U.S. patent application number 15/224113 was filed with the patent office on 2017-02-23 for cell culture media and methods of antibody production.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Lauren BROWN, Veronica CARVALHAL, Thomas DIROCCO, Nathan MCKNIGHT, Natarajan VIJAYASANKARAN.
Application Number | 20170051049 15/224113 |
Document ID | / |
Family ID | 51537877 |
Filed Date | 2017-02-23 |
United States Patent
Application |
20170051049 |
Kind Code |
A1 |
CARVALHAL; Veronica ; et
al. |
February 23, 2017 |
CELL CULTURE MEDIA AND METHODS OF ANTIBODY PRODUCTION
Abstract
Cell culture media are provided herein as are methods of using
the media for cell culture and antibody production from cells.
Compositions comprising antibodies and fragments thereof, produced
by the methods herein are also provided.
Inventors: |
CARVALHAL; Veronica; (South
San Francisco, CA) ; VIJAYASANKARAN; Natarajan;
(South San Francisco, CA) ; BROWN; Lauren; (South
San Francisco, CA) ; DIROCCO; Thomas; (South San
Francisco, CA) ; MCKNIGHT; Nathan; (South San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
51537877 |
Appl. No.: |
15/224113 |
Filed: |
July 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14211467 |
Mar 14, 2014 |
9441035 |
|
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15224113 |
|
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61801247 |
Mar 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/14 20130101;
C12N 2501/33 20130101; C12N 2501/998 20130101; C12N 5/0682
20130101; C12N 2500/32 20130101; C07K 16/22 20130101; C07K 2317/24
20130101; C12N 2500/20 20130101; C07K 2317/76 20130101; C12N
2510/02 20130101; C07K 16/00 20130101; A61P 35/00 20180101 |
International
Class: |
C07K 16/22 20060101
C07K016/22; C12N 5/071 20060101 C12N005/071 |
Claims
1. A method of producing bevacizumab, or a fragment thereof,
comprising the step of culturing a Chinese hamster ovary (CHO) cell
comprising a nucleic acid encoding bevacizumab or fragment thereof
in a cell culture medium, wherein the cell culture medium comprises
two or more components selected from the group consisting of
copper, insulin, and cystine, wherein the cell culture medium
further comprises a plant-derived hydrolysate and an animal-derived
hydrolysate, and wherein the cell produces bevacizumab, or a
fragment thereof.
2. The method of claim 1, wherein the cell culture medium comprises
copper and insulin.
3. The method of claim 1, wherein the cell culture medium comprises
copper and cystine.
4. The method of claim 1, wherein the cell culture medium comprises
insulin and cystine.
5. The method of claim 1, wherein the cell culture medium comprises
copper, insulin, and cystine.
6. (canceled)
7. The method of claim 1, wherein the cell culture medium comprises
insulin at a concentration of from about 1.0 mg/L to about 100.0
mg/L.
8. The method of claim 1, wherein the cell culture medium comprises
insulin at a concentration of from about 10.0 mg/L to about 100.0
mg/L.
9. The method of claim 1, wherein the cell culture medium comprises
insulin at a concentration of from about 10.0 mg/L to about 50.0
mg/L.
10. The method of claim 1, wherein the cell culture medium
comprises insulin at a concentration of from about 10.0 mg/L to
about 35.0 mg/L.
11. The method of claim 1, wherein the cell culture medium
comprises insulin at a concentration of from about 10.0 mg/L to
about 25.0 mg/L.
12. The method of claim 1, wherein the cell culture medium
comprises insulin at a concentration of about 25 mg/L.
13. The method of claim 1, wherein the cell culture medium
comprises copper at a concentration of from about 69 nM to about
1,000 nM.
14. The method of claim 1, wherein the cell culture medium
comprises copper at a concentration of from about 325 nM to about
375 nM.
15. The method of claim 1, wherein the cell culture medium
comprises copper at a concentration of from about 325 nM to about
350 nM.
16. The method of claim 1, wherein the cell culture medium
comprises copper at a concentration of about any one of 330 nM, 335
nM, 339 nM, 340 nM, 345 nM or 350 nM.
17. The method of claim 1, wherein the cell culture medium
comprises copper at a concentration of about 339 nM.
18. The method of claim 1, wherein the cell culture medium
comprises cystine at a concentration of from about 0.7 mM to about
2.0 mM.
19. The method of claim 1, wherein the cell culture medium
comprises cystine at a concentration of from about 1.0 mM to about
1.6 mM.
20. The method of claim 1, wherein the cell culture medium
comprises cystine at a concentration of from about 1.2 mM to about
1.4 mM
21. The method of claim 1, wherein the cell culture medium
comprises cystine at a concentration of about 1.3 mM.
22. The method of claim 1, wherein the cell culture medium
comprises the animal-derived hydrolysate at a concentration of from
about 6.0 g/L to about 20.0 g/L.
23. The method of claim 1, wherein the cell culture medium
comprises the animal-derived hydrolysate at a concentration of from
about 8.0 g/L to about 12.0 g/L.
24. The method of claim 1, wherein the cell culture medium
comprises the animal-derived hydrolysate at a concentration of from
about 9.0 g/L to about 11.0 g/L.
25. The method of claim 1, wherein the cell culture medium
comprises the animal-derived hydrolysate at a concentration of
about 13 g/L.
26. The method of claim 1, wherein the cell culture medium
comprises the plant-derived hydrolysate at a concentration of from
about 1.0 g/L to about 10.0 g/L.
27. The method of claim 1, wherein the cell culture medium
comprises the plant-derived hydrolysate at a concentration of from
about 2.0 g/L to about 3.0 g/L.
28. The method of claim 1, wherein the cell culture medium
comprises the plant-derived hydrolysate at a concentration of from
about 2.25 g/L to about 2.75 g/L
29. The method of claim 1, wherein the cell culture medium
comprises the plant-derived hydrolysate at a concentration of about
3.1 g/L.
30. (canceled)
31. The method of claim 1, wherein the cell culture medium
comprises insulin and the method further comprises the step of
adding an additional amount of insulin to the cell culture
medium.
32. The method of claim 31, wherein the additional amount of
insulin is added to the cell culture medium once during the cell
culture cycle.
33. The method of claim 31, wherein the additional amount of
insulin is added to the cell culture medium at least three times
during the cell culture cycle.
34. The method of claim 31, wherein the additional amount of
insulin is added to the cell culture medium at least six times
during the cell culture cycle.
35. The method of claim 31, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 1.0 mg/L to about
100.0 mg/L.
36. The method of claim 31, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 10.0 mg/L to about
100.0 mg/L.
37. The method of claim 31, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 10.0 mg/L to about
50.0 mg/L.
38. The method of claim 31, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 10.0 mg/L to about
35.0 mg/L.
39. The method of claim 31, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 10.0 mg/L to about
25.0 mg/L.
40. The method of claim 31, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of about 15 mg/L.
41. The method of claim 1, wherein the method further comprises the
step of adding cysteine to the cell culture medium.
42. The method of claim 41, wherein cysteine is added in an amount
to provide from about 0.5 to about 2.0 mM cysteine in the cell
culture medium.
43. The method of claim 41, wherein cysteine is added in an amount
to provide about 0.8 mM cysteine in the cell culture medium.
44. The method of claim 1, wherein the method further comprises the
step of adding cystine to the cell culture medium.
45. The method of claim 44, wherein cystine is added in an amount
to provide from about 0.1 to about 1.5 mM cystine in the cell
culture medium.
46. The method of claim 44, wherein cystine is added in an amount
to provide about 0.2 mM cystine in the cell culture medium.
47. The method of claim 1, wherein the cell is cultured at a
temperature ranging from about 28.degree. C. to about 37.degree.
C.
48. The method of claim 47, wherein the cell is cultured at a
temperature ranging from about 31.degree. C. to about 35.degree.
C.
49. The method of claim 1, wherein the cell is cultured at a first
temperature of about 35.degree. C. for a first period of time, is
cultured at a second temperature of about 33.degree. C. for a
second period of time, and is cultured at a third temperature of
about 31.degree. C. for a third period of time.
50. The method of claim 1, wherein bevacizumab, or a fragment
thereof, is secreted into the cell culture medium.
51. The method of claim 1, further comprising the step of
recovering the bevacizumab, or a fragment thereof, from the cell
culture.
52. Bevacizumab, or fragment thereof, produced by the method of
claim 1.
53. A composition comprising: (i) bevacizumab, or a fragment
thereof, produced by the method of claim 1 and (ii) a
pharmaceutically acceptable carrier.
54. A method of culturing a Chinese hamster ovary (CHO) cell
comprising a nucleic acid encoding bevacizumab, or a fragment
thereof, the method comprising the step of contacting the CHO cell
with a cell culture medium comprising two or more components
selected from the group consisting of copper, insulin and cystine,
wherein the cell culture medium further comprises a plant-derived
hydrolysate and an animal-derived hydrolysate.
55. The method of claim 54, wherein the cell culture medium
comprises copper and insulin.
56. The method of claim 54, wherein the cell culture medium
comprises copper and cystine.
57. The method of claim 54, wherein the cell culture medium
comprises insulin and cystine.
58. The method of claim 54, wherein the cell culture medium
comprises copper, insulin, and cystine.
59. (canceled)
60. The method of claim 54, wherein the cell culture medium
comprises insulin at a concentration of from about 1.0 mg/L to
about 100.0 mg/L.
61. The method of claim 54, wherein the cell culture medium
comprises insulin at a concentration of from about 10.0 mg/L to
about 100.0 mg/L.
62. The method of claim 54, wherein the cell culture medium
comprises insulin at a concentration of from about 10.0 mg/L to
about 50.0 mg/L.
63. The method of claim 54, wherein the cell culture medium
comprises insulin at a concentration of from about 10.0 mg/L to
about 35.0 mg/L.
64. The method of claim 54, wherein the cell culture medium
comprises insulin at a concentration of from about 10.0 mg/L to
about 25.0 mg/L.
65. The method of claim 54, wherein the cell culture medium
comprises insulin at a concentration of about 25 mg/L.
66. The method of claim 54, wherein the cell culture medium
comprises copper at a concentration of from about 69 nM to about
1,000 nM.
67. The method of claim 54, wherein the cell culture medium
comprises copper at a concentration of from about 325 nM to about
375 nM.
68. The method of claim 54, wherein the cell culture medium
comprises copper at a concentration of from about 325 nM to about
350 nM.
69. The method of claim 54, wherein the cell culture medium
comprises copper at a concentration of about any one of 330 nM, 335
nM, 339 nM, 340 nM, 345 nM or 350 nM.
70. The method of claim 54, wherein the cell culture medium
comprises copper at a concentration of about 339 nM.
71. The method of claim 54, wherein the cell culture medium
comprises cystine at a concentration of from about 0.7 mM to about
2.0 mM.
72. The method of claim 54, wherein the cell culture medium
comprises cystine at a concentration of from about 1.0 mM to about
1.6 mM.
73. The method of claim 54, wherein the cell culture medium
comprises cystine at a concentration of from about 1.2 mM to about
1.4 mM
74. The method of claim 54, wherein the cell culture medium
comprises cystine at a concentration of about 1.3 mM.
75. The method of claim 54, wherein the cell culture medium
comprises the animal-derived hydrolysate at a concentration of from
about 6.0 g/L to about 20.0 g/L.
76. The method of claim 54, wherein the cell culture medium
comprises the animal-derived hydrolysate at a concentration of from
about 8.0 g/L to about 12.0 g/L.
77. The method of claim 54, wherein the cell culture medium
comprises the animal-derived hydrolysate at a concentration of from
about 9.0 g/L to about 11.0 g/L.
78. The method of claim 54, wherein the cell culture medium
comprises the animal-derived hydrolysate at a concentration of
about 13 g/L.
79. The method of claim 54, wherein the cell culture medium
comprises the plant-derived hydrolysate at a concentration of from
about 1.0 g/L to about 10.0 g/L.
80. The method of claim 54, wherein the cell culture medium
comprises the plant-derived hydrolysate at a concentration of from
about 2.0 g/L to about 3.0 g/L.
81. The method of claim 54, wherein the cell culture medium
comprises the plant-derived hydrolysate at a concentration of from
about 2.25 g/L to about 2.75 g/L
82. The method of claim 54, wherein the cell culture medium
comprises the plant-derived hydrolysate at a concentration of about
3.1 g/L.
83. (canceled)
84. The method of claim 54, wherein the cell culture medium
comprises insulin and the method further comprises the step of
adding an additional amount of insulin to the cell culture
medium.
85. The method of claim 84, wherein the additional amount of
insulin is added to the cell culture medium once during the cell
culture cycle.
86. The method of claim 84, wherein the additional amount of
insulin is added to the cell culture medium at least three times
during the cell culture cycle.
87. The method of claim 84, wherein the additional amount of
insulin is added to the cell culture medium at least six times
during the cell culture cycle.
88. The method of claim 84, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 1.0 mg/L to about
100.0 mg/L.
89. The method of claim 84, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 10.0 mg/L to about
100.0 mg/L.
90. The method of claim 84, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 10.0 mg/L to about
50.0 mg/L.
91. The method of claim 84, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 10.0 mg/L to about
35.0 mg/L.
92. The method of claim 84, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 10.0 mg/L to about
25.0 mg/L.
93. The method of claim 84, wherein the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of about 15 mg/L.
94. The method of claim 54, wherein the method further comprises
the step of adding cysteine to the cell culture medium.
