invention may be orally
administered, e.g., with an inert diluent or an assimilable edible
carrier. The compound (and other ingredients, if desired) may also
be enclosed in a hard or soft shell gelatin capsule, compressed
into tablets, or incorporated directly into the subject's diet. For
oral therapeutic administration, the compounds may be incorporated
with excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. To administer a compound of the invention by other
than parenteral administration, it may be necessary to coat the
compound with, or co-administer the compound with, a material to
prevent its inactivation.
[0283] Supplementary active compounds can also be incorporated into
the compositions. In certain aspects, a protein of the invention is
co-formulated with and/or co-administered with one or more
additional therapeutic agents that are useful for treating
disorders. For example, an antibody or antibody portion of the
invention may be co-formulated and/or co-administered with one or
more additional antibodies that bind other targets (e.g.,
antibodies that bind other cytokines or that bind cell surface
molecules). Furthermore, one or more antibodies of the invention
may be used in combination with two or more of the foregoing
therapeutic agents. Such combination therapies may advantageously
utilize lower dosages of the administered therapeutic agents, thus
avoiding possible toxicities or complications associated with the
various monotherapies. It will be appreciated by the skilled
practitioner that when the protein of the invention are used as
part of a combination therapy, a lower dosage of protein may be
desirable than when the protein alone is administered to a subject
(e.g., a synergistic therapeutic effect may be achieved through the
use of combination therapy which, in turn, permits use of a lower
dose of the protein to achieve the desired therapeutic effect).
[0284] It should be understood that the protein of the invention
can be used alone or in combination with an additional agent, e.g.,
a therapeutic agent, said additional agent being selected by the
skilled artisan for its intended purpose. For example, the
additional agent can be a therapeutic agent art-recognized as being
useful to treat the disease or condition being treated by the
protein of the present invention. The additional agent also can be
an agent which imparts a beneficial attribute to the therapeutic
composition, e.g., an agent which effects the viscosity of the
composition.
[0285] 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.
[0286] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a protein of the invention is
0.01-20 mg/kg, or 1-10 mg/kg, or 0.3-1 mg/kg. 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.
6. EXAMPLES
6.1 Method for Reducing the Extent of Acidic Species in Cell
Culture by the Addition of Medium Components
[0287] Production of recombinant proteins by host cells can result
in product-related charge heterogeneities present in the population
of proteins produced by the cells. The presence of acidic species
in the population of proteins is an example of a product-related
charge heterogeneity. Control of the amount of acidic species
present in the population of proteins produced by the host cells
can be accomplished by modifying the culture conditions of the host
cells.
[0288] 6.1.1 Materials and Methods
[0289] Cell Source and Adaptation Cultures
[0290] Three adalimumab producing cell lines, one mAb1 producing
cell line and one mAb2 producing were employed in the studies
covered here. 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.
[0291] 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.
[0292] 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.
[0293] Cell Culture Media
[0294] 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, a proprietary basal media
formulation from Invitrogen) 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, both growth and
production medium were also supplemented with insulin. For mAB1 and
mAB2 producing cell lines, the growth medium were also supplemented
with insulin.
[0295] For the hydrolysate-based formulation (media 2), the growth
media was composed of PFCHO (proprietary chemically defined
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.
[0296] 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.
[0297] 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 unsupplemented (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.
[0298] Calcium Chloride Dihydrate (Sigma or Fluka) used for the
experiments were reconstituted in Milli-Q water to make a stock
solution, which was subsequently supplemented to the production
basal media. After addition of calcium chloride, media was brought
to a pH similar to non-supplemented (control) media using 6N
hydrochloric acid/5N NaOH, and it was brought to an osmolality
similar to non-supplemented (control) media by adjusting the
concentration of sodium chloride.
[0299] Niacinamide (Sigma or Calbiochem) used for the experiments
were reconstituted in Milli-Q water to make a stock solution, which
was subsequently supplemented to the production basal media. After
addition of niacinamide, media was brought to a pH similar to
non-supplemented (control) media using 6N hydrochloric acid/5N
NaOH, and it was brought to an osmolality similar to
non-supplemented (control) media by adjusting the concentration of
sodium chloride.
[0300] 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 2 List of medium additives supplemented to
culture media Catalog No./Source Medium of medium additive
supplements Arginine Sigma, A8094 Lysine Calbiochem, 4400 Histidine
Sigma, H5659 Ornithine Sigma, 06503 Calcium Fulka, 21097 Chloride
Sigma, C8106 Niacinamide Calbiochem, 481907 Sigma, N0636
[0301] Production Cultures
[0302] 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, for 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 35.degree. C., 30% 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 1:5.
[0303] Cultures were run in either batch or fed-batch mode. In the
batch mode, cells were cultured in the respective production
medium. 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
(proprietary chemically defined feed formulation from Invitrogen)
as per the following feed schedule--(4% (v/v)--day 6, day 7, and
day 8, respectively) along with 10.times. Ex-Cell PFCHO feed
(proprietary chemically defined formulation)--3% (v/v) on day 3.
The cultures were also fed with 1.25% (v/v) of 40% glucose stock
solution when the glucose concentration was below 3.0 g/l.
[0304] Retention samples for titer analysis, of 2.times.1.5 mL,
were collected daily for the bioreactor experiments (section 2.2.4)
beginning on Day 8, and frozen at -80.degree. C. The samples taken
from each were later submitted for titer analysis.
[0305] 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.
[0306] WCX-10 Assay
[0307] This method is employed towards the quantification of the
acidic species and other charge variants present in cell culture
harvest samples. Cation exchange chromatography was performed on a
Dionex ProPac WCX-10, Analytical column (Dionex, CA).
[0308] For 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 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.
[0309] 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 Chloride 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.
[0310] Quantitation is based on the relative area percent of
detected peaks. The peaks that elute at relative residence time
earlier than the main peak corresponding to the drug product are
together represented as the acidic peaks (FIG. 1).
[0311] Lysine-C Peptide Mapping for MGO Quantification
[0312] Typical trypsin digestion employed almost universally for
peptide mapping cuts a denatured, reduced and alkylated protein at
the carboxyl side of the two basic amino acids, lysine and
arginine. Methylglyoxal is a small molecule metabolite derived as a
glycolysis byproduct which can modify arginine residues. A
modification of an arginine prevents trypsin from cutting this site
and results in a mis-cleavage. The challenge of quantifying the
amount of MGO modified peptide is that it is not compared to an
equivalent non-modified peptide but rather two parental cleaved
peptides which will likely have different ionization potential than
the modified peptide. In order to determine a truly accurate direct
measurement of an MGO-modified peptide, it must be compared to its
non-modified counterpart and expressed as a percent. Using
endoproteinase Lysine-C as an alternative enzyme, cleavages only
occur at lysine residues. The result is a direct comparison of the
same peptide with and without an MGO modification which provides a
high degree of accuracy in quantifying even trace levels of the
modified species.
[0313] Procedure: Samples are diluted to a nominal concentration of
4 mg/mL. 8 M guanidine-HCl is added to the sample in a 3:1 ratio
resulting in a 1 mg/mL concentration in 6M guanidine-HCl. The
samples are reduced with 10 mM final conc. DTT for 30 minutes at
37.degree. C. followed by an alkylation with 25 mM final conc.
iodoacetic acid for 30 minutes at 37.degree. C. in the dark. The
samples are then buffer exchanged into 10 mM Tris pH 8.0 using
NAP-5 columns. The samples are then digested for 4 hours at
37.degree. C. using endoproteinase Lys-C at an enzyme to protein
ratio of 1:20. The digest is quenched by adding 5 .mu.L of formic
acid to each sample. Samples are analyzed by LC/MS peptide mapping.
Briefly, 50 .mu.L of sample is loaded onto a Waters BEH C18 1.7.mu.
1.0.times.150 mm UPLC column with 98% 0.08% formic acid, 0.02% TFA
in water and 2% 0.08% formic acid, 0.02% TFA in acetonitrile. The
composition is changed to 65% 0.08% formic acid, 0.02% TFA in water
and 35% 0.08% formic acid, 0.02% TFA in acetonitrile in 135 minutes
using a Waters Acquity UPLC system. Eluting peaks are monitored
using a Thermo Scientific LTQ-Orbitrap Mass Spectrometer. Specific
mass traces are extracted for both modified and non-modified
peptides in order to accurately quantify the total amount of MGO
modification at each site. Mass spectra are also analyzed for the
specific region of the chromatogram to confirm the peptide
identity. An example data set is shown in FIG. 140.
[0314] 6.1.2 Results
[0315] Effect of Arginine Supplementation to Cell Culture Media
[0316] The addition of arginine was tested in several experimental
systems covering multiple cell lines, media and monoclonal
antibodies. Following is a detailed description of two
representative experiments where two different adalimumab producing
cell lines were cultured in a chemically defined media (media
1).
[0317] Cell line 2 was cultured in media 1 with different total
amounts 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 test conditions, although a slight decrease in viable
cell density profile was observed in samples with the 9 g/l
arginine test condition (FIGS. 1, 2). The harvest titers were
comparable between the conditions (FIG. 3). 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 post protein
A purification and the percentages of total peak(s) area
corresponding to the acidic species were quantified (FIG. 4, 5).