95. The method of claim 94, wherein cysteine is added in an amount
to provide from about 0.5 to about 2.0 mM cysteine in the cell
culture medium.
96. The method of claim 94, wherein cysteine is added in an amount
to provide about 0.8 mM cysteine in the cell culture medium.
97. The method of claim 54, wherein the method further comprises
the step of adding cystine to the cell culture medium.
98. The method of claim 97, wherein cystine is added in an amount
to provide from about 0.1 to about 1.5 mM cystine in the cell
culture medium.
99. The method of claim 97, wherein cystine is added in an amount
to provide about 0.2 mM cystine in the cell culture medium.
100. (canceled)
101. The method of claim 54, wherein the cell is cultured at a
temperature ranging from about 28.degree. C. to about 37.degree.
C.
102. The method of claim 101, wherein the cell is cultured at a
temperature ranging from about 28.degree. C. to about 35.degree.
C.
103. The method of claim 54, wherein the cell is cultured at a
first temperature of about 35.degree. C. for a first period of
time, is cultured at a second temperature of about 33.degree. C.
for a second period of time, and is cultured at a third temperature
of about 31.degree. C. for a third period of time.
104. The method of claim 54, wherein bevacizumab, or a fragment
thereof, is secreted into the cell culture medium.
105. The method of claim 54, wherein the CHO cell is contacted with
the cell culture medium during the cell's growth phase.
106. The method of claim 54, wherein the CHO cell is contacted with
the cell culture medium during the cell's production phase.
107. A kit for supplementing a cell culture medium for use in
culturing a Chinese hamster ovary (CHO) cell comprising a nucleic
acid encoding bevacizumab, or a fragment thereof, the kit
comprising at least two of components (i)-(iii): (i) insulin in an
amount to provide from about 1.0 mg/L to about 100.0 mg/L insulin
in the cell culture medium; (ii) cystine in an amount to provide
from about 0.7 mM to about 2.0 mM cystine in the cell culture
medium; (iii) and copper in an amount to provide from about 69.0 nM
to about 1,000.0 nM copper in the cell culture medium, wherein the
cell culture medium further comprises a plant-derived hydrolysate
and an animal-derived hydrolysate.
108-111. (canceled)
112. A cell culture medium for use in culturing a Chinese hamster
ovary (CHO) cell comprising a nucleic acid encoding bevacizumab, or
a fragment thereof, the cell culture medium comprising at least two
of components (i)-(iii): (i) from about 1.0 mg/L to about 100.0
mg/L insulin; (ii) from about 69.0 nM to about 1,000.0 nM copper;
and (iii) from about 0.7 mM to about 2.0 mM cystine, wherein the
cell culture medium further comprises a plant-derived hydrolysate
and an animal-derived hydrolysate.
113-120. (canceled)
121. A composition comprising (a) a Chinese hamster ovary (CHO)
cell comprising a nucleic acid encoding bevacizumab, or a fragment
thereof; and (b) a cell culture medium according to claim 112.
122. A composition comprising: (a) bevacizumab, or a fragment
thereof; and (b) a cell culture medium according to claim 112.
123. (canceled)
124. A method of enhancing the amount of bevacizumab, or a fragment
thereof, produced from a Chinese hamster ovary (CHO) cell
comprising a nucleic acid encoding bevacizumab, or a fragment
thereof, the method comprising the step of culturing the CHO cell
in a cell culture medium comprising at least two of insulin, copper
and cystine, wherein the cell culture medium further comprises a
plant-derived hydrolysate and an animal-derived hydrolysate, and
wherein the amount of bevacizumab, or a fragment thereof, produced
from the CHO cell is enhanced relative to culturing the CHO cell in
a cell culture medium without at least two of insulin, copper and
cystine.
125-174. (canceled)
175. A method of culturing a Chinese hamster ovary (CHO) cell
comprising a nucleic acid encoding bevacizumab, or a fragment
thereof, in a cell culture medium comprising at least two of
insulin, copper and cystine, wherein the cell culture medium
further comprises a plant-derived hydrolysate and an animal-derived
hydrolysate, and wherein the amount of bevacizumab, or a fragment
thereof, produced from the CHO cell is enhanced relative to
culturing the CHO cell in a cell culture medium without at least
two of insulin, copper and cystine.
176-225. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Patent Application Ser. No. 61/801,247, filed Mar. 15,
2013, the contents of which are incorporated herein by reference in
its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to cell culture media for use
in culturing a mammalian cell comprising a nucleic acid encoding
bevacizumab, or a fragment thereof, and to methods of using the
media in bevacizumab production as well as compositions and kits
comprising the bevacizumab, or a fragment thereof, produced by the
methods provided herein.
BACKGROUND OF THE INVENTION
[0003] Cell culture manufacturing technology is widely used for the
production of protein-based therapeutics, such as antibodies, for
use in pharmaceutical formulations. Commercial production of
protein-based products, such as an antibody product, requires
optimization of cell culture parameters in order for the cell to
produce enough of the protein product to meet manufacturing
demands. However, when cell culture parameters are optimized for
improving productivity of the protein product it is also necessary
to maintain the desired quality attributes of the product such as
the glycosylation profile, aggregate levels, charge heterogeneity,
and amino acid sequence integrity (Li, et al., 2010, mAbs.,
2(5):466-477).
[0004] Bevacizumab, also known as "Avastin.RTM.", is a recombinant
humanized monoclonal antibody that binds vascular endothelial
growth factor in in vitro and in vivo assay systems (U.S. Pat. No.
7,227,004; U.S. Pat. No. 6,884,879; U.S. Pat. No. 7,060,269; U.S.
Pat. No. 7,169,901; U.S. Pat. No. 7,297,334) and is used in the
treatment of cancer, where it inhibits tumor growth by blocking the
formation of new blood vessels. Bevacizumab has an approximate
molecular weight of 149,000 daltons, is glycosylated, and is
produced in a mammalian cell (Chinese Hamster Ovary) expression
system in a nutrient cell culture medium.
[0005] Improved and cost-effective methods of producing
bevacizumab, or a fragment thereof, are desirable. Cell culture
media comprising components that enable a cell to produce a desired
amount of bevacizumab, or a fragment thereof, while maintaining
acceptable product quality attributes of bevacizumab, or a fragment
thereof, would be beneficial. Cell culture media for use in
producing manufacturing-scale amounts of bevacizumab, or a fragment
thereof, would be particularly advantageous.
BRIEF SUMMARY OF THE INVENTION
[0006] The invention provided herein discloses, inter alia, methods
of producing bevacizumab, or a fragment thereof, in a cell culture
medium comprising at least two of copper, insulin, and cystine, and
optionally comprises an animal-derived hydrolysate and/or a
plant-derived hydrolysate. Also provided are methods for culturing
a mammalian cell (e.g., a CHO cell) comprising a nucleic acid
encoding bevacizumab, or a fragment thereof, using a cell culture
medium provided herein. Further disclosed herein are cell culture
media compositions that enhance the amount (e.g., enhance the
titer) of bevacizumab, or a fragment thereof, produced from a
mammalian cell in cell culture, as well as compositions comprising
bevacizumab, or a fragment thereof, produced by the methods
described herein.
[0007] Accordingly, in one aspect, the invention provides a method
of producing bevacizumab, or a fragment thereof, comprising the
step of culturing a mammalian cell comprising a nucleic acid
encoding bevacizumab or fragment thereof in a cell culture medium,
wherein the cell culture medium comprises two or more components
selected from the group consisting of copper, insulin, and cystine,
and wherein the cell produces bevacizumab, or a fragment thereof.
In a further embodiment, the cell culture medium comprises copper
and insulin. In another further embodiment, the cell culture medium
comprises copper and cystine. In yet another further embodiment,
the cell culture medium comprises insulin and cystine. In still
another further embodiment, the cell culture medium comprises
copper, insulin, and cystine. In any of the embodiments herein, the
cell culture medium can further comprise a plant-derived
hydrolysate, an animal-derived hydrolysate or both a plant-derived
hydrolysate and an animal-derived hydrolysate. In some of the
embodiments herein, the cell culture medium comprises copper at a
concentration selected from the concentrations listed in Table 1.
In some of the embodiments herein, the cell culture medium
comprises insulin at a concentration selected from the
concentrations listed in Table 1. In some of the embodiments
herein, the cell culture medium comprises cystine at a
concentration selected from the concentrations listed in Table 1.
It is understood that any combination of amounts of copper, insulin
and/or cystine, e.g., the amounts provided in Table 1, are intended
the same as if each and every combination of amounts were
specifically and individually listed. In any of the embodiments
herein, the cell culture medium can comprise insulin at a
concentration of from about 7.0 mg/L to about 11.0 mg/L. In any of
the embodiments herein, the cell culture medium can comprise copper
at a concentration of from about 69.0 nM to about 400.0 nM. In any
of the embodiments herein, the cell culture medium can comprise
cystine at a concentration of from about 0.8 mM to about 2.5 mM. In
any of the embodiments herein, the cell culture medium can comprise
an animal-derived hydrolysate at a concentration of from about 5.6
g/L to about 38.0 g/L. In any of the embodiments herein, the cell
culture medium can comprise a plant-derived hydrolysate at a
concentration of from about 1.4 g/L to about 6.2 g/L. In some
embodiments herein, the cell culture medium is a basal cell culture
medium. In some embodiments herein, the cell culture medium is a
feed cell culture medium. In some embodiments herein, the cell
culture medium is a basal cell culture medium comprising at least
one of copper, insulin, and cystine, and where the basal cell
culture medium is supplemented (e.g., at a period of time following
initiation of a cell culture cycle, such as any one of at least one
time, two times, at least three times, at least four times, at
least five times, at least six times, at least seven times, etc. of
a cell culture cycle) with a feed cell culture medium comprising
any one or more of insulin, an animal-derived hydrolysate and a
plant-derived hydrolysate. In another variation, a feed cell
culture medium comprises any one or more of insulin, an
animal-derived hydrolysate, a plant-derived hydrolysate, cysteine
and cystine. In another variation, a feed cell culture medium
comprises insulin, an animal-derived hydrolysate, a plant-derived
hydrolysate and cysteine. In another variation, a feed cell culture
medium comprises any one or more of insulin, an animal-derived
hydrolysate, a plant-derived hydrolysate and cystine. The feed cell
culture medium may comprise the any one or more of insulin, an
animal-derived hydrolysate, a plant-derived hydrolysate, cysteine
and cystine in any amount provided herein. In some embodiments
herein, the cell culture medium comprises insulin and the method
further comprises the step of adding an additional amount of
insulin to the cell culture medium (e.g., such as via a feed medium
introduced to the basal cell culture medium at a period of time
following initiation of the cell culture cycle). In some
embodiments, the additional amount of insulin is added in an amount
to provide insulin in the cell culture medium at a concentration
selected from the concentrations listed in Table 1. In a further
embodiment, the additional amount of insulin is added to the cell
culture medium at least once during the cell culture cycle. In
another further embodiment, the additional amount of insulin is
added to the cell culture medium at least three times during the
cell culture cycle. In yet another further embodiment, the
additional amount of insulin is added to the cell culture medium at
least six times during the cell culture cycle. In some of the
embodiments herein, the additional amount of insulin is added in an
amount to provide insulin in the cell culture medium at a
concentration of from about 5.6 mg/L to about 66.0 mg/L. In some of
the embodiments herein, the method further comprises the step of
adding an additional amount of animal-derived hydrolysate and/or
plant-derived hydrolysate to the cell culture medium provided
herein (e.g., such as via a feed medium introduced to the basal
cell culture medium at a period of time following initiation of the
cell culture cycle). In some of the embodiments herein, the
additional amount of animal-derived hydrolysate and/or
plant-derived hydrolysate added to the cell culture is added in an
amount to provide animal-derived hydrolysate and/or plant-derived
hydrolysate in the cell culture medium at a concentration selected
from the concentrations listed in Table 1. In any of the
embodiments herein, the cell (e.g., a CHO cell capable of producing
bevacizumab, or a fragment thereof) can be cultured at a
temperature ranging from about 28.degree. C. to about 37.degree. C.
or about 31.degree. C. to about 37.degree. C. In any of the
embodiments herein, bevacizumab, or a fragment thereof, can be
secreted into the cell culture medium. In any of the embodiments
herein, the method can further comprise the step of recovering the
bevacizumab, or a fragment thereof, from the cell culture. In a
particular variation, the recovered bevacizumab is purified.
[0008] Also provided herein are methods of producing bevacizumab,
or a fragment thereof, in a cell culture medium comprising an
animal-derived hydrolysate and a plant-derived hydrolysate and
optionally further comprising copper, insulin and/or cystine. In
one such aspect, the an animal-derived hydrolysate is present in a
greater amount than the plant-derived hydrolysate. In one such
variation, the animal-derived hydrolysate is present in the cell
culture media at a concentration of from about 5.6 g/L to about
38.0 g/L or from about 7.0 g/L to about 35.0 g/L or from about 7.0
g/L to about 25.0 g/L or from about 7.0 g/L to about 15.0 g/L or
from about 8.0 g/L to about 12.0 g/L or from about 7.0 g/L to about
11.0 g/L or about any one of 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L,
30 g/L, 35 g/L, 40 g/L, 45 g/L or 50 g/L or about any one of 5 g/L,
6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, or 12 g/L or about 10
g/L. In another variation, the plant-derived hydrolysate is present
in the cell culture media at a concentration of from about 1.4 g/L
to about 6.2 g/L or from about 1.5 g/L to about 5.5 g/L or from
about 1.5 g/L to about 4.5 g/L or from about 1.5 g/L to about 3.5
g/L or from about 1.5 g/L to about 2.5 g/L or from about 1.75 g/L
to about 2.75 g/L or from about 2.0 g/L to about 3.0 g/L or from
about 2.25 g/L to about 2.75 g/L or about any one of 1.75 g/L, 2.0
g/L, 2.25 g/L, 2.5 g/L, 3.0 g/L, 3.25, 3.5 g/L, 3.75 g/L, or 4.0
g/L or about any one of 2.0 g/L, 2.25 g/L, 2.5 g/L or 3.0 g/L or
about 2.5 g/L. It is understood that each and every combination of
amount of animal-derived hydrolysate and plant-derived hydrolysate
is described the same as if each and every combination were
specifically and individually listed.