The percentage of acidic species in the control sample was as high
as 19.7% on day 10. In the sample with the highest total
concentration of arginine in this experiment (9 g/l), the
percentage of acidic species was reduced to 12.2%. A dose dependent
decrease in acidic species was observed in test conditions with
arginine concentrations beyond 2 g/l (FIG. 4). A similar trend in
reduction of acidic species with arginine increase was also
observed in the day 12 harvest samples (FIG. 5). Further, while the
extent of acidic species in the 1 g/l arginine samples increased
from 19.7% (day 10 harvest) to 25.5% (day 12 harvest), this
increase in the 9 g/l arginine test condition was significantly
smaller from 12.2% (day 10 harvest) to 13.9% (day 12 harvest).
Thus, the increase of total arginine led to a reduction in the
extent of total acidic species at a particular time point in
culture as well the rate of increase of acidic species with time of
culture.
[0318] Cell line 3 was cultured in media 1 with different total
amounts 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. 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. 6, 7). The product titer was
also comparable between all conditions (FIG. 8). 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 acidic species were quantified (FIG. 9). The
percentage of acidic species in the control sample was as high as
23.3% on day 10. In the sample with the highest total concentration
of arginine in this experiment (9 g/l), the percentage of acidic
species was reduced to 17.0%. A dose dependent decrease in acidic
species was observed in conditions with higher concentrations of
arginine.
[0319] 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 summarized in FIGS. 10,
11, 12. A reduction in acidic species with increased arginine
concentration was also observed in each case.
[0320] In addition to adalimumab, the utility of this method for
acidic species reduction was also demonstrated for processes
involving two other mAB producing cell lines. The experimental
setup for each of these experiments was similar to that described
in section above and in the materials and methods. The reduction of
acidic species with increased arginine concentration for
experiments corresponding to each mAB is summarized in FIG. 13, 14.
For mAB2, a significant reduction in acidic species was only
observed at arginine concentration of 9 g/l.
[0321] In U.S. patent application Ser. No. 13/830,976 we describe
the utility of arginine supplementation to culture media towards
modulation of the lysine variant distribution. It is possible that
a fraction of acidic species also shifted along with shift in
lysine variants (from Lys 0 to Lys1 and Lys2), in addition to the
fraction of acidic species that is completely removed from the
entire protein population. To estimate the acidic species reduction
that is independent of this redistribution of lysine variants,
protein A eluate samples from a representative set of arginine
supplementation experiments were pre-treated with the enzyme
carboxypeptidase before WCX-10. One set of samples from adalimumab
experiment and another set of samples from a mAB2 experiment were
used for this analysis. The carboxypeptidase treatment of the
samples resulted in the cleavage of the C-terminal lysine residues
as demonstrated by the complete conversion of Lys1/Lys2 to Lys 0 in
each of these samples (data not shown here). As a result of this
conversion, the acidic species quantified in these samples
corresponded to an aggregate sum of acidic species that would be
expected to also include those species that may have previously
shifted corresponding to the lysine variant shift and perhaps gone
unaccounted for in the samples that were not treated with
carboxypeptidase prior to WCX-10. A dose dependent reduction in
acidic species was observed in the carboxypeptidase treated samples
with increasing concentration arginine (FIG. 15, 16). This suggests
that the acidic species reduction described here is not completely
attributed to a probable shift of the acidic species corresponding
to the lysine variant redistribution.
[0322] Effect of Lysine Supplementation to Cell Culture Media
[0323] The addition of lysine was tested in several experimental
systems covering multiple cell lines, media and monoclonal
antibodies. Following is a detailed description of two
representative experiments where two different cell lines were
cultured in a chemically defined media (media 1) for the production
of adalimumab.
[0324] Cell line 2 was cultured in media 1 with different total
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. 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 samples with respect to the control sample (FIG.
17). The viability profiles were comparable between the conditions
(FIG. 18). On Days 10 and 11 of culture samples were collected for
titer analysis (FIG. 19). The titers for all conditions were
comparable. 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 acidic species were
quantified (FIG. 20). The percentage of acidic species in the
control was as high as 26.5%. In the sample with the highest tested
concentration of lysine in this experiment (11 g/l), the percentage
of acidic species was reduced to 15.0%. A dose dependent decrease
in acidic species was observed in test conditions with higher total
concentrations of lysine.
[0325] Cell line 3 was cultured in media 1 with different total
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. 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 test conditions, although a slight decrease in viable
cell density and viability profiles was observed in samples with
higher lysine concentrations than that in the control sample (FIG.
21, 22). On Days 10, 11 and 12 of culture samples were collected
for titer analysis (FIG. 23). The titers for all conditions were
comparable. 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 acidic species were
quantified (FIG. 24). The percentage of acidic species in the
control sample was as high as 26.6%. In the sample with the highest
tested concentration of lysine in this experiment (11 g/l) the
percentage of acidic species was reduced to 18.1%. A dose dependent
decrease in acidic species was observed in test conditions with
higher total concentrations of lysine.
[0326] 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 and in materials and methods section. The
summaries of results of the different experiments performed for
adalimumab are summarized in FIGS. 25, 26, 27. A reduction in
acidic species with increased lysine concentration was also
observed in each case.
[0327] 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 above and in the
materials and methods section. The reduction of acidic species with
lysine addition for experiments corresponding to each mAB is
summarized in FIGS. 28, 29. For mAB2, a significant reduction in
acidic species was only observed at lysine concentration of 11
g/l.
[0328] In U.S. patent application Ser. No. 13/830,976 we describe
the utility of lysine supplementation to culture media towards
modulation of the lysine variant distribution. To estimate the
acidic species reduction that is independent of this redistribution
of lysine variants, protein A eluate samples from a representative
set of lysine supplementation experiments were pre-treated with the
enzyme carboxypeptidase before WCX-10. One set of samples from
adalimumab experiment and another set of samples from a mAB2
experiment were used for this analysis. The carboxypeptidase
treatment of the samples resulted in the cleavage of the C-terminal
lysine residues as demonstrated by the conversion of Lys1/Lys2 to
Lys 0 in each of these samples (data not shown here). As a result
of this conversion, the acidic species quantified in these samples
corresponded to an aggregate sum of acidic species that would be
expected to also include those species that may have previously
shifted corresponding to the lysine variant shift and perhaps gone
unaccounted for in the samples that were not treated with
carboxypeptidase prior to WCX-10. A dose dependent reduction in
acidic species was observed in the carboxypeptidase treated samples
with increasing concentration of lysine for the adalimumab samples
from 26.8% in the non-supplemented sample to 21.1% in the 10 g/l
Lysine supplemented sample, a reduction of 5.7% in total acidic
species (FIG. 30). Similar results were also observed for the mA2
samples (FIG. 31). This suggests that the acidic species reduction
described here is not completely attributed to a probable shift of
the acidic species corresponding to the lysine redistribution.
[0329] Effect of Histidine Supplementation to Cell Culture
Media
[0330] The addition of histidine was tested in several experimental
systems covering multiple cell lines, media and monoclonal
antibodies. Following is a detailed description of two
representative experiments where two different cell lines were
cultured in a chemically defined media (media 1) for the production
of adalimumab.
[0331] Cell line 2 was cultured in media 1 with different total
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. 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 with the 10 g/l histidine condition having significant
reduction in growth (FIG. 32). A corresponding effect on viability
was also observed (FIG. 33). On Days 10, 11 and 12 of culture
samples were collected for titer analysis and reported for the
harvest day for each sample (FIG. 34). There was a small dose
dependent decrease in titers for conditions with histidine
supplementation. On Days 11-12, 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 acidic species were quantified (FIG. 35). The
percentage of acidic species in the control sample was as high as
26.5%. In the sample with the highest tested concentration of
histidine in this experiment (10 g/l), the percentage of acidic
species was reduced to 15.6%. A dose dependent decrease in acidic
species was observed in test conditions with increased histidine
concentrations.
[0332] Cell line 3 was cultured in media 1 with different total
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. 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 conditions with histidine concentrations higher than that in
the control (FIG. 36). The viability profiles were more comparable
between conditions with this cell line (FIG. 37). On Day 12 of
culture, samples were collected for titer analysis (FIG. 38). The
titers for all conditions were comparable. 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 acidic species were quantified (FIG. 39). The
percentage of acidic species in the control sample was 26.2%. In
the sample with the highest tested concentration of histidine in
this experiment (8 g/l), the percentage of acidic species was
reduced to 20.0%. A dose dependent decrease in acidic species was
observed in test conditions with increased histidine
concentration.
[0333] Additional experiments were performed with multiple cell
lines in chemically defined or hydrolysate based media to evaluate
the wide range of applicability of this method. The experimental
setup for each of these experiments was similar to that described
above and in the materials and methods section. The summaries of
results of the different experiments performed for adalimumab are
summarized in FIGS. 40, 41, 42. A reduction in acidic species with
increased histidine concentration was observed with cell line 1 in
media 1 (FIG. 40) and with cell line 2 in media 3 (FIG. 42). For
cell line 2 in media 3, a dose dependent reduction in acidic
species was observed upto 4 g/l histidine, with no further
significant reduction at higher concentrations of histidine (FIG.
42). For cell line 1, media 2, no significant reduction of acidic
species was observed within the histidine concentration range (0-4
g/l) (FIG. 41).
[0334] 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 above and in the
materials and methods section. The reduction of acidic species with
increased histidine concentration for experiments corresponding to
each mAB is summarized in FIGS. 43, 44. For mAB2, in contrast with
the results reported with arginine and lysine supplementation shown
previously, a clear significant dose dependent reduction in total
acidic species from 28.1% in the control to 21.5% in 4 g/l
histidine sample was observed.
[0335] In U.S. patent application Ser. No. 13/830,976 we also
describe the utility of increased histidine to culture media
towards modulation of the lysine variant distribution. To estimate
the acidic species reduction that is independent of this
redistribution of lysine variants, protein A eluate samples from a
representative set of histidine supplementation experiments were
also pre-treated with the enzyme carboxypeptidase before WCX-10.
One set of samples from adalimumab experiment and another set of
samples from a mAB2 experiment were used for this analysis. The
carboxypeptidase treatment of the samples resulted in the cleavage
of the C-terminal lysine residues as demonstrated by the complete
conversion of Lys1/Lys2 to Lys 0 in each of these samples (data not
shown here). A dose dependent reduction in acidic species was
observed in the carboxypeptidase treated samples with increasing
concentration of histidine (FIG. 45, 46). This suggests that the
acidic species reduction described here is not completely
attributed to a probable shift of the acidic species corresponding
to the lysine redistribution.
[0336] Effect of Ornithine Supplementation to Cell Culture
Media
[0337] The addition of ornithine was tested in several experimental
systems covering multiple cell lines, media and monoclonal
antibodies. Following is a detailed description of two
representative experiments where two different cell lines were
employed in a chemically defined media (media 1) for the production
of adalimumab.
[0338] Cell line 2 was cultured in media 1 with different total
concentrations of ornithine (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. The cells
grew to maximum viable cell densities (VCD) in the range of
15-22.times.10.sup.6 cells/ml for the different conditions tested.
A slight decrease in viable cell density with ornithine
supplementation was observed (FIG. 47). Corresponding differences
in the viability profiles were also observed (FIG. 48). On Day 11
of culture, samples were collected for titer analysis (FIG. 49).
The titers for all conditions were comparable. On Day 11, duplicate
shake flasks were harvested for each condition and then
subsequently analyzed using WCX-10 post protein A purification and
the percentages of total peak(s) area corresponding to the acidic
species were quantified (FIG. 50). The percentage of acidic species
in the control sample was 26.5%. In the sample with the highest
tested concentration of ornithine in this experiment (10 g/l), the
percentage of acidic species was reduced to 16.1%. A dose dependent
decrease in acidic species was observed in test conditions with
increased ornithine concentration.
[0339] Cell line 3 was cultured in media 1 supplemented with
different total concentrations of ornithine (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.
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 viable cell density and viability profiles were
comparable (FIG. 51, 52). On Day 12 of culture, samples were
collected for titer analysis (FIG. 53). The titers for all
conditions were comparable. 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 acidic
species were quantified (FIG. 54). The percentage of acidic species
in the control sample was 24.8%. In the sample with the highest
tested concentration of ornithine in this experiment (8 g/l), the
percentage of acidic species was reduced to 20.5%. A dose dependent
decrease in acidic species was observed in test conditions with
increased ornithine concentration.
[0340] Additional experiments were performed with multiple cell
lines in chemically defined or hydrolysate based media to evaluate
the wide range of applicability of this method. The experimental
setup for each of these experiments was similar to that described
above and in the materials and methods section. The summaries of
results of the different experiments performed for adalimumab are
summarized in FIGS. 55, 56 and 57. For cell line 1 in media 1, a
dose dependent reduction was observed (FIG. 55). However, for cell
line 1 in media 2, a hydrolysate media, no significant reduction in
acidic species was observed across the conditions (FIG. 56). For
cell line 2 in media 3, a reduction in acidic species from 22.1% in
the control sample to 18.7% in the 2 g/l ornithine sample with no
further reduction at higher ornithine concentrations was observed
(FIG. 57).
[0341] 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 section above and in
the materials and method section. The reduction of acidic species
with ornithine addition for experiments corresponding to each mAB
is summarized in FIG. 58, 59. In the case of mAB1, a 7.3% dose
dependent reduction in total acidic species was observed within the
concentration range tested. For mAB2, about 2% reduction was
observed in the 1 g/l ornithine concentration sample with minimum
further reduction at higher ornithine concentrations.
[0342] Similar to the analysis conducted with the other amino
acids, protein A eluate samples from a representative set of
ornithine experiments were also pre-treated with the enzyme
carboxypeptidase before WCX-10. One set of samples from adalimumab
experiment and another set of samples from a mAB2 experiment were
used for this analysis. A dose dependent reduction in acidic
species was observed in the carboxypeptidase treated samples with
increasing concentration of ornithine (FIG. 60, 61). The percentage
of acidic species was also comparable between an untreated and a
carboxypeptidase treated sample for a particular concentration of
ornithine. This suggests that the acidic species reduction is
independent of any probable shift of the acidic species that may be
corresponding to any lysine redistribution.
[0343] Effect of Increasing a Combination of Arginine, Lysine,
Histidine, Ornithine to Cell Culture Media
[0344] In this experiment, the combined use of the four amino acids
arginine, lysine, histidine and ornithine for acidic species
reduction 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 a
single amino acid concentration was increased, a further reduction
in total acidic species was observed in conditions where
combinations of amino acids were increased in the media (FIG. 62).
A progressive decrease was observed in total acidic species when
more amino acids were increased in combination. The percentage of
acidic species was reduced from 21.9% in the lowest concentration
sample to 12.3% in the sample with high concentrations of all four
amino acids.
[0345] Control of Acidic Species Through Cell Culture with
Increased Arginine and Lysine and Choice of Harvest Criterion
and/or Modulation of pH
[0346] The increase of the amino acid (arginine, lysine)
concentration in basal media may also be combined with choice of
when to harvest a culture to achieve optimal reduction in total
acidic species. In this example, a study was carried out in 3 L
bioreactors with cell line 1 (producing adalimumab) in media 1. Two
sets of conditions were tested: Control condition (Arginine 1 g/l,
Lysine 1 g/l); Test condition 1 (Arginine 3 g/l, Lysine 5 g/l).
Cell growth, viability and titer profiles were comparable between
the conditions (FIG. 63, 64, 65). A small amount of cell culture
harvests were collected every day from day 4 to day 10 from each of
the reactors and submitted for protein A purification and WCX-10
analysis. The percentage of acidic species in the control condition
increased from 12.1% (on day 4) to 24.6% (on day 10) (FIG. 66). The
percentage of acidic species in the test condition 1 was lower than
that observed in the control condition at each corresponding
culture day. The percentage of acidic species in the test condition
also increased from 8.7% (day 4) to 18.8% (day 10). The rate of
increase in acidic species with culture duration also correlated
with the drop in viability for both conditions, with a sharp
increase on day 8. Thus, along with increasing arginine and lysine
concentrations in culture media, choice of harvest day/harvest
viability can be used in combination to achieve a desired acidic
species reduction.
[0347] The increase of the amino acid (arginine, lysine)
concentration in basal media may be combined with process pH
modulation to achieve further reduction in total acidic species. In
this example, a study was carried out in 3 L bioreactors with cell
line 1 (producing adalimumab) in media 1. Three sets of conditions
were tested in duplicates: Control condition (Arginine (1 g/l),
Lysine (1 g/l), pH 7.1->6.9 in 3 days, pH 6.9 thereafter); Test
condition 1 (Arginine (3 g/l), Lysine (3 g/l), pH 7.1->6.9 in 3
days, pH 6.9 thereafter); Test condition 2 (Arginine (3 g/l),
Lysine (3 g/l), pH 7.1->6.8 in 3 days, pH 6.8 thereafter). In
comparison to the control, a slight decrease in VCD profile and
harvest titer was observed for condition 2 (FIG. 67, 68, 69). The
cultures were harvested when the viability was less than 50% and
the culture harvests were submitted for protein A and WCX-10
analysis. The percentage of acidic species in the control sample
was 19.1%. The percentage of acidic species was reduced to 14.3% in
test condition 1 and to 12.8% in test condition 2 (FIG. 70). Thus,
this demonstrates that the increase of amino acid concentration
along with choice of lower final process pH can be used in
combination for further reducing the extent of acidic species.
[0348] Effect of Supplementation of CaCl.sub.2 to Cell Culture
Media
[0349] The addition of calcium chloride was tested in several
experimental systems covering multiple cell lines, media and
monoclonal antibodies. Following is a detailed description of two
representative experiments where two different cell lines were
cultured in a chemically defined media (media 1) for the production
of adalimumab.