[0009] In some aspects, the invention provides bevacizumab, or
fragment thereof, produced by any of the methods described
herein.
[0010] In other aspects, the invention provides a composition
comprising: (i) bevacizumab, or a fragment thereof, produced by any
of the methods described herein and (ii) a pharmaceutically
acceptable carrier.
[0011] In some aspects, the invention also provides a method of
culturing a mammalian cell comprising a nucleic acid encoding
bevacizumab, or a fragment thereof, the method comprising the step
of contacting the mammalian cell with a cell culture medium
comprising two or more components selected from the group
consisting of copper, insulin and cystine. In a further embodiment,
the cell culture medium comprises copper and insulin. In another
further embodiment, the cell culture medium comprises copper and
cystine. In yet another further embodiment, the cell culture medium
comprises insulin and cystine. In still yet another further
embodiment, the cell culture medium comprises copper, insulin, and
cystine. In some embodiments herein, the cell culture medium
further comprises a plant-derived hydrolysate, an animal-derived
hydrolysate or both a plant-derived hydrolysate and an
animal-derived hydrolysate. In some of the embodiments herein, the
cell culture medium comprises insulin at a concentration of from
about 7.0 mg/L to about 11.0 mg/L. In some of the embodiments
herein, the cell culture medium comprises copper at a concentration
of from about 69.0 nM to about 400.0 nM. In some of the embodiments
herein, the cell culture medium comprises cystine at a
concentration of from about 0.8 mM to about 2.5 mM. In some of the
embodiments herein the cell culture medium comprises an
animal-derived hydrolysate at a concentration of from about 5.6 g/L
to about 38.0 g/L. In some of the embodiments herein, the cell
culture medium comprises a plant-derived hydrolysate at a
concentration of from about 1.4 g/L to about 6.2 g/L. In some of
the embodiments herein, the cell culture medium is a basal cell
culture medium. In some embodiments herein, the cell culture medium
is a feed cell culture medium. In some embodiments herein, the cell
culture medium is a basal cell culture medium comprising at least
one of copper, insulin, and cystine, and where the basal cell
culture medium is supplemented (e.g., at a period of time following
initiation of a cell culture cycle, such as any one of at least two
times, at least three times, at least four times, at least five
times, at least six times, at least seven times, etc. of a cell
culture cycle) with a feed cell culture medium comprising any one
or more of insulin, an animal-derived hydrolysate and a
plant-derived hydrolysate. In some embodiments herein, the cell
culture medium comprises insulin and the method further comprises
the step of adding an additional amount of insulin to the cell
culture medium. In a further embodiment, the additional amount of
insulin is added to the cell culture medium at least once during
the cell culture cycle. In another further embodiment, the
additional amount of insulin is added to the cell culture medium at
least three times during the cell culture cycle. In yet another
further embodiment, the additional amount of insulin is added to
the cell culture medium at least six times during the cell culture
cycle. In some embodiments, the additional amount of insulin is
added in an amount to provide insulin in the cell culture medium at
a concentration of from about 5.6 mg/L to about 66.0 mg/L. In some
of the embodiments herein, the method further comprises the step of
adding an additional amount of animal-derived hydrolysate and/or
plant-derived hydrolysate to the cell culture medium provided
herein (e.g., such as via a feed medium introduced to the basal
cell culture medium at a period of time following initiation of the
cell culture cycle). In some of the embodiments herein, the
additional amount of animal-derived hydrolysate and/or
plant-derived hydrolysate added to the cell culture is added in an
amount to provide animal-derived hydrolysate and/or plant-derived
hydrolysate in the cell culture medium at a concentration selected
from the concentrations listed in Table 1. In some of the
embodiments herein, the cell is cultured at a temperature ranging
about 28.degree. C. to about 37.degree. C. or from about 31.degree.
C. to about 37.degree. C. In any of the embodiments herein,
bevacizumab, or a fragment thereof, can be secreted into the cell
culture medium. In some embodiments herein, the mammalian cell is
contacted with the cell culture medium during the cell's growth
phase. In some embodiments herein, the mammalian cell is contacted
with the cell culture medium during the cell's production
phase.
[0012] In other aspects, the invention provides a kit for
supplementing a cell culture medium for use in culturing a
mammalian cell comprising a nucleic acid encoding bevacizumab, or a
fragment thereof, the kit comprising at least two of components
(i)-(iii): (i) insulin in an amount to provide from about 7.0 mg/L
to about 11.0 mg/L insulin in the cell culture medium; (ii) cystine
in an amount to provide from about 0.8 mM to about 2.5 mM cystine
in the cell culture medium; (iii) and copper in an amount to
provide from about 25.0 nM to about 400.0 nM copper in the cell
culture medium. In some embodiments, the kit further comprises a
plant-derived hydrolysate. In a further embodiment, the kit
comprises the plant-derived hydrolysate in an amount to provide
from about 1.4 g/L to about 6.2 g/L plant-derived hydrolysate in
the cell culture medium. In any of the embodiments herein, the kit
can further comprise an animal-derived hydrolysate. In some
embodiments, the kit comprises the animal-derived hydrolysate in an
amount to provide from about 5.6 g/L to about 38.0 g/L
animal-derived hydrolysate in the cell culture medium. The kit may
additionally contain instructions for use, such as instructions for
use in supplementing a cell culture medium.
[0013] In another aspect, the invention provides a cell culture
medium for use in culturing a mammalian cell comprising a nucleic
acid encoding bevacizumab, or a fragment thereof, the cell culture
medium comprising at least two of components (i)-(iii): (i) from
about 7.0 mg/L to about 11.0 mg/L insulin; (ii) from about 25.0 nM
to about 400.0 nM copper; and (iii) from about 0.8 mM to about 2.5
mM cystine. In some embodiments, the cell culture medium comprises
from about 7.0 mg/L to about 11.0 mg/L insulin; and from about 25.0
nM to about 400.0 nM copper. In some embodiments, the cell culture
medium comprises: from about 7.0 mg/L to about 11.0 mg/L insulin;
and from about 0.8 mM to about 2.5 mM cystine. In some embodiments,
the cell culture medium comprises from about 25.0 nM to about 400.0
nM copper; and from about 0.8 mM to about 2.5 mM cystine. In any of
the embodiments herein, the cell culture medium can further
comprise from about 1.4 g/L to about 6.2 g/L plant-derived
hydrolysate. In any of the embodiments herein, the cell culture
medium can further comprise from about 5.6 g/L to about 38.0 g/L
animal-derived hydrolysate.
[0014] In yet another aspect, the invention also provides a
composition comprising (a) a mammalian cell comprising a nucleic
acid encoding bevacizumab, or a fragment thereof; and (b) any cell
culture medium provided herein.
[0015] In another aspect, the invention provides a composition
comprising: (a) bevacizumab, or a fragment thereof; and (b) any
cell culture medium provided herein. In a further embodiment,
bevacizumab, or a fragment thereof, is secreted into the medium by
a mammalian cell comprising a nucleic acid encoding bevacizumab, or
a fragment thereof.
[0016] In some aspects, also provided herein is a method of
enhancing titer of bevacizumab, or a fragment thereof, from a
mammalian cell comprising a nucleic acid encoding bevacizumab, or a
fragment thereof, the method comprising the step of culturing the
mammalian cell in a cell culture medium comprising at least two of
insulin, copper and cystine, wherein titer is enhanced relative to
culturing the mammalian cell in a cell culture medium without at
least two of insulin, copper and cystine. In some embodiments, the
cell culture medium comprises copper and insulin. In some
embodiments, the cell culture medium comprises copper and cystine.
In some embodiments, the cell culture medium comprises insulin and
cystine. In some embodiments, the cell culture medium comprises
copper, insulin, and cystine. In any of the embodiments herein, the
cell culture medium can further comprise a plant-derived
hydrolysate, an animal-derived hydrolysate or both a plant-derived
hydrolysate and an animal-derived hydrolysate. In any of the
embodiments herein, the cell culture medium can comprise insulin at
a concentration of from about 7.0 mg/L to about 11.0 mg/L. In any
of the embodiments herein, the cell culture medium can comprise
copper at a concentration of from about 69.0 nM to about 400.0 nM.
In any of the embodiments herein, the cell culture medium can
comprise cystine at a concentration of from about 0.8 mM to about
2.5 mM. In any of the embodiments herein, the cell culture medium
can comprise an animal-derived hydrolysate at a concentration of
from about 5.6 g/L to about 38.0 g/L. In any of the embodiments
herein, the cell culture medium can comprise a plant-derived
hydrolysate at a concentration of from about 1.4 g/L to about 6.2
g/L. In some embodiments, the cell culture medium is a basal cell
culture medium. In some embodiments herein, the cell culture medium
is a feed cell culture medium. In some embodiments herein, the cell
culture medium is a basal cell culture medium comprising at least
one of copper, insulin, and cystine, and where the basal cell
culture medium is supplemented (e.g., at a period of time following
initiation of a cell culture cycle, such as any one of at least two
times, at least three times, at least four times, at least five
times, at least six times, at least seven times, etc. of a cell
culture cycle) with a feed cell culture medium comprising any one
or more of insulin, an animal-derived hydrolysate and a
plant-derived hydrolysate. In some embodiments, the cell culture
medium comprises insulin and the method further comprises the step
of adding an additional amount of insulin to the cell culture
medium. In a further embodiment, the additional amount of insulin
is added to the cell culture medium at least once during the cell
culture cycle. In another further embodiment, the additional amount
of insulin is added to the cell culture medium at least three times
during the cell culture cycle. In yet another further embodiment,
the additional amount of insulin is added to the cell culture
medium at least six times during the cell culture cycle. In some
embodiments herein, the additional amount of insulin is added in an
amount to provide insulin in the cell culture medium at a
concentration of from about 5.6 mg/L to about 66.0 mg/L. In some of
the embodiments herein, the method further comprises the step of
adding an additional amount of animal-derived hydrolysate and/or
plant-derived hydrolysate to the cell culture medium provided
herein (e.g., such as via a feed medium introduced to the basal
cell culture medium at a period of time following initiation of the
cell culture cycle). In some of the embodiments herein, the
additional amount of animal-derived hydrolysate and/or
plant-derived hydrolysate added to the cell culture is added in an
amount to provide animal-derived hydrolysate and/or plant-derived
hydrolysate in the cell culture medium at a concentration selected
from the concentrations listed in Table 1. In any of the
embodiments herein, the cell may be cultured at a temperature
ranging from about 28.degree. C. to about 37.degree. C. or from
about 31.degree. C. to about 37.degree. C. In any of the
embodiments herein, bevacizumab, or a fragment thereof, can be
secreted into the cell culture medium. In any of the embodiments
herein, the method may further comprise the step of recovering the
bevacizumab, or a fragment thereof, from the cell culture. In a
further aspect, the recovered bevacizumab, or a fragment thereof,
is purified.
[0017] In another aspect, the invention herein provides a method of
culturing a mammalian cell comprising a nucleic acid encoding
bevacizumab, or a fragment thereof, in a cell culture medium
comprising at least two of insulin, copper and cystine, wherein
titer of bevacizumab, or a fragment thereof, is enhanced relative
to culturing the mammalian cell in a cell culture medium without at
least two of insulin, copper and cystine. In some embodiments, the
cell culture medium comprises copper and insulin. In some
embodiments, the cell culture medium comprises copper and cystine.