[0350] Cell line 2 was cultured in media 1 with different
concentrations of calcium (0.14, 0.84 and 1.54 mM). 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
22-24.5.times.10.sup.6 cells/ml for the different conditions
tested. The viable cell density and viability profiles for all test
conditions were comparable (FIG. 71, 72). On Day 10 of culture
samples were collected for titer analysis (FIG. 73). The titers for
all conditions were comparable. On Day 10 duplicate shake flasks
were harvested for each condition and then subsequently analyzed
using WCX-10 post protein A purification and the percentages of
total peak(s) area corresponding to the acidic species were
quantified (FIG. 74). The percentage of acidic species in the 0.14
mM calcium condition was 23.8%. In the sample with the highest
tested concentration of calcium in this experiment (1.54 mM), the
percentage of acidic species was reduced to 21.6%. A dose dependent
decrease in acidic species was observed in test conditions with
increased calcium concentration.
[0351] Cell line 3 was cultured in media 1 with different total
concentrations of calcium (0.14, 0.49, 0.84, 1.19, 1.54, 1.89 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
9.5-10.5.times.10.sup.6 cells/ml for the different conditions
tested. The viable cell density and viability profiles for all test
conditions were comparable (FIG. 75, 76). On Day 11 of culture,
samples were collected for titer analysis. The harvest titers for
all conditions were comparable (FIG. 77). 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 acidic species were quantified (FIG. 78). The
percentage of acidic species in the 0.14 mM calcium condition was
23.7%. In the sample with the highest tested concentration of
calcium in this experiment (1.89 mM), the percentage of acidic
species was reduced to 20.7%. A dose dependent decrease in acidic
species was observed in test conditions with increased calcium
concentration.
[0352] Additional experiments were performed with multiple cell
lines in chemically defined or hydrolysate based media to evaluate
the wide range of applicability of this method. The experimental
setup for each of these experiments was similar to that described
in section above and in the materials and methods section. The
summaries of results of the different experiments performed for
adalimumab are summarized in FIGS. 79, 80 and 81. A reduction in
acidic species with increased calcium concentration was also
observed in each case.
[0353] 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 section above. The
dose dependent reduction of acidic species with ornithine addition
for experiments corresponding to each mAB is summarized in FIGS.
82, 83. For mAB1, a small yet significant acidic species reduction
from 15.4% (0.14 mM calcium sample) to 11.8% (1.54 mM calcium
chloride supplemented sample) was observed. For mAB2, a larger dose
dependent reduction from 28.9% (0.14 mM calcium sample) to 23.1%
(1.40 mM calcium chloride supplemented sample) was observed.
[0354] Effect of Increased Concentration of Arginine, Lysine,
Calcium Chloride, Niacinamide in Combination
[0355] In this experiment, the effect of the combined use of the
amino acids arginine, lysine, inorganic salt calcium chloride and
vitamin niacinamide for acidic species reduction was evaluated. The
experiment described here was performed using cell line 2
(producing adalimumab) in chemically defined media (media 1)
supplemented with 3% (v/v) PFCHO (proprietary chemically defined
medium formulation from SAFC). A central composite DOE experimental
design was used in this experiment. The basal media for each
condition was supplemented with different concentrations of the
four supplements. Cell cultures were carried out in duplicates for
each condition. Upon harvest, WCX-10 analysis was performed post
protein A purification. In Table 3, the experimental conditions
from DOE design, including the concentration of each component
supplemented, and the % total acidic species (or AR) obtained for
each condition is summarized. Reduction of acidic species through
the increased concentration of these components in combination was
observed. For instance, condition (#24), where all four components
were at their maximum concentration, the % total AR was reported to
be reduced to 9.7%. Using the data from the experiment, a model
predicting the effects of addition of these components to media for
AR reduction (R.sup.2: 0.92, P<0.0001) is described in FIG. 84.
The model predicted a contribution from each of the four components
towards acidic species reduction. 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 total AR.
TABLE-US-00002 TABLE 3 Experimental design and summary for the
combined addition of arginine, lysine, calcium chloride and
niacinamide Calcium Arginine Lysine Chloride Niacinamide % Total
Conditions (g/l) (g/l) (mM) (mM) AR 1 0.0 4.0 0.7 0.8 13.0 2 0.0
6.0 1.4 0.0 12.6 3 4.0 2.0 0 1.6 12.3 4 4.0 6.0 0 1.6 11.6 5 2.0
4.0 0.7 0.8 11.2 6 0.0 6.0 0 0.0 15.0 7 0.0 6.0 1.4 1.6 10.7 8 0.0
2.0 0 0.0 16.7 9 2.0 4.0 0.7 0.8 11.0 10 4.0 6.0 1.4 1.6 11.0 11
2.0 2.0 0.7 0.8 12.9 12 2.0 4.0 1.4 0.8 11.1 13 0.0 6.0 0 1.6 13.2
14 4.0 2.0 0 0.0 12.3 15 2.0 4.0 0.7 0.0 13.0 16 2.0 4.0 0.7 1.6
11.4 17 0.0 2.0 1.4 1.6 12.0 18 2.0 4.0 0 0.8 12.0 19 4.0 4.0 0.7
0.8 12.0 20 0.0 2.0 1.4 0.0 14.0 21 4.0 6.0 1.4 0.0 11.0 22 0.0 2.0
0 1.6 13.6 23 2.0 6.0 0.7 0.8 11.0 24 4.0 2.0 1.4 1.6 9.7 25 4.0
6.0 0 0.0 11.8 26 4.0 2.0 1.4 0.0 10.4 27 2.0 4.0 0 0.0 12.7
[0356] Use of Niacinamide Supplementation to Cell Culture Media for
Acidic Species Reduction
[0357] In addition to the use of niacinamide in combination with
other supplements described in the previous section, niacinamide
addition may also be used independent of the other supplements as
demonstrated in the experiments below for two mAbs: adalimumab and
mAb1.
[0358] For the experiment corresponding to adalimumab, Cell line 1
was cultured in media 1 supplemented with different amounts of
niacinamide (0, 0.2, 0.4, 0.8 and 1.6 mM). 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 8.5-11.times.10.sup.6
cells/ml for the different conditions tested. A slight decrease in
the viable cell density profile was observed with the maximum
niacinamide supplementation (1.6 mM for this experiment) (FIG. 85).
The viability profile for the test conditions were comparable (FIG.
86). On Day 12 of culture, samples were collected for titer
analysis. The titers for all conditions were comparable (FIG. 87).
On Day 11 and day 12, duplicate shake flasks were harvested for
each condition and then subsequently analyzed using WCX-10 post
protein A purification and the percentages of total peak(s) area
corresponding to the acidic species were quantified (FIG. 88, 89).
The percentage of acidic species in the day 10 control sample
(without niacinamide supplementation) was 19.6%. In the day 10
sample with the highest tested concentration of niacinamide in this
experiment (1.6 mM), the percentage of acidic species was reduced
to 15.9%. Similar acidic species reduction with niacinamide
supplementation was also observed in the day 12 samples.
[0359] For the experiment corresponding to mAb2, a mAB2 producing
cell line was cultured in media 1 supplemented with different
amounts of niacinamide (0, 0.1, 0.5, 1.0, 3.0 and 6.0 mM). 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
14-21.5.times.10.sup.6 cells/ml for the different conditions
tested. A slight decrease in the viable cell density profile was
observed for the conditions with 3.0 mM and 6.0 mM niacinamide
concentrations (FIG. 90). The viability profiles for all test
conditions were comparable (FIG. 91). On Day 12 of culture samples
were collected for titer analysis (FIG. 92). The titers for all
conditions were comparable. On Day 12 duplicate shake flasks were
harvested for each condition and then subsequently analyzed using
WCX-10 post protein A purification and the percentages of total
peak(s) area corresponding to the acidic species were quantified
(FIG. 93). The percentage of acidic species in the control sample
(without niacinamide supplementation) was 27.0%. In the sample with
the highest tested concentration of niacinamide in this experiment
(6.0 mM), the percentage of acidic species was reduced to 19.8%. A
dose dependent decrease in acidic species was observed in test
conditions with niacinamide supplementation.
[0360] Types of Acidic Species Variants Reduced by Supplementation
of Culture Medium with Additives
[0361] The addition of medium additives may be used to specifically
reduce particular acidic variants within the larger fraction of
total acidic species. In Table 4, a summary of the extent of some
of the sub-species of the acidic species fraction have been
presented for a representative set of experiments for adalimumab.
Along with the reduction in total acidic species, the methods
presented in this section may also be used for reduction of
sub-species that include, but not limited to, AR1, AR2 and MGO
(methylglyoxal) modified product variants.
TABLE-US-00003 TABLE 4 Summary of types of acidic species variants
reduced in cultures supplemented with medium additives % MGO
modified species LIGHT CHAIN HEAVY CHAIN Sample % AR % AR1 % AR2
Arg 30 Arg 93 Arg 108 Arg 16 (19) Arg 259 Arg 359 Arg 420 TOTAL
Control 26.9 9.7 17.3 1.63 1.21 0.33 0.6 0.06 3.96 3.31 11.1 Lysine
(10 g/l) 15.0 4.5 10.4 1.29 0.91 0 0.46 0.04 1.77 2.09 6.56
Histidine (10 g/l) 14.7 5.3 9.4 1.21 0.61 0 0.49 0.02 1.42 1.47
5.22 Ornithine (10 g/l) 16.5 4.4 12.1 1.17 0.71 0 0.37 0.03 1.11
1.29 4.68 Control 22.5 7.5 15.0 1.13 0.69 0 0.17 0.02 0.03 0 2.04
Arginine (8 g/l) 17.1 4.6 12.5 1.05 0.63 0 0.16 0.04 0.04 0 1.92
Control 23.1 6.6 16.6 1.43 0.82 0 0.38 0.05 1 1.35 5.03 Calcium
Chloride 20.8 5.9 14.9 1.28 0.83 0 0.2 0.04 1.07 1.52 4.94 (1.75
mM)
[0362] 6.1.3 Conclusion
[0363] The different experiments above demonstrate that
supplementation of cell culture medium with supplemental amounts of
amino acids, calcium chloride and niacinamide enhances product
quality by decreasing the amount of acidic species in the culture
harvest. The amino acids included in the study were arginine,
lysine, ornithine and histidine and belong to group of amino acids
that are basic. The study covered examples from multiple cell
lines/molecules, in shake flasks and bioreactors and in batch and
fed-batch culture formats. A dose dependent effect in the extent of
reduction of acidic species with increasing concentrations of the
supplements was observed. In addition, the possibility to
supplement these medium additives individually or in suitable
combinations for acidic species reduction was also
demonstrated.
6.2 Method for Reducing the Extent of Acidic Species in Cell
Culture by Adjusting Process Parameters
[0364] 6.2.1 Materials and Methods
[0365] Cell Source and Adaptation Cultures
[0366] Two adalimumab producing CHO cell lines and a mAB2 producing
cell line were employed in the studies covered here. Upon thaw,
adalimumab producing cell line 3 was cultured in chemically defined
growth media (media 1) in a combination of vented shake flasks on a
shaker platform @140 rpm and 20 L wave bags. 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.
[0367] Upon thaw, adalimumab producing cell line 1 was cultured in
a hydrolysate based growth media (media 2) in a combination of
vented shake flasks on a shaker platform @ 110 rpm and 20 L
wavebags in a 35.degree. C., 5% CO.sub.2 incubator. In some cases,
the culture might be transferred into a seed reactor with pH 7.1,
35.degree. C. and 30% DO. The culture would be adapted to either
media for 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.
[0368] Upon thaw, mAb2 producing cells were cultured in 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.
[0369] Cell Culture Media
[0370] Media 1, the chemical defined growth or production media,
was prepared from basal IVGN CD media (proprietary formulation).
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 cell line 1 and mAb2, the medium was also supplemented with
insulin. In addition, 10 mM or 5 mM of Galactose (Sigma, G5388) and
0.2 .mu.M or 1004 of Manganese (Sigma, M1787) were supplemented
into production medium for cell line 3 or 1, respectively.
Osmolality was adjusted by the concentration of sodium chloride.
All media was filtered through filter systems (0.22 .mu.m PES) and
stored at 4.degree. C. until usage.
[0371] Media 2 is the hydrolysate based media, which contains basal
proprietary media, Bacto TC Yeastolate and Phytone Peptone.
[0372] Production Cultures
[0373] 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 (except mAb2 with a
ratio of 1:5). When the media glucose concentration reduced to less
than 3 g/L, approximately 1.25% (v/v) of 40% glucose stock solution
was fed
[0374] 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.
[0375] WCX-10 Assay
[0376] 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). For adalimumab producing cell lines, 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 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. The WCX-10 method used for mAb B
used different buffers. 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 Chloride 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.
[0377] Quantitation is based on the relative area percent of
detected peaks. The peaks that elute at relative residence time
earlier than the main peak corresponding to the drug product are
together represented as the acidic peaks.
[0378] 6.2.2 Results
[0379] Effect of Process pH in Media 1 with Cell Line 1
[0380] 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 the same maximum viable cell
density on day 8, except for the culture at pH 6.7 condition, in
which the maximum cell density was much lower than the other
cultures (FIG. 94). In addition, the viability of the culture at pH
7.1 and pH 7.0 dropped much earlier than the other cultures. 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 (FIG.
95). Samples taken in the last three days of the cultures were
measured for IgG concentration. 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. 96). The cultures were harvested either on day 10 or on day
12. The harvest was protein A purified, then analyzed using WCX-10.
The resulting peak areas from WCX-10 analysis were quantified (FIG.
97). The percentage of acidic species decreased corresponding to
the decrease in pH, from 56.0% in the pH 7.1 condition to 14.0% in
the pH 6.7 condition. Since the cultures at pH 6.9, 6.8 and 6.7
were at 70% viability on day 10, additional samples were taken on
day 12 for these cultures, when viability reached .about.50%.
WCX-10 analysis was also performed for these samples. The
percentage of acidic species on day 12 was increased for these
three conditions (i.e., pH 6.9, 6.8 and 6.7) comparing to day 10;
however, the increase in the percentage of acidic species was
smaller at lower pH. The percentage of acidic species increased
18.8% (pH 6.9), 8.1% (pH6.8) and 3.5% (pH6.7), respectively from
day 10 (70% viability) to day 12 (50% viability). Therefore, the
percentage of acidic species was lower at lower pH on day 12 too.
The percent acidic species decreased with decrease in pH from 39.1%
in the pH 6.9 condition to 17.5% in the pH6.7 condition, for a
total reduction of 21.6%.
[0381] The effect of process pH to specifically reduce particular
acidic variants within the larger fraction of total acidic species
was also evaluated. In Table 5, a summary of the extent of some of
the sub-species of the acidic species fraction have been presented.
Along with the reduction in total acidic species, the methods
presented in this section may also be used for reduction of
sub-species that include, but not limited to, AR1, AR2 and MGO
(methylglyoxal) modified product variants.
TABLE-US-00004 TABLE 5 Effect of process pH on reduction of
sub-species of acidic variants % MGO modified species Sample LIGHT
CHAIN HEAVY CHAIN Final pH % AR % AR1 % AR2 Arg 30 Arg 93 Arg 108
Arg 16 (19) Arg 259 Arg 359 Arg 420 TOTAL 7.1 56.0 32.8 23.3 26.1
10.6 0.2 6.1 2.7 3.5 0.5 49.7 6.9 39.1 18.9 20.2 9.5 3.8 0.0 2.2
0.9 1.2 0.2 18.8 6.7 17.5 5.2 12.2 1.2 0.5 0.0 0.2 0.1 0.1 0.0
2.0
[0382] Effect of Process pH in Media 2 with Cell Line 1
[0383] 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 with lower
pH set points (FIGS. 98 and 99). The cultures were harvested on
.about.50% viability. The product titer was slightly higher at pH
6.8 comparing to pH 6.9 and 7.0 (FIG. 100). The resulting peak
areas from WCX-10 analysis were quantified (FIG. 101). The
percentage of acidic species decreased with decrease in pH from
20.7% in the pH 7.0 condition to 18.0% in the pH6.8 condition, for
a total reduction of 2.7%.
[0384] Effect of Process pH in Media 1 with Cell Line 3
[0385] 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 (FIGS. 102 and 103). The cells were
harvested either on day 10 or when viability dropped to equal or
less than 50%. The titer was slightly increased as the pH was
reduced, reached the highest titer at pH 6.8 condition (FIG. 104).
The resulting peak areas from WCX-10 analysis were quantified (FIG.
105). The percent acidic species decreased with decrease in pH from
29.7% in the pH 7.1 condition to 21.5% in the pH6.7 condition, for
a total reduction of 8.2%.
[0386] 6.2.3 Conclusion
[0387] The experiments described in the instant Example demonstrate
that altering cell culture process parameters on-line can be used
to modulate/reduce the acidic species of a protein of interest,
e.g., the antibody adalimumab or mAB2. For example, a decrease in
final pH set points can lead to reductions in Acidic Regions.
6.3 Method for Reducing Acidic Species in Cell Culture by the
Addition of Amino Acids to Clarified Cell Culture Harvest and by
Modifying the pH of the Clarified Harvest
[0388] The present study describes a process for reducing and
controlling levels of acidic species in antibody preparations.
Specifically, the invention provides a method for reducing the
acidic variant content in clarified harvest, as well as a method
for reducing the formation rate of acidic species in clarified
harvest. The method involves adding additives like various amino
acids to clarified harvest or adjusting the pH of the clarified
harvest using acidic substances.
[0389] 6.3.1 Materials and Methods
[0390] Clarified Harvest Material
[0391] Different batches of adalimumab clarified harvest material
were employed in the following experiments described below.