In some embodiments, the cell culture medium comprises insulin and
cystine. In some embodiments, the cell culture medium comprises
copper, insulin, and cystine. In some of the embodiments herein,
the cell culture medium further comprises a plant-derived
hydrolysate, an animal-derived hydrolysate or both a plant-derived
hydrolysate and an animal-derived hydrolysate. In some of the
embodiments herein, the cell culture medium comprises insulin at a
concentration of from about 7.0 mg/L to about 11.0 mg/L. In some of
the embodiments herein, the cell culture medium comprises copper at
a concentration of from about 69.0 nM to about 400.0 nM. In some of
the embodiments herein, the cell culture medium comprises cystine
at a concentration of from about 0.8 mM to about 2.5 mM. In some of
the embodiments herein, the cell culture medium comprises an
animal-derived hydrolysate at a concentration of from about 5.6 g/L
to about 38.0 g/L. In some of the embodiments herein, the cell
culture medium comprises a plant-derived hydrolysate at a
concentration of from about 1.4 g/L to about 6.2 g/L. In some
embodiments, the cell culture medium is a basal cell culture
medium. In some embodiments herein, the cell culture medium is a
feed cell culture medium. In some embodiments herein, the cell
culture medium is a basal cell culture medium comprising at least
one of copper, insulin, and cystine, and where the basal cell
culture medium is supplemented (e.g., at a period of time following
initiation of a cell culture cycle, such as any one of at least two
times, at least three times, at least four times, at least five
times, at least six times, at least seven times, etc. of a cell
culture cycle) with a feed cell culture medium comprising any one
or more of insulin, an animal-derived hydrolysate and a
plant-derived hydrolysate. In any of the embodiments herein, the
cell culture medium can comprise insulin and the method can further
comprise the step of adding an additional amount of insulin to the
cell culture medium. In a further embodiment, the additional amount
of insulin is added to the cell culture medium at least once during
the cell culture cycle. In another further embodiment, the
additional amount of insulin is added to the cell culture medium at
least three times during the cell culture cycle. In yet another
further embodiment, the additional amount of insulin is added to
the cell culture medium at least six times during the cell culture
cycle. In some of the embodiments herein, the additional amount of
insulin is added in an amount to provide insulin in the cell
culture medium at a concentration of from about 5.6 mg/L to about
66.0 mg/L. In some of the embodiments herein, the method further
comprises the step of adding an additional amount of animal-derived
hydrolysate and/or plant-derived hydrolysate to the cell culture
medium provided herein (e.g., such as via a feed medium introduced
to the basal cell culture medium at a period of time following
initiation of the cell culture cycle). In some of the embodiments
herein, the additional amount of animal-derived hydrolysate and/or
plant-derived hydrolysate added to the cell culture is added in an
amount to provide animal-derived hydrolysate and/or plant-derived
hydrolysate in the cell culture medium at a concentration selected
from the concentrations listed in Table 1. In some of the
embodiments herein, the cell is cultured at a temperature ranging
from about 28.degree. C. to about 37.degree. C. or from about
31.degree. C. to about 37.degree. C., such as a temperature of
about 31.degree. C., 33.degree. C. or 35.degree. C. It is
understood that the temperature may vary (either up or down)
throughout the cell culture process, e.g., within a temperature
ranging from about 28.degree. C. to about 37.degree. C. In one
aspect, the cell is cultured at a first temperature of about
35.degree. C. for a first period of time (such as about 1-10 or 1-8
or 1-7 days), is cultured at a second temperature of about
33.degree. C. for a second period of time (such as about 1-5 or 1-4
or 1-3 or 1-2 days), and is cultured at a third temperature of
about 31.degree. C. for a third period of time (such as about 1-5
or 1-4 or 1-3 or 1-2 days). In any of the embodiments herein,
bevacizumab, or a fragment thereof, can be secreted into the cell
culture medium. In some embodiments, the method further comprises
the step of recovering the bevacizumab, or a fragment thereof, from
the cell culture. In one aspect, the recovered bevacizumab, or a
fragment thereof, is purified.
[0018] The specification is considered to be sufficient to enable
one skilled in the art to practice the invention. Various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description and fall within the scope of the
appended claims. All publications, patents, and patent applications
cited herein are hereby incorporated by reference in their entirety
for all purposes.
DETAILED DESCRIPTION
[0019] Improved and cost-effective methods of producing
bevacizumab, or a fragment thereof, are provided. Cell culture
media comprising components that enable a cell to consistently
produce a desired amount of bevacizumab, or a fragment thereof,
while maintaining acceptable product quality attributes of
bevacizumab, or a fragment thereof, are described. The cell culture
media provided herein may find use in producing manufacturing-scale
amounts of bevacizumab, or a fragment thereof.
[0020] The methods provided herein, including: (i) a method of
producing bevacizumab, or a fragment thereof; (ii) a method of
culturing a mammalian cell comprising a nucleic acid encoding
bevacizumab, or a fragment thereof; and (iii) a method of enhancing
production of bevacizumab, or a fragment thereof, (e.g., enhancing
titer yields of bevacizumab, or a fragment thereof) from a
mammalian cell comprising a nucleic acid encoding bevacizumab, or a
fragment thereof, in one aspect utilize a cell culture medium
comprising two or more of copper, insulin, and cystine (such as a
cell culture medium comprising copper and insulin, or a cell
culture medium comprising copper and cystine, or a cell culture
medium comprising insulin and cystine, or a cell culture medium
comprising copper, insulin and cystine). Bevacizumab, or fragment
thereof, produced by any of the methods detailed herein is also
provided, as are compositions comprising bevacizumab, or fragment
thereof. In one aspect, bevacizumab, or fragment thereof, produced
by any of the methods detailed herein exhibits acceptable product
quality attributes of bevacizumab, or a fragment thereof, such as
N-glycosylation profile, charge heterogeneity, and sequence
integrity. In a particular variation, the product quality
attributes of bevacizumab, or a fragment thereof, are acceptable if
they are substantially similar to bevacizumab, or a fragment
thereof produced by a method that does not use a cell culture
medium comprising at least two of copper, insulin, and cystine. A
cell culture medium comprising two or more of copper, insulin, and
cystine (e.g., a cell culture medium comprising: (i) copper and
insulin; (ii) copper and cystine; (iii) insulin and cystine; or
(iv) copper, insulin and cystine) is also provided. In one
variation, the cell culture medium comprising two or more of
copper, insulin, and cystine enhances production of bevacizumab, or
a fragment thereof, (e.g., enhances titer yields of bevacizumab, or
a fragment thereof) by a mammalian cell cultured in the medium
relative to culturing the mammalian cell in a cell culture medium
without at least two of insulin, copper and cystine. Also provided
herein is a composition comprising a cell culture medium comprising
at least two of copper, insulin, and cystine and (i) a mammalian
cell comprising a nucleic acid encoding bevacizumab, or a fragment
thereof and/or (ii) bevacizumab, or a fragment thereof. A culturing
vessel comprising any of the cell culture media provided herein are
provided. In one aspect, the culturing vessel is a manufacturing
scale culturing vessel, such as a culturing vessel capable of
containing at least 2 liters, at least 10 liters, at least 100
liters, at least 500 liters, at least 1,000 liters, at least 2,500
liters, at least 5,000 liters, at least 7,500 liters, at least
10,000 liters, at least 12,000 liters or more of a cell culture
medium provided herein as is required for producing manufacturing
scale amounts of bevacizumab from cell culture. Thus, the methods
provided herein may find use in a manufacturing-scale production of
bevacizumab, or a fragment thereof.
I. Definitions
[0021] The term "bevacizumab" refers to a recombinant humanized
anti-VEGF monoclonal antibody generated according to Presta et al.
(1997) Cancer Res. 57:4593-4599, also known as "rhuMAb VEGF" or
"AVASTIN.RTM.". It comprises mutated human IgGl framework regions
and antigen-binding complementarity-determining regions from the
murine anti-human VEGF monoclonal antibody A.4.6.1 that blocks
binding of human VEGF to its receptors. Approximately 93% of the
amino acid sequence of bevacizumab, including most of the framework
regions, is derived from human IgGl, and about 7% of the sequence
is derived from the murine antibody A4.6.1. bevacizumab binds to
the same epitope as the monoclonal anti-VEGF antibody A4.6.1
produced by hybridoma ATCC HB 10709. Bevacizumab and other
humanized anti-VEGF antibodies are further described in U.S. Pat.
No. 6,884,879.
[0022] The terms "medium" and "cell culture medium" refer to a
nutrient source used for growing or maintaining cells. As is
understood by a person of skill in the art, the nutrient source may
contain components required by the cell for growth and/or survival
or may contain components that aid in cell growth and/or survival.
Vitamins, essential or non-essential amino acids (e.g., cysteine
and cystine), and trace elements (e.g., copper) are examples of
medium components. Any media provided herein may also be
supplemented with any one or more of insulin, plant hydrolysates
and animal hydrolysates.
[0023] "Culturing" a cell refers to contacting a cell with a cell
culture medium under conditions suitable to the survival and/or
growth and/or proliferation of the cell.
[0024] "Batch culture" refers to a culture in which all components
for cell culturing (including the cells and all culture nutrients)
are supplied to the culturing vessel at the start of the culturing
process.
[0025] "Fed batch cell culture," as used herein refers to a batch
culture wherein the cells and culture medium are supplied to the
culturing vessel initially, and additional culture nutrients are
fed, continuously or in discrete increments, to the culture during
the culturing process, with or without periodic cell and/or product
harvest before termination of culture.
[0026] "Perfusion culture" is a culture by which the cells are
restrained in the culture by, e.g., filtration, encapsulation,
anchoring to microcarriers, etc., and the culture medium is
continuously or intermittently introduced and removed from the
culturing vessel.
[0027] "Culturing vessel" refers to a container used for culturing
a cell. The culturing vessel can be of any size so long as it is
useful for the culturing of cells.
[0028] The term "titer" as used herein refers to the total amount
of recombinantly expressed antibody produced by a cell culture
divided by a given amount of medium volume. Titer is typically
expressed in units of milligrams of antibody per milliliter of
medium. Titer can be expressed or assessed in terms of a relative
measurement, such as a percentage increase in titer as compared
obtaining the protein product under different culture
conditions.
[0029] A "nucleic acid" refers to polymers of nucleotides of any
length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or
bases, and/or their analogs, or any substrate that can be
incorporated into a polymer by DNA or RNA polymerase, or by a
synthetic reaction. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and their analogs. If
present, modification to the nucleotide structure may be imparted
before or after assembly of the polymer.
[0030] An "isolated nucleic acid" means and encompasses a
non-naturally occurring, recombinant or a naturally occurring
sequence outside of or separated from its usual context. An
isolated nucleic acid molecule is other than in the form or setting
in which it is found in nature. Isolated nucleic acid molecules
therefore are distinguished from the nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
includes a nucleic acid molecule contained in cells that ordinarily
express the protein where, for example, the nucleic acid molecule
is in a chromosomal location different from that of natural
cells.
[0031] An "isolated" protein (e.g., an isolated antibody) is one
which has been identified and separated and/or recovered from a
component of its natural environment. Contaminant components of its
natural environment are materials which would interfere with
research, diagnostic or therapeutic uses for the protein, and may
include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. Isolated protein includes the protein in
situ within recombinant cells since at least one component of the
protein's natural environment will not be present. Ordinarily,
however, isolated protein will be prepared by at least one
purification step.
[0032] A "purified" protein (e.g., antibody) means that the protein
has been increased in purity, such that it exists in a form that is
more pure than it exists in its natural environment and/or when
initially produced and/or synthesized and/or amplified under
laboratory conditions. Purity is a relative term and does not
necessarily mean absolute purity.
[0033] "Contaminants" refer to materials that are different from
the desired protein product (e.g., different from an antibody
product). A contaminant may include, without limitation: host cell
materials, such as CHOP; nucleic acid; a variant, fragment,
aggregate or derivative of the desired protein; another
polypeptide; endotoxin; viral contaminant; cell culture media
components, etc.
[0034] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired biological activity. An antibody
can be human, humanized and/or affinity matured.
[0035] The terms "full length antibody," "intact antibody" and
"whole antibody" are used herein interchangeably to refer to an
antibody in its substantially intact form, not antibody fragments
as defined below. The terms particularly refer to an antibody with
heavy chains that contain an Fc region.
[0036] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0037] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen. The Fab
fragment contains the heavy- and light-chain variable domains and
also contains the constant domain of the light chain and the first
constant domain (CH1) of the heavy chain. Fab' fragments differ
from Fab fragments by the addition of a few residues at the carboxy
terminus of the heavy chain CH1 domain including one or more
cysteines from the antibody hinge region. Fab'-SH is the
designation herein for Fab' in which the cysteine residue(s) of the
constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0038] "Fv" is the minimum antibody fragment which contains a
complete antigen-binding site. In one embodiment, a two-chain Fv
species consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent association. In a
single-chain Fv (scFv) species, one heavy- and one light-chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv species. It
is in this configuration that the three HVRs of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six HVRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three HVRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0039] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315, 1994.
[0040] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, e.g., the individual antibodies comprising the
population are identical except for possible mutations, e.g.,
naturally occurring mutations, that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. In
certain embodiments, such a monoclonal antibody typically includes
an antibody comprising a polypeptide sequence that binds a target,
wherein the target-binding polypeptide sequence was obtained by a
process that includes the selection of a single target binding
polypeptide sequence from a plurality of polypeptide sequences. For
example, the selection process can be the selection of a unique
clone from a plurality of clones, such as a pool of hybridoma
clones, phage clones, or recombinant DNA clones. It should be
understood that a selected target binding sequence can be further
altered, for example, to improve affinity for the target, to
humanize the target binding sequence, to improve its production in
cell culture, to reduce its immunogenicity in vivo, to create a
multispecific antibody, etc., and that an antibody comprising the
altered target binding sequence is also a monoclonal antibody of
this invention. In contrast to polyclonal antibody preparations,
which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a
monoclonal antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity,
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins.
[0041] The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),
phage-display technologies (see, e.g., Clackson et al., Nature,
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132 (2004), and technologies for
producing human or human-like antibodies in animals that have parts
or all of the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad.
Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258
(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg
et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813
(1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996);
Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and
Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0042] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art, including
phage-display libraries. Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also
available for the preparation of human monoclonal antibodies are
methods described in Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr.
Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be
prepared by administering the antigen to a transgenic animal that
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled,
e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and
6,150,584 regarding XENOMOUSE.TM. technology). See also, for
example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562
(2006) regarding human antibodies generated via a human B-cell
hybridoma technology.
[0043] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.,
Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0044] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0045] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0046] "Framework" or "FR" residues are those variable domain
residues other than the HVR residues as herein defined.
[0047] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat," and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., supra. Using this numbering system,
the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or HVR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence
[0048] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., Sequences of Immunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The "EU numbering system" or "EU index" is generally used
when referring to a residue in an immunoglobulin heavy chain
constant region (e.g., the EU index reported in Kabat et al.,
supra). The "EU index as in Kabat" refers to the residue numbering
of the human IgGl EU antibody.
[0049] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of the active ingredient to be effective, and which
contains no additional components which are unacceptably toxic to a
subject to which the formulation would be administered. Such
formulations are sterile.
[0050] "Pharmaceutically acceptable" carriers, excipients, or
stabilizers are ones which are nontoxic to the cell or mammal being
exposed thereto at the dosages and concentrations employed
(Remington's Pharmaceutical Sciences (20.sup.th edition), ed. A.
Gennaro), 2000, Lippincott, Williams & Wilkins, Philadelphia,
Pa.). Often the physiologically acceptable carrier is an aqueous pH
buffered solution. Examples of physiologically acceptable carriers
include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as Tween.TM., polyethylene glycol (PEG), and
Pluronics.TM..
[0051] A "sterile" formulation is aseptic or free or essentially
free from all living microorganisms and their spores.
[0052] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the content clearly dictates otherwise. Thus, for example,
reference to "a compound" optionally includes a combination of two
or more such compounds, and the like.
[0053] It is understood that aspects and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
[0054] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X." Numeric ranges are inclusive of the
numbers defining the range.
[0055] Where aspects or embodiments of the invention are described
in terms of a Markush group or other grouping of alternatives, the
present invention encompasses not only the entire group listed as a
whole, but each member of the group individually and all possible
subgroups of the main group, but also the main group absent one or
more of the group members. The present invention also envisages the
explicit exclusion of one or more of any of the group members in
the claimed invention.
II. Cell Culture Media
[0056] Cell culture media provided herein may find use in methods
(e.g., a method of producing bevacizumab, or a fragment thereof; a
method of culturing a mammalian cell comprising a nucleic acid
encoding bevacizumab, or a fragment thereof; and/or a method of
enhancing production of bevacizumab, or a fragment thereof, such as
by enhancing titer yields of bevacizumab, from a mammalian cell
comprising a nucleic acid encoding bevacizumab) and in compositions
(e.g., a composition comprising a cell culture medium comprising at
least two of copper, insulin, and cystine and (i) a mammalian cell
comprising a nucleic acid encoding bevacizumab, or a fragment
thereof and/or (ii) bevacizumab, or a fragment thereof) as detailed
herein.
[0057] In some aspects, the cell culture medium provided herein
comprises components (e.g., at least two of copper, insulin, and
cystine) that may be used in culturing a cell that produces
bevacizumab, or a fragment thereof, wherein the cell, when cultured
in the presence of the media components (e.g., at least two of
copper, insulin, and cystine), produces bevacizumab, or a fragment
thereof, in a desired amount, which may be in an amount that is
greater than the amount of bevacizumab produced by a cell cultured
in a cell culture medium that does not contain the media components
(e.g., a cell culture medium that does not contain at least two of
copper, insulin, and cystine). In one aspect, the cell culture
media provided herein is used in culturing a cell that produces
bevacizumab, or a fragment thereof, wherein the cell, when cultured
in the presence of the media components (e.g., at least two of
copper, insulin, and cystine), produces bevacizumab, or a fragment
thereof, in a desired amount and with an acceptable quality
attribute, such as an acceptable molecular weight. As used herein,
"an acceptable quality attribute" of bevacizumab can refer to a
chemical and/or physical attribute required for regulatory approval
or marketing of bevacizumab and may be the chemical and/or physical
attribute used in assessing lot-to-lot consistency of batches of
bevacizumab, or a fragment thereof, produced by a cell.
[0058] In other aspects of the invention, cell culture media
components (e.g., at least two of copper, insulin, and cystine)
have been identified as capable of providing antibody-producing
cells with improved or acceptable quality attributes that
contribute to higher production of bevacizumab (e.g., results in
higher titer of bevacizumab) as compared to cells that produce
bevacizumab and are cultured in a cell culture medium that does not
contain these components (a cell culture medium that does not
contain at least two of copper, insulin and cystine). Certain
identified media components (e.g., at least two of copper, insulin,
and cystine) can be used to provide an antibody-producing cell
(e.g., a CHO cell) with the capability of producing bevacizumab, or
a fragment thereof, with an acceptable titer, which in one aspect
is a titer greater than the titer obtained when the cells produce
bevacizumab, or a fragment thereof, in a cell culture medium that
does not comprise at least two of copper, insulin, and cystine. As
used herein, "an acceptable titer" of an antibody produced from a
cultured cell (e.g., bevacizumab produced from a CHO cell) can as a
non-limiting example refer to the amount of antibody required to
meet manufacturing-scale production of the antibody or to the
amount of antibody required to assess consistency in lot-to-lot
batches of the antibody product. The cell culture media provided
herein may improve the amount of bevacizumab that is produced from
a cell comprising a nucleic acid encoding bevacizumab as compared
to the amount of bevacizumab produced from the cell cultured in a
different media.
[0059] A cell culture medium for use in culturing a cell for use in
culturing a mammalian cell comprising a nucleic acid encoding
bevacizumab, or a fragment thereof, is provided, wherein the cell
culture medium comprises any one or more of: (a) copper; (b)
insulin; (c) cystine; (d) an animal-derived hydrolysate; and (e) a
plant-derived hydrolysate. In some embodiments, the cell culture
medium comprises 2 or 3 or 4 or 5 of components (a), (b), (c), (d)
and (e). It is understood that the cell culture medium provided
herein may contain any combination of components (a), (b), (c), (d)
and (e) the same as if each and every combination were specifically
and individually listed. For example, it is understood that a cell
culture medium comprising three of components (a), (b), (c), (d)
and (e) may comprise any combination of the components so long as
at least three of the components are present (e.g., a cell culture
medium comprising components (a), (b) and (c) or comprising
components (a), (d) and (e) or comprising components (c), (d) and
(e) are contemplated). In some embodiments, a cell culture medium
provided herein comprises components (a), (b), (c), (d) and (e). In
some embodiments, a cell culture medium provided herein comprises
(a) and (b). In some embodiments, a cell culture medium provided
herein comprises (a) and (c). In some embodiments, a cell culture
medium provided herein comprises (b) and (c).
[0060] In some aspects, a cell culture medium as provided herein
contains one or more media components selected from the group
consisting of copper, insulin, cystine in amounts as described in
Table 1. In some embodiments, the cell culture medium further
comprises an animal-derived hydrolysate in amounts as described in
Table 1. In other embodiments, the cell culture medium further
comprises a plant-derived hydrolysate in amounts as described in
Table 1. In some embodiments, the cell culture medium further
comprises both an animal-derived hydrolysate and a plant-derived
hydrolysate in amounts as described in Table 1.
[0061] It is also understood that a cell culture medium provided
herein may comprise any one or more of the cell culture medium
components of Table 1 (any one or more of copper, insulin, cystine,
an animal-derived hydrolysate and a plant-derived hydrolysate) in
any of the amounts listed in Table 1, the same as if each and every
combination of components and amounts were specifically and
individually listed. In one variation, the cell culture medium
provided herein comprises two or three or four or each of copper,
insulin, cystine, an animal-derived hydrolysate and a plant-derived
hydrolysate in any of the amounts listed in Table 1, the same as if
each and every combination of components and amounts were
specifically and individually listed. In one aspect, the cell
culture medium comprises at least two of copper, insulin and
cystine in any of the amounts listed in Table 1, and an in further
variation further comprises an animal-derived hydrolysate and/or a
plant-derived hydrolysate in any of the amounts listed in Table
1.
TABLE-US-00002 TABLE 1 Exemplary Amounts of Media Components
Component Amount of Component in Medium (a) Insulin from about 1.0
mg/L to about 100.0 mg/L; from about 5.0 mg/L to about 80.0 mg/L;
from about 5.0 mg/L to about 60.0 mg/L; from about 5.0 mg/L to
about 50.0 mg/L; from about 5.0 mg/L to about 40.0 mg/L; from about
5.0 mg/L to about 30.0 mg/L; from about 5.0 mg/L to about 25.0
mg/L; from about 10.0 mg/L to about 25.0 mg/L; from about 10.0 mg/L
to about 30.0 mg/L; from about 15.0 mg/L to about 20.0 mg/L; from
about 5.0 mg/L to about 15.0 mg/L; from about 6.0 mg/L to about
12.0 mg/L; from about 7.0 mg/L to about 11.0 mg/L; from about 8.0
mg/L to about 10.0 mg/L; from about 10 mg/L to about 100 mg/L; from
about 10 mg/L to about 50 mg/L; from about 10 mg/L to about 35
mg/L; from about 10 mg/L to about 250 mg/L; from about 1.0 mg/L to
about 66 mg/L; from about 1.0 mg/L to about 60 mg/L; from about 1.0
mg/L to about 50 mg/L; from about 1.0 mg/L to about 40 mg/L; from
about 1.0 mg/L to about 30 mg/L; from about 1.0 mg/L to about 20
mg/L; from about 1.0 mg/L to about 10 mg/L; from about 10 mg/L to
about 66 mg/L; from about 20 mg/L to about 66 mg/L; from about 30
mg/L to about 66 mg/L; from about 40 mg/L to about 66 mg/L; from
about 50 mg/L to about 66 mg/L; from about 60 mg/L to about 66
mg/L; from about 5.6 mg/L to about 66 mg/L; from about 10 mg/L to
about 60 mg/L; from about 20 mg/L to about 50 mg/L; from about 30
mg/L to about 40 mg/L; from about 1 mg/L to about 14 mg/L; from
about 1.3 mg/L to about 13 mg/L; from about 1.6 mg/L to about 12
mg/L; from about 1.4 mg/L to about 11 mg/L; from about 5.6 mg/L to
about 14 mg/L; from about 5.9 mg/L to about 13 mg/L; from about 6.2
mg/L to about 12 mg/L; from about 7 mg/L to about 11 mg/L; about
any of 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 6.0 or 7.0 or 8.0 or 9.0
or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20
or 21 or 22 or 23 or 24 or 25 or 26 or 27 or27 or 28 or 29 or 30 or
31 or 32 or 33 or 34 or 35 or 36 or 37 or 38 or 39 or 40 or 41 or
42 or 43 or 45 mg/L; any of 5.6 mg/L, 6 mg/L, 6.2 mg/L, 7 mg/L, 8
mg/L, 9 mg/L, 10 mg/L, 11 mg/L, 12 mg/L, 13 mg/L, 14 mg/L or 15
mg/L or 16 mg/L or 17 mg/L or 18 mg/L or 19 mg/L or 20 mg/L or 21
mg/L or 22 mg/L or 23 mg/L or 24 mg/L or 25 mg/L; at least about
any of 1.0 or 3.0 or 5.0 or 7.0 or 10 or 11 or 12 or 13 or 14 or 15
or 16 or 17 or 18 or 19 or 20 mg/L and no more than about 44 or 24
or 14 or 11 mg/L. (b) Cystine from about 0.5 mM to about 2.5 mM;
from about 0.5 mM to about 2.0 mM; from about 0.5 mM to about 1.75
mM; from about 0.5 mM to about 2.5 mM; from about 0.8 mM to about