Clarified harvest is liquid material containing a monoclonal
antibody of interest that has been extracted from a fermentation
bioreactor after undergoing centrifugation to remove large solid
particles and subsequent filtration to remove finer solid particles
and impurities from the material. Clarified harvest was used for
low pH treatment studies described herein. Clarified harvest was
also used for the experiments to study the effect of amino acid
concentration on the presence of acidic species in clarified
harvest, and for acid type-pH treatment studies described herein.
Different batches of mAB-B and mAb-C clarified harvest material
were employed for experiments to study the effect of amino acid and
low pH treatment studies on the presence of acidic species
described herein.
[0392] Preparation of Study Materials
[0393] The clarified harvest material was first adjusted to pH 4
using 3M citric acid. The material at pH 4 was then agitated for 60
minutes before adjusting the pH to a target pH of 5, 6 or 7 with 3M
sodium hydroxide. The material was then agitated for a further 60
minutes. The samples were then subjected to centrifugation at
7300.times.g for 15 minutes in a Sorvall Evolution RC with an
SLA-3000 centrifuge bowl. The supernatants obtained from the
centrifuged material were then depth filtered using B1HC depth
filters (Millipore) followed by 0.22 .mu.m sterile filters. The
filtrates of different pH were then subjected to holding for
different period of time for evaluating the formation rate of
acidic variants. After the holding, the material was purified with
Protein A affinity column and the eluate was sampled and analyzed
using the WCX 10 method. The preparation scheme is shown below in
FIG. 106.
[0394] The material to study the effect of arginine on acidic
species was prepared in two ways. For lower target arginine
concentrations of 5 mM, 10 mM, 30 mM and 100 mM, they were made by
adding the appropriate amount of 0.5M arginine stock buffer at pH 7
(pH adjusted with acetic acid) to attain the target arginine
concentrations needed. For higher target arginine concentrations of
50 mM, 100 mM, 300 mM, 500 mM, 760 mM, 1M and 2M, they were made by
adding the appropriate amount of arginine (solid) to the samples to
attain the target arginine concentrations, with subsequent
titration to a final pH of 7 using glacial acetic acid. Arginine
was adjusted to a final concentration of 100 mM using the two
methods to determine if the method of preparation would result in
different effects. For all the experiments, following the arginine
addition, treated clarified harvests were held at room temperature
for the indicated duration followed by purification with Protein A
column and analysis of charge variants. This study provided two
results; (1) data of samples from Day 0 gave the effects of
arginine on reducing acidic species in clarified harvest, (2) data
of samples with different holding days gave effect of arginine on
reducing the formation rate of acidic species. The preparation
scheme is shown in FIG. 107.
[0395] The material to study the effect of histidine was prepared
with target concentrations of 5 mM, 10 mM, 30 mM 50 mM, 100 mM, 200
mM and 250 mM. The samples were prepared by adding the appropriate
amount of histidine (solid) to the samples to attain the target
histidine concentrations, with subsequent titration to a final pH
of 7 using glacial acetic acid. The sample preparation scheme is
shown in FIG. 108.
[0396] The material to study the effect of Lysine was prepared with
target concentrations of 5 mM, 10 mM, 30 mM 50 mM, 100 mM, 200 mM,
300 mM, 500 mM and 1000 mM. The samples were prepared by adding the
appropriate amount of lysine hydrochloride (solid) to the samples
to attain the target Lysine concentrations, with subsequent
titration to a final pH of 7 using hydrochloric acid. The sample
preparation scheme is shown below in FIG. 109.
[0397] The material to study the effect of methionine was prepared
with target concentrations of 5 mM, 10 mM, 30 mM 50 mM, 100 mM, 200
mM and 300 mM. The samples were prepared by adding the appropriate
amount of methionine (solid) to the samples to attain the target
methionine concentrations, with subsequent titration to a final pH
of 7 using glacial acetic acid. The sample preparation scheme is
shown in FIG. 110.
[0398] The material to study the effect of different amino acids
was prepared with different target concentrations for each of the
20 amino acids evaluated as well as two controls using sodium
acetate in place of an amino acid, and the other simply bringing
the pH of the clarified harvest down to pH 7 using glacial acetic
acid. The target concentrations for the amino acids are shown below
in Table 6.
TABLE-US-00005 TABLE 6 Amino Acid Target Concentrations
Concentration Amino Acid (mM) Alanine 100 Arginine 100 Asparagine
100 Aspartic Acid 30 Cysteine 100 Glutamic Acid 30 Glutamine 100
Glycine 100 Histidine 100 Isoleucine 100 Leucine 100 Lysine 100
Methionine 100 Phenylalanine 100 Proline 100 Serine 100 Threonine
100 Tryptophan 30 Tyrosine 2 Valine 100 NaAc 100
[0399] The samples were prepared by adding the appropriate amount
of amino acid (solid) to the samples to attain the target amino
acid concentrations as shown in Table 6, with subsequent titration
to a final pH of 7 using glacial acetic acid. The sample
preparation scheme is shown below in FIG. 111.
[0400] The material to study the effect of additives other than
amino acids was prepared with different target concentrations for
each of the additives evaluated as well as a control in which
sodium hydroxide was used in place of arginine to bring the pH of
the material to pH 10 before neutralizing it back to pH 7 with
glacial acetic acid. The target concentrations for the additives
are shown below in Table 7.
TABLE-US-00006 TABLE 7 Alternative Additive Target Concentrations
Additive Low Conc High Conc Sucrose 0.1M 1M Trehalose 0.1M 1M
Mannitol 4% w/v 10% w/v Glycerol 1% v/v 10% v/v PEG 1% w/v 2% w/v
Tween80 0.5% v/v 2% v/v
[0401] The samples were prepared by adding the appropriate amount
of additive to the samples to attain the target amino acid
concentrations as shown in Table 2, with subsequent titration to a
final pH of 7 using glacial acetic acid.
[0402] The material to study the effect of the aforementioned
methods on CDM clarified harvest was prepared using the following
scheme shown in FIG. 112.
[0403] The mAb B hydrolysate clarified harvest was used to study
the effect of the aforementioned methods.
[0404] The mAb C hydrolysate clarified harvest was used to study
the effect of the aforementioned methods.
[0405] Hold Studies for Treated Clarified Harvest
[0406] After the aforementioned sample preparations, the samples
were placed in separate sterile stainless steel containers for the
purpose of holding at either 4.degree. C. or at room temperature.
For each material, different containers were used for each day of
holding evaluated. For the acidified samples, the acidic variant
compositions of the samples were evaluated on days 0, 3, 7 and 14
of holding at either temperature. For the arginine containing
materials, the acidic variant compositions of the samples were
evaluated on days 0, 5 and 8 of holding at room temperature. For
the histidine containing materials, the acidic variant compositions
of the samples were evaluated on days 0, 3 and 7 of holding at room
temperature.
[0407] Acid Type and pH Effects on Clarified Harvest
[0408] The effects of acid type, clarified harvest pH and arginine
content on acidic variant reduction were evaluated in this study.
The samples were prepared in triplicates on 3 consecutive days to
target arginine concentrations of either 0 mM (no arginine added)
or 500 mM, then titrated with either glacial acetic acid,
phosphoric acid, 3M citric acid or 6M hydrochloric acid to target
pH values of either 5, 6 or 7. One other sample was prepared by
adding a 2M arginine acetate pH 7 stock buffer to clarified harvest
to attain a target arginine concentration of 500 mM. The sample
preparation scheme is shown in FIG. 113.
[0409] Protein a Purification
[0410] Protein A purification of the samples was performed using a
5 mL rProtein A FF Hitrap column (GE Healthcare) at 10 g D2E7/L
resin loading and a operating flow rate of 3.4 mL/min. 5 column
volumes (CVs) of equilibration (1.times.PBS pH 7.4) is followed by
loading of the sample, then washing of the column with
equilibration buffer to remove non-specifically bound impurities,
followed by elution of the protein with 0.1M Acetic acid, 0.15M
sodium chloride.
[0411] The eluate samples were collected and neutralized to pH
6.9-7.2 with 1M Tris pH 9.5 at 45-75 minutes after collection. The
samples were then frozen at -80.degree. C. for at least one day
before thawing and subjecting to WCX-10 analysis.
[0412] Effects Purification Method, Acid Concentration and
Neutralization on Clarified Harvest
[0413] The effects of purification methods with different types of
chromatography resins, acid concentration and pH neutralization on
acidic variant reduction were evaluated in this study. The
following samples were prepared, shown below in Table 8.
TABLE-US-00007 TABLE 8 Acid Concentration Sample Treatments Sample
Treatment Control None 3M Citric Acid pH 6 Titrate to pH 6 with 3M
Citric Acid 1M Citric Acid pH 6 Titrate to pH 6 with 1M Citric Acid
Glacial Acetic Acid pH 6 Titrate to pH 6 with Glacial Acetic Acid
3M Acetic Acid pH 6 Titrate to pH 6 with 3M Acetic Acid 3M Citric
Acid pH 5 Titrate to pH 5 with 3M Citric Acid 3M Acetic Acid pH 5
Titrate to pH 5 with 3M Acetic Acid 3M Citric Acid pH 5 to 7
Titrate to pH 5 with 3M Citric Acid, then 3M Tris to pH 7 3M Acetic
Acid pH 5 to 7 Titrate to pH 5 with 3M Acetic Acid, then 3M Tris to
pH 7
[0414] Each of the material made was then subjected to either
Mabselect Sure or Fractogel S capture in duplicate. The eluate
samples are collected and neutralized to pH 6.9-7.2 with 1M Tris pH
9.5 at 45-75 minutes after collection. The samples are then frozen
at -80.degree. C. for at least one day before thawing and
subjecting to WCX-10 analysis.