2.5 mM; from about 0.8 mM to about 2.25 mM; from about 0.8 mM to
about 2.0 mM; from about 0.8 mM to about 1.75 mM; from about 0.8 mM
to about 1.6 mM; from about 0.8 mM to about 1.25 mM; from about 0.8
mM to about 1.0 mM; from about 1.0 mM to about 1.6 mM; from about
1.0 mM to about 2.5 mM; from about 1.25 mM to about 2.5 mM; from
about 1.5 mM to about 2.5 mM; from about 1.75 mM to about 2.5 mM;
from about 2.0 mM to about 2.5 mM; from about 2.25 mM to about 2.5
mM; from about 0.9 mM to about 2.0 mM; from about 0.8 mM to about
1.75 mM; from about 0.9 mM to about 1.5 mM; from about 1.0 mM to
about 1.25 mM; from about 1.0 mM to about 2.0 mM; from about 1.0 mM
to about 1.5 mM; from about 1.2 mM to about 1.4 mM; about any of
0.8 or 0.9 or 1.0 or 1.1 or 1.2 or 1.3 or 1.4 or 1.5 or 1.6 mM; any
of 0.8 mM, 0.85 mM, 0.9 mM, 0.95 mM, 1.0 mM, 1.05 mM, 1.1 mM, 1.15
mM, 1.2 mM, 1.25 mM, 1.3 mM, 1.35 mM, 1.4 mM, 1.5 mM, 1.55 mM, 1.6
mM, 1.65 mM, 1.7 mM, or 1.75 mM; at least about any of 0.8 or 0.9
or 1.0 or 1.1 mM and no more than about 1.75 or 1.6 or 1.5 or 1.4
mM. (c) Copper from about 69 nM to about 1,000.0 nM; from about 20
nM to about 480.0 nM; from about 20 nM to about 400 nM; from about
20 nM to about 350 nM; from about 20 nM to about 300 nM; from about
20 nM to about 250 nM; from about 20 nM to about 200 nM; from about
20 nM to about 150 nM; from about 20 nM to about 100 nM; from about
20 nM to about 50 nM; from about 50 nM to about 480 nM; from about
100 nM to about 480 nM; from about 150 nM to about 480 nM; from
about 200 nM to about 480 nM; from about 250 nM to about 480 nM;
from about 300 nM to about 480 nM; from about 325 nM to about 375
nM; from about 350 nM to about 480 nM; from about 400 nM to about
480 nM; from about 50 nM to about 450 nM; from about 100 nM to
about 400 nM; from about 150 nM to about 350 nM; from about 200 nM
to about 300 nM; from about 22 nM to about 440 nM; from about 26 nM
to about 400 nM; from about 30 nM to about 360 nM; from about 54 nM
to about 480 nM; from about 62 nM to about 440 nM; from about 69 nM
to about 400 nM; from about 80 nM to about 400 nM; from about 100
nM to about 400 nM; from about 125 nM to about 400 nM; from about
150 nM to about 400 nM; from about 200 nM to about 400 nM; from
about 250 nM to about 400 nM; from about 300 nM to about 400 nM;
from about 325 nM to about 375 nM; from about 325 nM to about 350
nM; any of about 25 or 26 or 27 or 28 or 29 or 30 or 40 or 50 or 60
or 69 or 100 or 110 or 120 or 125 or 130 or 140 or 150 or 160 or
170 or 175 or 180 or 190 or 200 or 210 or 220 or 225 or 230 or 240
or 250 or 260 or 270 or 275 or 280 or 290 or 300 or 310 or 320 or
325 or 330 or 335 or 336 or 337 or 338 or 339 or 340 or 345 or 350
or 360 or 370 or 375 or 380 or 390 or 400 nM; any of 54 nM, 56 nM,
58 nM, 60 nM, 62 nM, 64 nM, 66 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72
nM, 73 nM, 74 nM, 75 nM, 100 nM, 125 nM, 150 nM, 175 nM, 200 nM,
225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM, or 400 nM;
at least any of about 20 or 25 or 30 or 35 or 40 or 45 or 50 or 55
or 60 or 65 or 70 or 80 nM and no more than about 420 or 400 or 380
or 360 nM. (d) Animal- from about 6.0 g/L to about 20 g/L; from
about 5.6 g/L to derived about 38 g/L; from about 5.6 g/L to about
30 g/L; from hydrolysate about 5.6 g/L to about 25 g/L; from about
5.6 g/L to about 20 g/L; from about 7.0 g/L to about 20 g/L; from
about 9.0 to about 11.0 g/L; from about 5.6 g/L to about 10 g/L;
from about 5.6 to about 38 g/L; from about 10 g/L to about 38 g/L;
from about 15 g/L to about 38 g/L; from about 20 g/L to about 38
g/L; from about 25 g/L to about 38 g/L; from about 30 g/L to about
38 g/L; from about 35 g/L to about 38 g/L; from about 10 g/L to
about 30 g/L; from about 15 g/L to about 25 g/L; from about 5.6 g/L
to about 14 g/L; from about 5.9 g/L to about 13 g/L; from about 6.2
g/L to about 12 g/L; from about 7.0 g/L to about 11.0 g/L; from
about 7.0 g/L to about 35.0 g/L; from about 7.0 g/L to about 25.0
g/L; from about 7.0 g/L to about 15.0 g/L; from about 8.0 g/L to
about 12.0 g/L; any of about 2.8 or 3.0 or 3.2 or 3.4 or 3.6 or 3.8
or 4.0 or 4.2 or 4.4 or 4.6 or 4.8 or 5.0 or 5.2 or 5.4 or 5.6 or 6
or 6.2 or 7 or 7.4 or 7.8 or 8 or 9 or 10 or 11 or 12 or 13 or 14
or 15 or 20 or 25 or 30 or 35 or 40 or 45 or 50 g/L; any of 2.8
g/L, 3.0 g/L, 3.2 g/L, 3.4 g/L, 3.6 g/L, 3.8 g/L, 4.0 g/L, 4.2 g/L,
4.4 g/L, 4.6 g/L, 4.8 g/L, 5.0 g/L or 6 g/L, 6.2 g/L, 7 g/L, 8 g/L,
9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, or 14 g/L; at least any of
about 2.8 or 3.2 or 3.4 or 3.6 or 3.8 or 4.0 or 4.2 or 4.6 or 5.0
or 5.6 or 6 or 6.2 or 7 or 8 or 9 g/L and no more than about 14 or
13 or 12 or 11 g/L. (e) Plant- from about 1.0 g/L to about 10.0
g/L; from about 1.4 g/L derived to about 6.2 g/L; from about 1.4
g/L to about 6.0 g/L; from hydrolysate about 1.4 g/L to about 5.5
g/L; from about 1.4 g/L to about 5.0 g/L; from about 1.4 g/L to
about 4.5 g/L; from about 1.4 g/L to about 4.0 g/L; from about 1.4
g/L to about 3.5 g/L; from about 1.4 g/L to about 3.0 g/L; from
about 1.4 g/L to about 2.5 g/L; from about 1.4 g/L to about 2.0
g/L; from about 2.0 g/L to about 6.2 g/L; from about 2.5 g/L to
about 6.2 g/L; from about 3.0 g/L to about 6.2 g/L; from about 3.5
g/L to about 6.2 g/L; from about 4.0 g/L to about 6.2 g/L; from
about 4.5 g/L to about 6.2 g/L; from about 5.0 g/L to about 6.2
g/L; from about 5.5 g/L to about 6.2 g/L; from about 1.5 g/L to
about 6.0 g/L; from about 2.0 g/L to about 5.5 g/L; from about 2.5
g/L to about 5.0 g/L; from about 3.0 g/L to about 4.5 g/L; from
about 3.0 g/L to about 3.2 g/L; from about 3.5 g/L to about 4.0
g/L; from about 1.4 g/L to about 3.4 g/L; from about 1.5 g/L to
about 3.0 g/L; from about 1.75 g/L to about 2.8 g/L; from about 1.5
g/L to about 5.5 g/L; from about 1.5 g/L to about 4.5 g/L; from
about 1.5 g/L to about 3.5 g/L; from about 1.5 g/L to about 2.5 g/L
from about 1.75 g/L to about 2.75 g/L; from about 2.0 g/L to about
3.0 g/L; from about 2.25 g/L to about 2.75 g/L; any of about 1.4 or
1.5 or 1.75 or 2.0 or 2.25 or 2.5 or 2.8 or 3.0 or 3.1 or 3.25 or
3.4 or 3.5 or 3.75 or 4.0 g/L; any of 1.4 g/L, 1.5 g/L, 1.75 g/L,
2.0 g/L, 2.25 g/L, 2.5 g/L, 2.8 g/L, 3.0 g/L, 3.2 g/L, 3.25 g/L,
3.4, 3.75, or 4.0 g/L; at least any of about 1.4 or 1.5 or 1.75 g/L
and no more than about 3.4 or 3 or 2.8 g/L.
[0062] In one variation, insulin is present in the cell culture
media at a concentration of from about 1.0 mg/L to about 100.0 mg/L
or from about 5.0 mg/L to about 80.0 mg/L or from about 5.0 mg/L to
about 60.0 mg/L or from about 5.0 mg/L to about 50.0 mg/L or from
about 5.0 mg/L to about 40.0 mg/L or from about 5.0 mg/L to about
30.0 mg/L or from about 5.0 mg/L to about 25.0 mg/L or from about
10.0 mg/L to about 25.0 mg/L or from about 10.0 mg/L to about 30.0
mg/L or from about 15.0 mg/L to about 20.0 mg/L or from about 5.0
mg/L to about 15.0 mg/L or from about 6.0 mg/L to about 12.0 mg/L
or from about 7.0 mg/L to about 11.0 mg/L or from about 8.0 mg/L to
about 10.0 mg/L or at a concentration of about any one of 5.0 mg/L,
6.0 mg/L, 7.0 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0
mg/L, 13.0 mg/L, 14.0 mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0
mg/L, 19.0 mg/L, 20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0
mg/L, 25.0 mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0
mg/L or at a concentration of about any one of 7 mg/L, 8.0 mg/L,
9.0 mg/L, 10.0 mg/L or 11.0 mg/L.
[0063] In one variation, copper is present in the cell culture
media at a concentration of from about 69.0 nM to about 400.0 nM or
from about 80 nM to about 400 nM or from about 100 nM to about 400
nM or from about 125 nM to about 400 nM or from about 150 nM to
about 400 nM or from about 200 nM to about 400 nM or from about 250
nM to about 400 nM or from about 300 nM to about 400 nM or from
about 325 nM to about 375 nM or from about 325 nM to about 350 nM
or at a concentration of about any one of 100 nM, 125 nM, 150 nM,
175 nM, 200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375
nM or 400 nM or at a concentration of about any one of 330 nM, 335
nM, 340 nM, 345 nM or 350 nM or at a concentration of about 335 nM,
336 nM, 337 nM, 338 nM, 339 nM or 400 nM or at a concentration of
about 339 nM.
[0064] In one variation cystine is present in the cell culture
medium at a concentration of from about 0.8 mM to about 2.5 mM or
from about 0.8 mM to about 2.0 mM or from about 0.8 mM to about
1.75 mM or from about 0.8 mM to about 1.5 mM or from about 1.0 mM
to about 2.0 mM or from about 1.0 mM to about 1.5 mM or from about
1.2 mM to about 1.4 mM or at a concentration of about any one of
0.8 mM or 0.9 mM or 1.0 mM or 1.1 mM or 1.2 mM or 1.3 mM or 1.4 mM
or 1.5 mM or at a concentration of about any one of 1.1 mM, 1.3 mM
or 1.5 mM or at a concentration of about 1.3 mM.
[0065] In one variation, an animal-derived hydrolysate is present
in the cell culture media at a concentration of from about 5.6 g/L
to about 38.0 g/L or from about 7.0 g/L to about 35.0 g/L or from
about 7.0 g/L to about 25.0 g/L or from about 7.0 g/L to about 15.0
g/L or from about 8.0 g/L to about 12.0 g/L or from about 7.0 g/L
to about 11.0 g/L or about any one of 5 g/L, 10 g/L, 15 g/L, 20
g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, 45 g/L or 50 g/L or about any
one of 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, or 12 g/L
or about 10 g/L.
[0066] In one variation, an plant-derived hydrolysate is present in
the cell culture media at a concentration of from about 1.4 g/L to
about 6.2 g/L or from about 1.5 g/L to about 5.5 g/L or from about
1.5 g/L to about 4.5 g/L or from about 1.5 g/L to about 3.5 g/L or
from about 1.5 g/L to about 2.5 g/L or from about 1.75 g/L to about
2.75 g/L or from about 2.0 g/L to about 3.0 g/L or from about 2.25
g/L to about 2.75 g/L or about any one of 1.75 g/L, 2.0 g/L, 2.25
g/L, 2.5 g/L, 3.0 g/L, 3.25, 3.5 g/L, 3.75 g/L, or 4.0 g/L or about
any one of 2.0 g/L, 2.25 g/L, 2.5 g/L or 3.0 g/L or about 2.5
g/L.
[0067] In a further variation, cysteine is present in the cell
culture medium. Cysteine may in one aspect be added to a basal cell
culture medium (e.g., by supplementing the basal cell culture
medium with a feed medium comprising cysteine). In one variation, a
cell culture medium comprises cysteine (which may be added to a
basal cell culture medium that does not comprise cysteine via a
feed medium comprising cysteine) in a concentration of from about
0.5 mM to about 5.0 mM or from about 1.0 mM to about 12.0 mM or
from about 2.0 mM to about 10.0 mM or from about 2.0 mM to about
8.0 mM or about 1.0 mM to about 10.0 mM or from about 1.0 mM to
about 8.0 mM or from about 2.0 mM to about 12.0 mM or from about
3.0 mM to about 12.0 mM or from about 4.0 mM to about 12.0 mM or
from about 5.0 mM to about 12.0 mM or from about 6.0 mM to about
12.0 mM or from about 6.0 mM to about 10.0 mM or from about 6.0 mM
to about 8.0 mM or about any one of 0.5 mM, 0.8 mM, 1.0 mM, 1.5 mM,
2.0 mM, 2.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0
mM, 8.5 mM or 9.0 mM. In one aspect, cysteine is added to a basal
cell culture media (which addition may be at any point in time of
the cell culture cycle and may be in one or more amounts, which may
be the same or different), in an amount such that cysteine is
present in the cell culture media at a concentration of about 7.5
mM.
[0068] In a further variation, cystine is present in the cell
culture medium. Cystine may in one aspect be added to a basal cell
culture medium (e.g., by supplementing the basal cell culture
medium which may or may not already comprise cystine with a feed
medium comprising cystine). In one variation, a cell culture medium
comprises cystine (which may be added to a basal cell culture
medium via a feed medium comprising cystine) in a concentration of
from about 0.5 mM to about 5.0 mM, such as in an amount to provide
about 0.8 mM cysteine in the cell culture medium.
[0069] In certain embodiments, the cell culture medium comprises
cystine but is free of cysteine.
[0070] The cell culture medium (e.g., a basal cell culture medium)
may further be supplemented with an additional cell culture medium
components (e.g., such as via a feed cell culture medium). In one
aspect, the additional cell culture medium component comprises
insulin. In another aspect, the additional cell culture medium
component comprises insulin and cysteine. A cell culture media
provided herein may be supplemented with any amount of insulin
and/or cysteine that is suitable for culturing a cell. In one
aspect, insulin is added to a cell culture medium (e.g., added to a
basal cell culture medium at one or more point in time of the cell
culture cycle) in an amount to provide a concentration of insulin
in the cell culture of about 15 mg/L or about 25 mg/L. In one
aspect, insulin is added to a cell culture medium (e.g., added to a
basal cell culture medium at one or more point in time of the cell
culture cycle) in an amount to provide a concentration of insulin
in the cell culture selected from the group consisting of: from
about 1.0 mg/L to about 100.0 mg/L; from about 10.0 mg/L to about
100.0 mg/L; from about 10.0 mg/L to about 50.0 mg/L; from about
10.0 mg/L to about 35.0 mg/L; from about 10.0 mg/L to about 25.0
mg/L; from about 5.0 mg/L to about 80.0 mg/L; from about 5.0 mg/L
to about 60.0 mg/L; from about 5.0 mg/L to about 50.0 mg/L; from
about 5.0 mg/L to about 30.0 mg/L; from about 5.0 mg/L to about
25.0 mg/L; from about 10.0 mg/L to about 25.0 mg/L; from about 10.0
mg/L to about 30.0 mg/L; from about 15.0 mg/L to about 20.0 mg/L;
from about 5.0 mg/L to about 15.0 mg/L; from about 6.0 mg/L to
about 12.0 mg/L; from about 7.0 mg/L to about 11.0 mg/L; from about
8.0 mg/L to about 10.0 mg/L; about any one of 5.0 mg/L, 6.0 mg/L,
7.0 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0 mg/L, 13.0
mg/L, 14.0 mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0 mg/L, 19.0
mg/L, 20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0 mg/L, 25.0
mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0 mg/L;
about any one of 7 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L and 11.0
mg/L. In another aspect, cysteine is added to a cell culture medium
(e.g., added to a basal cell culture medium at one or more point in
time of the cell culture cycle) in an amount to provide a
concentration of cysteine in the cell culture selected from the
group consisting of: from about 1.0 mM to about 12.0 mM or from
about 2.0 mM to about 10.0 mM or from about 2.0 mM to about 8.0 mM
or about 1.0 mM to about 10.0 mM or from about 1.0 mM to about 8.0
mM or from about 2.0 mM to about 12.0 mM or from about 3.0 mM to
about 12.0 mM or from about 4.0 mM to about 12.0 mM or from about
5.0 mM to about 12.0 mM or from about 6.0 mM to about 12.0 mM or
from about 6.0 mM to about 10.0 mM or from about 6.0 mM to about
8.0 mM or about any one of 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM,
7.5 mM, 8.0 mM, 8.5 mM or 9.0 mM. In another aspect, cystine is
added to a cell culture medium (e.g., added to a basal cell culture
medium at one or more point in time of the cell culture cycle) in
an amount to provide a concentration of cystine in the cell culture
of from about 0.1 mM to about 1.5 mM, such as a concentration of
about 0.2 mM.
[0071] In some aspects, a cell culture medium provided herein
comprises from about 5.0 mg/L to about 14.0 mg/L, from about 5.5
mg/L to about 13.0 mg/L, from about 6.0 mg/L to about 12.0 mg/L,
from about 7.0 mg/L to about 11.0 mg/L, from about 8.0 mg/L to
about 10.0 mg/L, or from about 8.5 mg/L to about 14.0 mg/L insulin.
It is understood that the cell culture medium comprising insulin
may further comprise any one or more of copper and cystine in any
amount provided herein. For example, it is understood that a cell
culture medium comprising from about 6.0 mg/L to about 12.0 mg/L
insulin may further comprise from about 70 nM to about 400 nM of
copper and/or from about 0.5 mM to about 2.5 mM cystine and may
further comprise an animal-derived hydrolysate and/or plant-derived
hydrolysate, for example an animal-derived hydrolysate from about
5.5 g/L to about 40.0 g/L and/or a plant-derived hydrolysate from
about 1.5 g/L to about 6.5 g/L.
[0072] In other aspects, a cell culture medium provided herein
comprises from about 65 nM to about 400 nM, from about 70 nM to
about 375 nM, from about 75 nM to about 350 nM, from about 80 nM to
about 325 nM, from about 85 nM to about 300 nM, or from about 90 nM
to about 275 nM copper. It is understood that the cell culture
medium comprising copper may further comprise any one or more of
insulin and cystine in any amount provided herein. For example, it
is understood that a cell culture medium comprising from about 85
nM to about 300 nM copper may further comprise from about 0.8 mM to
about 1.75 mM of cystine and/or from about 8.0 mg/L to about 12.0
mg/L insulin and may further comprise an animal-derived hydrolysate
and/or plant-derived hydrolysate, for example an animal-derived
hydrolysate from about 5.5 g/L to about 40.0 g/L and/or a
plant-derived hydrolysate from about 1.5 g/L to about 6.5 g/L.
[0073] In some aspects, a cell culture medium provided herein
comprises from about 0.8 mM to about 1.75 mM, from about 0.9 mM to
about 1.50 mM, from about 1.0 mM to about 1.40 mM, or from about
1.0 mM to about 1.30 mM cystine. It is understood that the cell
culture medium comprising cystine may further comprise any one or
more of insulin and copper in any amount provided herein. For
example, it is understood that a cell culture medium comprising
from about 0.8 mM to about 1.75 mM cystine may further comprise
from about 70 nM to about 375 nM of copper and/or from about 8.0
mg/L to about 12.0 mg/L insulin and may further comprise an
animal-derived hydrolysate and/or plant-derived hydrolysate, for
example an animal-derived hydrolysate from about 5.5 g/L to about
40.0 g/L and/or a plant-derived hydrolysate from about 1.5 g/L to
about 6.5 g/L.
[0074] In some embodiments, the cell culture medium further
comprises an animal-derived hydrolysate in amounts as described in
Table 1. In other embodiments, the cell culture medium further
comprises a plant-derived hydrolysate in amounts as described in
Table 1. In some embodiments, the cell culture medium further
comprises both an animal-derived hydrolysate and a plant-derived
hydrolysate in amounts as described in Table 1.
[0075] In some aspects, a cell culture medium provided herein
comprises from about 1.5 g/L to about 6.0 g/L, from about 2.0 g/L
to about 5.5 g/L, from about 2.5 g/L to about 5.0 g/L, from about
3.0 g/L to about 4.5 g/L, or from about 3.5 g/L to about 4.0 g/L
plant-derived hydrolysate. It is understood that the cell culture
medium comprising plant-derived hydrolysate may further comprise
any one or more of cystine, insulin and copper in any amount
provided herein. For example, it is understood that a cell culture
medium comprising from about 0.8 mM to about 1.75 mM cystine may
further comprise from about 70 nM to about 375 nM of copper and/or
from about 8.0 mg/L to about 12.0 mg/L insulin and may further
comprise an animal-derived hydrolysate, for example an
animal-derived hydrolysate from about 6.0 g/L to about 20.0
g/L.
[0076] In some aspects, a cell culture medium provided herein
comprises from about 6.0 g/L to about 35.0 g/L, from about 7.0 g/L
to about 30.0 g/L, from about 8.0 g/L to about 25.0 g/L, from about
9.0 g/L to about 20 g/L, or from about 10.0 g/L to about 15.0 g/L
animal-derived hydrolysate. It is understood that the cell culture
medium comprising animal-derived hydrolysate may further comprise
any one or more of cystine, insulin and copper in any amount
provided herein. For example, it is understood that a cell culture
medium comprising from about 0.8 mM to about 1.75 mM cystine may
further comprise from about 70 nM to about 375 nM of copper and/or
from about 8.0 mg/L to about 12.0 mg/L insulin and may further
comprise an plant-derived hydrolysate, for example an plant-derived
hydrolysate from about 1.5 g/L to about 3.0 g/L.
[0077] In some of the embodiments herein, the cell culture medium
comprises from about 0.9 mM to about 1.5 mM cystine. In some of the
embodiments herein, the cell culture medium comprises from about
1.4 mg/L to about 11.0 mg/L insulin or from about 1.44 mg/L to
about 66.0 mg/L insulin. In some of the embodiments herein, the
cell culture medium comprises from about 26.0 nM to about 400.0 nM
copper. In some aspects, a cell culture medium comprises two or
more components selected from: (a) about 69.0 nM to about 400.0 nM
copper, (b) from about 7.0 mg/L to about 11.0 mg/L insulin or from
about 1.44 mg/L to about 66 mg/L insulin, and (c) from about 0.8 mM
to about 2.5 mM cystine.
[0078] Cell culture media components described herein (e.g., a cell
culture media comprising any one or more of copper, insulin,
cystine, an animal-derived hydrolysate and a plant-derived
hydrolysate) may be added to a cell culture medium in a form that
is known in the art, such as a salt, a hydrate or combination
thereof. The cell culture media components can also be provided to
the cell culture media as a solution, an extract, or in solid form.
As a non-limiting example, cystine may be provided to the cell
culture medium as the disodium salt monohydrate powder. Protein
hydrolysates, also known as peptones, are typically manufactured by
enzymatic digestion of a variety of biologically based starting
materials such as animal tissues, milk-derived products,
microorganisms or plants. The hydrolysate used in the cell culture
medium provided herein can be derived from a plant or an animal
(e.g., plant-derived hydrolysate and/or animal-derived
hydrolysate). A plant hydrolysate as described herein can be
derived from, but not limited to, wheat gluten, maize, cereal, soy,
or cottonseed. An animal hydrolysate as described herein can be
derived from, but not limited to, bovine, chicken, caprine, equine,
human, ovine, porcine, or rabbit or other animals.
[0079] A method of preparing a cell culture medium for use in
culturing a mammalian cell comprising a nucleic acid encoding
bevacizumab, or a fragment thereof, is also provided, wherein the
method comprises combining any two or more of copper, insulin and
cystine in a composition suitable for cell culture. In one aspect,
the method comprises adding any two or more of copper, insulin and
cystine to a composition suitable for cell culture, wherein the two
or more of copper, insulin and cystine may be added to the
composition sequentially or simultaneously. In a further variation,
a method of preparing a cell culture medium for use in culturing a
mammalian cell comprising a nucleic acid encoding bevacizumab, or a
fragment thereof, is provided, wherein the method comprises
combining any two or more of copper, insulin and cystine in a
composition suitable for cell culture at a first period of time and
wherein the method further comprises adding an amount of insulin at
a second period of time, such as at least once, at least twice, at
least three times, at least four time, at least five times, at
least six times, at least seven times, etc. of a cell growth cycle.
In some embodiments, a cell growth cycle is at least 1 day, 2 days,
3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11
days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18,
days, 19 days, 20 days, or any amount of days wherein the cells may
remain in cell culture while still remaining viable. In one
variation of a method of preparing a cell culture medium, cystine
is added in an amount to provide about 0.9 mM to about 1.5 mM
cystine in the cell culture medium. In another variation of a
method of preparing a cell culture medium, insulin is added in an
amount to provide from about 1.4 mg/L to about 11.0 mg/L insulin or
from about 1.44 mg/L to about 66.0 mg/L insulin in the cell culture
medium. In another variation of a method of preparing a cell
culture medium, copper is added in an amount to provide from about
26.0 nM to about 400.0 nM copper in the cell culture medium. In
some aspects, a cell culture medium is prepared by combining two or
more components selected from: (a) copper in an amount to provide
about 69.0 nM to about 400.0 nM copper in the cell culture medium,
(b) insulin in an amount to provide from about 7.0 mg/L to about
11.0 mg/L or from about 1.44 mg/L to about 66 mg/L insulin in the
cell culture medium, and (c) cystine in an amount to provide from
about 0.8 mM to about 2.5 mM cystine in the cell culture
medium.
[0080] In some embodiments herein, the cell culture medium is a
basal cell culture medium. In other embodiments herein, the cell
culture medium is a feed cell culture medium. In some embodiments
herein, the cell culture medium is a basal cell culture medium
comprising at least one of copper, insulin, and cystine, and where
the basal cell culture medium is supplemented (e.g., at a period of
time following initiation of a cell culture cycle, such as any one
of at least two times, at least three times, at least four times,
at least five times, at least six times, at least seven times, etc.
of a cell culture cycle) with a feed cell culture medium comprising
any one or more of insulin, an animal-derived hydrolysate and a
plant-derived hydrolysate.
[0081] Individual media components provided herein may be present
in amounts that result in one or more advantageous properties for
culturing cells comprising a nucleic acid encoding bevacizumab, or
a fragment thereof, and/or for bevacizumab production from cell
culture. Advantageous properties include, but are not limited to,
increased cell viability, increase in the amount of bevacizumab
produced from the cell (e.g., enhanced bevacizumab titer) and/or
reduced oxidation of bevacizumab in cell culture. Advantageous
properties of the cell culture media provided herein may also
include maintaining or enhancing the amount of bevacizumab produced
by the cells (e.g., antibody titer) while maintaining the
N-glycosylation profile, the charge heterogeneity and/or the amino
acid sequence integrity of bevacizumab, or a fragment thereof.
These advantageous properties are applicable to methods of
culturing a cell comprising a nucleic acid encoding bevacizumab, or
a fragment thereof and to methods of producing bevacizumab, or a
fragment thereof in cell culture as described herein.
[0082] A cell culture medium provided herein in one aspect results
in one or more favorable product quality attribute or advantageous
property when used in a method of producing bevacizumab, or a
fragment thereof. In one variation, use of the cell culture medium
provided herein increases the amount of bevacizumab produced by the
cells (e.g., enhances antibody titer) as compared to the amount of
bevacizumab, or a fragment thereof, produced by culturing the cell
producing bevacizumab in a different cell culture medium.