[0415] Charge Variant Analysis (WCX-10 Assay)
[0416] Cation exchange chromatography was performed on a 4
mm.times.250 mm Dionex ProPac WCX-10 Analytical column (Dionex,
CA). A Shimadzu LC10A HPLC system was used to perform the HPLC
assay. 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.
[0417] Quantitation is based on the relative area percent of
detected peaks. The peaks that elute at relative residence time
less than that of the dominant Lysine 0 peak are together
represented as the acidic variant peaks (AR).
[0418] 6.3.2 Results
[0419] Effect of Low pH Treatment with Subsequent
Neutralization
[0420] The results of the low pH treatment with subsequent
neutralization are shown below in FIGS. 114 and 115. FIG. 115 shows
that the low pH treatment with subsequent neutralization to pH 5 or
6 reduces the rate of acidic variant formation over time. However,
there is no significant reduction in initial acidic variant content
as shown in FIG. 114.
[0421] Effect of Arginine Treatment
[0422] The results of the arginine treatment are shown in FIG. 116
and FIG. 117. FIG. 116, 117 shows that the sample preparation
method resulted in different levels of acidic species in clarified
harvest. Adding a 0.5M Arginine pH 7 stock buffer tends to increase
acidic species, while adding pure arginine with subsequent acetic
acid titration to pH 7 reduced acidic variants at arginine
concentrations of greater than 100 mM. Moreover, the effect due to
treatment method is demonstrated when comparing the two 100 mM
arginine samples, which show an absolute difference of 1% in acidic
variants between the two methods.
[0423] FIG. 118 shows that the rate of acidic variant formation
decreases with increasing arginine concentration in clarified
harvest, plateauing at around concentrations of 500 mM arginine and
higher. However, the two methods of sample preparation does not
result in significantly different formation rate of acidic
variants.
[0424] Effect of Histidine Treatment
[0425] The results of the histidine treatment are shown in FIG. 119
and FIG. 120. Similar to arginine treatment effect, as shown in
FIG. 128, when histidine was added to clarified harvest with
subsequent pH neutralization with acetic acid, acidic variants were
reduced at histidine concentrations higher than 50 mM. FIG. 120
shows that the rate of acidic variants formation decreases with
increasing Histidine concentration in clarified harvest, plateauing
at around concentrations of 200 mM Histidine and higher.
[0426] Effect of Lysine Treatment
[0427] The results of the lysine treatment are summarized in FIG.
121 and FIG. 122. Similar to arginine treatment effect, as shown in
FIG. 128, when lysine was added to clarified harvest with
subsequent pH neutralization with acetic acid, acidic variants were
significantly reduced by .about.1% or more. FIG. 132 shows that the
rate of acidic variants formation decreases with increasing lysine
concentration in clarified harvest.
[0428] Effect of Methionine Treatment
[0429] The results of the methionine treatment are summarized below
in FIGS. 133 and 144. Similar to arginine treatment effect, as
shown in FIG. 118, when methionine was added to clarified harvest
with subsequent pH neutralization with acetic acid, acidic variants
were significantly reduced by .about.1% or more at concentrations
of >10 mM. FIG. 124 shows that the rate of acidic variants
formation is not affected significantly by methionine presence in
clarified harvest.
[0430] Effect of Other Amino Acid Treatment
[0431] The results of the treatments with the various amino acids
are summarized below in FIGS. 125 and 146. As shown in FIG. 125,
the addition of 14 amino acids including arginine, histidine,
lysine and methionine resulted in lower amounts of acidic variant
content in clarified harvest. The addition of sodium acetate or the
use of acetic acid also caused a reduction in acidic variant
content as well. FIG. 126 shows that the rate of acidic variants
formation is reduced by several amino acids including arginine,
histidine, lysine, aspartic acid, glutamic acid, and leucine.
[0432] Effect of Alternative Additive Treatment
[0433] The results of the treatments with the other additives are
summarized below in FIGS. 127 and 128. As shown in FIG. 127, the
addition of any of the additives did not result in lower acidic
variant content in D2E7 hydrolysate clarified harvest. However,
FIG. 128 shows that the rate of acidic variants formation is
reduced by most of the additives.
[0434] Effect of Low pH/Arginine Treatment on D2E7 CDM Clarified
Harvest
[0435] The results of CDM clarified harvest study are summarized
below in FIGS. 129 and 130. As shown in FIG. 129, low pH/arginine
treatment did not result in lower acidic variant content in D2E7
CDM clarified harvest. However, FIG. 130 shows that the rate of
acidic variants formation is reduced significantly by all the
treatments.
[0436] Effect of Low pH/Arginine Treatment on mAb B Hydrolysate
Clarified Harvest
[0437] The results of mAb B hydrolysate clarified harvest study are
summarized below in FIGS. 131 and 132. As shown in FIGS. 131 and
132, low pH/arginine treatment results in both lower acidic variant
content and slower rates of acidic variants formation in mAb B
hydrolysate clarified harvest.
[0438] Effect of Low pH/Arginine Treatment on mAb C Hydrolysate
Clarified Harvest
[0439] The results of mAb C hydrolysate clarified harvest study are
summarized below in FIGS. 133 and 134. As shown in FIGS. 133 and
134, low pH/arginine treatment results in both lower acidic variant
content and slower rates of acidic variants formation in mAb C
hydrolysate clarified harvest.
[0440] Effect of Acid Type and pH
[0441] The results obtained from the acid type-pH study are
summarized in FIG. 135. Greater acidic species reduction is
obtained at lower pH. Arginine addition also reduces acidic species
content further, but not to a significant extent when taking the
high concentrations (500 mM) used into consideration. The results
also show that acidic species reduction of .about.1% can be
achieved with the usage of an arginine acetate stock buffer,
although using pure arginine powder with subsequent acid titration
performs slightly better. With regard to acid type used for pH
adjustment, there were no significant differences between different
acids observed.
[0442] Effect of Purification Method, Acid Concentration and
Neutralization
[0443] The results obtained from the study are summarized below in
FIGS. 136, 137, 138, and 139. FIGS. 136, 137 indicate that when the
acid used is of higher concentration, there is an decrease in
acidic variant content in hydrolysate clarified harvest as compared
to a lower concentration acid being used. FIGS. 138, 139 show that
when the clarified harvest is subjected to base neutralization to
pH 7 after being treated with low pH, there is an increase in
acidic variant content. The figures also show that the Fractogel
resin is better able to clear acidic variants than Mabselect
Sure.
[0444] 6.3.3 Conclusion
[0445] Antibody acidic species in clarified harvest can be reduced
by adding additives such as arginine or histidine to clarified
harvest at concentrations of more than 100 mM and 50 mM,
respectively. It can also be achieved by pH adjustment of the
clarified harvest to pH 6 or pH 5. In addition, the rate of acidic
variant formation can be reduced through the use of arginine or
histidine in a concentration dependent manner, or by low pH
treatment of the clarified harvest.
6.4 Method for Reducing Acidic Species in Cell Culture Use of a
Continuous Media Perfusion Technology
[0446] As demonstrated in section 6.3, generation or formation of
acidic species in the population of proteins may occur during the
hold of the antibody in clarified harvest or spent media. Thus, the
possibility of enhanced stability of the product antibody or a
reduction in acidic species generation was explored using a
continuous/perfusion based cell culture technology. Control or
reduction in the amount of acidic species present in the population
of proteins obtained at end of cell culture can be accomplished by
modifying the exchange rate of fresh medium into the bioreactor (or
removal of spent medium with product antibody out of the
bioreactor).
[0447] 6.4.1 Materials and Methods
[0448] Cell Source
[0449] One adalimumab producing CHO cell line was employed in the
study covered here. Upon thaw, the vial was cultured in a
chemically defined growth media (media 1) in a series of vented
shake flasks on a shaker platform at 110 rpm in a 35.degree. C., 5%
CO.sub.2 incubator. Cultures were propagated to obtain a sufficient
number of cells for inoculation of the perfusion cultibag.
[0450] Cell Culture Media
[0451] A chemically defined growth or production media was used in
this study. For preparation of the media formulation, the
proprietary media (Invitrogen) was supplemented with L-glutamine,
sodium bicarbonate, sodium chloride, recombinant human insulin and
methotrexate solution. Perfusion stage media consisted of all the
components in the growth medium, with the exception of a higher
concentration of recombinant human insulin and the exclusion of
methotrexate solution.
[0452] Perfusion Culture
[0453] The perfusion culture was carried out with the Sartorius
BIOSTAT RM 20 optical perfusion system (SN#00582|12) in a Sartorius
Cultibag RM 10L perfusion pro 1.2my (lot 1205-014) perfusion bag.
The perfusion bag was run with a working culture volume of 1.5 L
and operation conditions of; pH: 7.00, dissolved oxygen 30%, 25
rpm, 35.degree. C., an air overlay of 0.3 slpm and a CO.sub.2
overlay of 15 sccm. pH control was initiated on day three of the
culture. pH was controlled with 0.5M sodium hydroxide and CO.sub.2
additions.
[0454] Perfusion was carried out by `harvesting` spent culture
through an integrated 1.2 .mu.m filter integrated into the
perfusion cultibag. Fresh media was added to the culture through a
feed line at the same rate as the harvest. Perfusion began on day
four of the process at a rate of 1.0 exchanges per day (ex/day).
The perfusion rate was adjusted throughout the run to accommodate
glucose needs, lactate accumulation and sampling plans. Perfusion
cell-free harvest samples were collected at perfusion rates of 1.5,
3.0 and 6.0 exchange volumes/day on day 5-6 of perfusion. A fresh
harvest bag was used for each harvest sample. The samples were then
purified using protein A and analyzed using WCX-10 assay.
[0455] The perfusion culture was ended on day 8 of the process.
[0456] WCX-10 Assay
[0457] 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).
[0458] 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. The
WCX-10 method used for mAb2 samples used different buffers. 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 Chloride 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. Quantitation is based on the relative
area percent of detected peaks, as described above.
[0459] 6.4.2 Results
[0460] Effect of Use of Perfusion Technology and Choice of Medium
Exchange Rates on Acidic Species
[0461] Adalimumab producing cell line 1 was cultured in media 1 and
the cultures were carried out as described in the materials and
methods. As described in table 9, the exchange rates were modified
over a period of 24 hrs between day 5 and day 6 to explore the
influence of medium exchange rates on the extent of acidic species.
At a continuous medium exchange rate of 1.5 volumes/day, the
product antibody in spent medium was collected in a harvest bag
over a period of 17 hrs. The harvest bag was then exchanged with a
new bag and the old bag was transferred to 4 C. Subsequently and in
succession, the medium exchange rates were increased to 3 and 6
volumes/day and the product harvest was collected over a time
period of 5 and 2 hrs, respectively. After an overnight hold at 4
C, the three harvest samples were processed through protein A and
analyzed for acidic species using WCX-10. The percentage of acidic
species in the sample with a medium exchange rate of 1.5
volumes/day was 8.1%. In the sample with the highest tested
exchange rate in this experiment (6 volumes/day), the percentage of
acidic species was reduced to 6%. An exchange rate dependent
reduction in acidic species was observed in the three samples
(Table 9). Reductions in different sub-species within the acidic
variants (AR1 and AR2) were also noted. An increase in volumetric
productivity, with exchange rate, was also observed.
TABLE-US-00008 TABLE 9 Effect of medium exchange rates in a
perfusion bioreactor on acidic species Harvest bag Exchange rate
Exchange time Volumetric Start Time (day, (no. of working (for
collection in Productivity hrs:min) volumes/day) harvest bag) (hrs)
(mg/l-hr) % Total AR % AR1 % AR2 Day 5, 16:00 1.5 17 10.94 8.1 2.0
6.1 Day 6, 10:25 3 5 39.80 6.9 1.7 5.2 Day 6, 15:25 6 2 69.50 6.0
1.3 4.7
6.5. Utility of AR Reduction
[0462] The current invention provides a method for reducing acidic
species for a given protein of interest. In this example adalimumab
was prepared using a combination of supplementation of arginine and
lysine to cell culture as shown in this invention along with AEX
and CEX purification technologies (described in U.S. patent
application Ser. No. 13/829,989) to produce a Low-AR and High-AR
sample with a final AR of 2.5% and 6.9%, respectively. Both samples
were incubated in a controlled environment at 25.degree. C. and 65%
relative humidity for 10 weeks, and the AR measured every two
weeks. FIG. 142 shows the growth of AR for each sample over the 10
week incubation. It is evident from FIG. 142 the growth rate of AR
is linear and similar between both the Low-AR and High-AR samples.
Based on these results the reduced AR material can be stored 3 fold
longer before reaching the same AR level as the High-AR sample.
This is a significant utility as this can be very beneficial in
storage handling and use of the antibody or other proteins for
therapeutic use.
6.6 Process Combinations to Achieve Target % AR or AR
Reductions
[0463] Upstream and Downstream process technologies, e.g., cell
culture and chromatographic separations, of the inventions
disclosed in the following applications can be combined together or
combined with methods in the art to provide a final target AR value
or achieve a % AR reduction, as well as to, in certain embodiments,
reduce product related substances and/or process related
impurities. Upstream methods for AR reduction include, but are not
limited to those described in the instant application. Downstream
methods for AR reduction include, but are not limited to, those
described in U.S. patent application Ser. No. 13/829,989. Exemplary
technologies disclosed in the referenced applications include, but
are not limited to: cell culture additives & conditions;
clarified harvest additives and pH/salt conditions; mixed mode
media separations; anion exchange media separations; and cation
Exchange media separations.
[0464] The instant example demonstrates the combined effect of one
or more of these technologies in achieving a target AR value or AR
reduction, thereby facilitating the preparation of an antibody
material having a specific charge heterogeneity. Additional
examples of combinations of downstream technologies and upstream
technologies are provided in the referenced applications.
[0465] In this example, the combination of upstream and downstream
methods involves the reduction of acidic species in 3 L bioreactor
cell cultures supplemented with arginine (2 g/l) and lysine (4 g/l)
as has been previously demonstrated in the instant application. The
results of that strategy are summarized in Table 10. The total
acidic species was reduced from 20.5% in the control sample to
10.2% in sample from cultures that were supplemented with the
additives. In this study, Adalimumab producing cell line 1 was
cultured in media 1 (chemically defined media) supplemented with
amino acid arginine (2 g/l) and lysine (4 g/l) in a 300 L
bioreactor. On Day 12 of culture, the culture was harvested and
then subsequently analyzed using WCX-10 post protein A purification
and the percentages of total peak(s) area corresponding to the
acidic species were quantified. The percentage of acidic species
was estimated to be 9.1% in the 300 L harvest sample
TABLE-US-00009 TABLE 10 AR levels achieved with use of upstream
technologies 3 L Bioreactor 300 L Bioreactor Arginine (2 g/l) +
Arginine (2 g/l) + Lysine (4 g/l) Lysine (4 g/l) Total Total
Control AR AR AR1 (%) AR2 (%) Total AR (%) AR1 (%) AR2 (%) (%) AR1
(%) AR2 (%) (%) 6.3 14.2 20.5 2.6 7.6 10.2 2.4 6.7 9.1
[0466] The material produced by the 300 L Bioreactor employing
Arginine and Lysine additions, that effectively reduced the AR
levels to 9.1% was purified using a downstream process employing
Mixed Mode chromatography as the primary AR Reduction method.
[0467] Adalimumab was purified by a Protein A chromatography step
followed with a low pH viral inactivation step. The filtered viral
inactivated material was buffer exchanged and loaded onto a Capto
Adhere column. The flowthrough of Capto Adhere material was then
purified with a HIC column with bind/elute mode as well as Flow
Through mode. As shown in Table 11, AR reduction was achieved
primarily with MM step, with some contribution from other steps.
The table also shows that additional product related substances
such as aggregates and process related impurities such as HCP can
be effectively reduced employing these combined technologies.
TABLE-US-00010 TABLE 11 Complete Downstream Process Train with
Protein A Capture - AR, HMW and HCP reduction Yield % AR % HMW
Process (%) reduction reduction HCP LRF Clarified Harvest 97.0% n/a
n/a n/a Prt-A Eluate Pool 89.6% 0.06 1.87 Viral Inactivated 99.7%
No reduction 0.07 0.39 Filtrate MM FT pool 91.9% 2.26 0.83 1.63 HIC
(B/E) Eluate 90.1% 0.40 0.22 1.41 Nanofiltrate Filtrate 90.7% No
reduction No reduction 0.15 BDS (B/E) 102.0% No reduction No
reduction 0.22 HIC FT-pool 98.5% 0.16 0.23 0.46 VF (FT) Filtrate
96.1% No reduction No reduction 0.10 BDS (FT) 103.8% No reduction
No reduction No reduction
[0468] As is evident from the above example, the MM method further
reduced the AR levels, by 2.26%. Therefore upstream technologies
for reduction can be combined with downstream technologies to
achieve AR levels/AR reduction.
[0469] 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.
[0470] Patents, patent applications, publications, product
descriptions, GenBank Accession Numbers, and protocols that may be
cited throughout this application, the disclosures of which are
incorporated herein by reference in their entireties for all
purposes. For example, but not by way of limitation, the following
U.S. patent applications designated by the following U.S. patent
applications are incorporated herein by reference in their
entireties for all purposes: U.S. patent application Ser. Nos.
13/803,808, 13/829,989, 13/830,976, 13/831,181, and 13/804,220.
* * * * *