[0083] As would be understood by the skilled artisan, the cell
culture media detailed herein may comprise other components (e.g.,
besides the one or more of copper, insulin, and cystine, and
optionally peptone hydrolysate) that are useful for cell culture.
For example, it is understood that the cell culture media may
comprise additional components such as amino acids (e.g.,
glutamine, arginine, or asparagine), vitamins (including but not
limited to B vitamins such as any one or more of vitamin B1,
vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin B9, or
vitamin B12), trace elements, transition metals (including but not
limited to nickel, iron (e.g., ferric iron or ferrous iron), or
zinc), and other media components. Any media provided herein may
also be supplemented with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), ions
(such as sodium, chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleosides (such as adenosine and
thymidine), trace elements and glucose or an equivalent energy
source. Additional cell culture media components, such as those
listed herein, may be included in the cell culture medium at
appropriate concentrations that would be known to those skilled in
the art.
III. Methods and Uses of the Invention
[0084] Provided herein are methods of culturing cells used in the
production bevacizumab, or a fragment thereof, and use of cell
culture media that comprise one or more of copper, insulin and
cystine. In some aspects, a method is provided for culturing a
mammalian cell comprising a nucleic acid encoding bevacizumab or
fragment thereof, wherein the method comprises the step of
contacting the mammalian cell with a cell culture medium comprising
at least two of copper, insulin and cystine, wherein the cell
culture medium may additionally comprise a plant-derived
hydrolysate, an animal-plant-derived hydrolysate or both a
plant-derived hydrolysate and an animal-derived hydrolysate. In
some embodiments, the cell culture media comprises insulin. In some
of the embodiments herein, the cell culture media comprises copper.
In some of the embodiments herein, the cell culture media comprises
cystine. In some of the embodiments herein, the cell culture media
comprises copper and cystine. In some of the embodiments herein,
the cell culture media comprises copper and insulin. In some of the
embodiments herein, the cell culture media comprises insulin and
cystine. In some of the embodiments herein, the amount of the
components in the cell culture medium (e.g., the amount of copper,
insulin, cystine, plant-derived hydrolysate and/or animal-derived
hydrolysate) is in an amount selected from a value provided in
Table 1. In some embodiments, the method further comprises the step
of adding an additional amount of insulin to the medium. The
additional amount of insulin can be added to the cell culture
medium at least once, at least three times, at least 6 times or at
least 12 times during the cell culture cycle. In some of the
embodiments herein, the additional amount of insulin added to the
cell culture is added in an amount to provide insulin in the cell
culture medium at a concentration selected from Table 1 such as
from about 1 mg/L to about 44 mg/L. In some aspects, the cell
culture medium further comprises an animal-derived hydrolysate, a
plant-derived hydrolysate, or both an animal-derived hydrolysate
and a plant-derived hydrolysate.
[0085] In some other aspects, a method is provided for culturing a
cell comprising a nucleic acid encoding bevacizumab or fragment
thereof, wherein the method comprises the step of contacting the
cell with a cell culture medium comprising two or more components
selected from the group consisting of copper, insulin, and cystine.
In some embodiments herein, the cell culture medium comprises
insulin at a concentration of from about 7.0 mg/L to about 11.0
mg/L. In some embodiments herein, the cell culture medium comprises
copper at a concentration of from about 69.0 nM to about 400.0 nM.
In some embodiments herein, the cell culture medium comprises
cystine at a concentration of from about 0.8 mM to about 2.5 mM. In
some embodiments herein the cell culture medium comprises insulin
at a concentration of from about 7.0 mg/L to about 11.0 mg/L and
copper at a concentration of from about 69.0 nM to about 400.0 nM.
In some embodiments herein the cell culture medium comprises
insulin at a concentration of from about 7.0 mg/L to about 11.0
mg/L and cystine at a concentration of from about 0.8 mM to about
2.5 mM. In some embodiments herein the cell culture medium
comprises copper at a concentration of from about 69.0 nM to about
400.0 nM and cystine at a concentration of from about 0.8 mM to
about 2.5 mM. In some embodiments herein the cell culture medium
comprises insulin at a concentration of from about 7.0 mg/L to
about 11.0 mg/L, copper at a concentration of from about 69.0 nM to
about 400.0 nM and cystine at a concentration of from about 0.8 mM
to about 2.5 mM. In any of the embodiments herein, the cell culture
medium may comprise cystine, insulin and/or copper in an amount
selected from Table 1. In some of the embodiments herein, the
method further comprises the step of adding an additional amount of
insulin to the cell culture medium provided herein (e.g., such as
via a feed medium introduced to the basal cell culture medium at a
period of time following initiation of the cell culture cycle). The
additional amount of insulin can be added to the cell culture
medium at least once, at least two times, at three times, at least
six times, at least nine times, at least twelve times, or at least
fourteen times during the cell culture cycle. In some of the
embodiments herein, the additional amount of insulin added to the
cell culture is added in an amount to provide insulin in the cell
culture medium at a concentration selected from Table 1 such as 5.6
mg/L to about 66 mg/L. In some aspects, the cell culture medium
further comprises an animal-derived hydrolysate, a plant-derived
hydrolysate, or both an animal-derived hydrolysate and a
plant-derived hydrolysate. In some of the embodiments herein, the
method further comprises the step of adding an additional amount of
animal-derived hydrolysate and plant-derived hydrolysate to the
cell culture medium provided herein (e.g., such as via a feed
medium introduced to the basal cell culture medium at a period of
time following initiation of the cell culture cycle). In some of
the embodiments herein, the additional amount of animal-derived
hydrolysate and plant-derived hydrolysate added to the cell culture
is added in an amount to provide animal-derived hydrolysate and
plant-derived hydrolysate in the cell culture medium at a
concentration selected from the concentrations listed in Table
1.
[0086] In another aspect, provided herein are methods of producing
bevacizumab or a fragment thereof, wherein the method comprises the
step of contacting a cell capable of producing bevacizumab or a
fragment thereof with a cell culture medium comprising two or more
components selected from the group consisting of copper, insulin,
and cystine. In some embodiments herein, the cell culture medium
comprises insulin at a concentration of from about 7.0 mg/L to
about 11.0 mg/L. In some embodiments herein, the cell culture
medium comprises copper at a concentration of from about 69.0 nM to
about 400.0 nM. In some embodiments herein, the cell culture medium
comprises cystine at a concentration of from about 0.8 mM to about
2.5 mM. In any of the embodiments herein, the cell culture medium
may comprise cystine, insulin or copper in an amount selected from
Table 1. In some of the embodiments herein, the method further
comprises the step of adding an additional amount of insulin to the
cull culture medium provided herein. Insulin may be added to the
cell culture medium in any amount that is suitable for cell
culture. In one aspect, insulin is added to the cell culture medium
in an amount to provide insulin in the cell culture medium at a
concentration selected from the concentrations listed in Table 1.
In a particular aspect, insulin is added to the cell culture medium
in an amount to provide insulin in the cell culture medium at a
concentration selected from the group consisting of: from about 1.0
mg/L to about 100.0 mg/L; from about 5.0 mg/L to about 80.0 mg/L;
from about 5.0 mg/L to about 60.0 mg/L; from about 5.0 mg/L to
about 50.0 mg/L; from about 5.0 mg/L to about 40.0 mg/L; from about
5.0 mg/L to about 30.0 mg/L; from about 5.0 mg/L to about 25.0
mg/L; from about 10.0 mg/L to about 25.0 mg/L; from about 10.0 mg/L
to about 30.0 mg/L; from about 15.0 mg/L to about 20.0 mg/L; from
about 5.0 mg/L to about 15.0 mg/L; from about 6.0 mg/L to about
12.0 mg/L; from about 7.0 mg/L to about 11.0 mg/L and from about
8.0 mg/L to about 10.0 mg/L. In another aspect, insulin is added to
the cell culture medium in an amount to provide insulin in the cell
culture medium at a concentration of about any one of 5.0 mg/L, 6.0
mg/L, 7.0 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0
mg/L, 13.0 mg/L, 14.0 mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0
mg/L, 19.0 mg/L, 20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0
mg/L, 25.0 mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0
mg/L. In a further aspect, insulin is added to the cell culture
medium in an amount to provide insulin in the cell culture medium
at a concentration of about any one of 7 mg/L, 8.0 mg/L, 9.0 mg/L,
10.0 mg/L or 11.0 mg/L. The additional amount of insulin can be
added to the cell culture medium at any time during the cell
culture cycle. For example, insulin may be added at any one or more
of days 1-20 for a 20 day cell culture cycle (e.g., at any one or
more of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20). When an additional amount of insulin is added,
it may be added in any amount, which amount may be the same or
different when insulin is added more than once during a cell
culture cycle. It is therefore appreciated that for a 14 day cell
culture cycle, insulin may be added at any one or more of days 1-14
(e.g., at any one or more of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13 or 14) in any amount, which amount may be the same or
different when insulin is added more than one during a cell culture
cycle. The additional amount of insulin can be added to the cell
culture medium at least once, at least two times, at three times,
at least six times, at least nine times, at least twelve times, or
at least fourteen times during the cell culture cycle. In some of
the embodiments herein, the additional amount of insulin added to
the cell culture is added in an amount to provide insulin in the
cell culture medium at a concentration selected from Table 1 such
as 5.6 mg/L to about 66 mg/L. In some aspects, the cell culture
medium further comprises an animal-derived hydrolysate, a
plant-derived hydrolysate, or both an animal-derived hydrolysate
and a plant-derived hydrolysate. In some of the embodiments herein,
the method further comprises the step of adding an additional
amount of animal-derived hydrolysate and plant-derived hydrolysate
to the cell culture medium provided herein (e.g., such as via a
feed medium introduced to the basal cell culture medium at a period
of time following initiation of the cell culture cycle). In some of
the embodiments herein, the additional amount of animal-derived
hydrolysate and plant-derived hydrolysate added to the cell culture
is added in an amount to provide animal-derived hydrolysate and
plant-derived hydrolysate in the cell culture medium at a
concentration selected from the concentrations listed in Table
1.
[0087] In some of the embodiments herein, the method increases the
amount of bevacizumab or fragment thereof produced by the mammalian
cell as compared to a the amount of bevacizumab or fragment thereof
the mammalian cell produces when cultured in a cell culture medium
that does not comprise one or more of components listed in Table 1.
In some embodiments, the amount of bevacizumab or fragment thereof
produced by a cell cultured in a cell culture medium comprising at
least two of copper, insulin and cystine is increased by at least
5%, 6,%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 19%,
20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to the amount of
bevacizumab or fragment thereof produced by the cell when cultured
in a cell culture medium that does not comprise at least two of
copper, insulin and cystine.
[0088] The cell culture medium provided herein can be used a basal
cell culture medium and/or as a feed cell culture medium. In some
embodiments, a cell culture medium provided herein is used in a
method for culturing the cell during the cell's growth phase. In
some embodiments, a cell culture medium provided herein is used in
a method for culturing the cell during the cell's production
phase.
[0089] It is understood that any of the methods detailed herein
including: (i) a method of producing bevacizumab, or a fragment
thereof; (ii) a method of culturing a mammalian cell comprising a
nucleic acid encoding bevacizumab, or a fragment thereof; and (iii)
a method of enhancing production of bevacizumab, or a fragment
thereof, (e.g., enhancing titer yields of bevacizumab, or a
fragment thereof) from a mammalian cell comprising a nucleic acid
encoding bevacizumab, or a fragment thereof, may be carried out in
any suitable scale (e.g., any scale that produces bevacizumab, or a
fragment thereof). In one aspect, any of the methods detailed
herein are performed on a scale that is commensurate in scope with
commercial production of bevacizumab, or a fragment thereof. For
example, in one variation, a cell capable of producing bevacizumab
may be cultured in a cell culture medium provided herein wherein
the culturing occurs in a culturing vessel that is capable of
holding a commercial batch of bevacizumab, such as in a culturing
vessel capable of holding at least 10,000 L of cell culture (e.g.,
the methods in one aspect are carried out on at least a 10,000 L
cell culture scale, such as a 12,000 L cell culture scale).
[0090] In further embodiments of the methods provided herein, the
bevacizumab or a fragment thereof is recovered from the cell
culture. A composition comprising the recovered bevacizumab or a
fragment thereof can be subjected to at least one purification step
before assessment of, e.g., a quality attribute. In a further
embodiment, the composition is a pharmaceutical composition
comprising bevacizumab or a fragment thereof and a pharmaceutically
acceptable carrier.
[0091] Other methods and cell culture media are provided
throughout, such as in the Brief Summary of the Invention and
elsewhere.
[0092] Polypeptide Production
[0093] The cell culture media detailed herein can be used in a
method of culturing cells to produce bevacizumab or a fragment
thereof. The medium may be used in a method of culturing cells
capable of producing bevacizumab or a fragment thereof, whether by
batch culture, fed batch culture or perfusion culture. In one
embodiment, bevacizumab or a fragment thereof is directly secreted
into the medium by the host cell. In another embodiment,
bevacizumab or a fragment thereof is released into the medium by
lysis of a cell comprising a nucleic acid encoding the antibody or
fragment thereof.
[0094] Bevacizumab or a fragment thereof that is expressible in a
host cell may be produced in accordance with the present disclosure
and may be present in the compositions provided.
[0095] Methods for producing antibodies and fragments thereof, in
cell culture are well known in the art. Provided herein are
non-limiting exemplary methods for producing an antibody (e.g.,
full length antibodies, antibody fragments and multispecific
antibodies) in cell culture. See Molecular Cloning: A Laboratory
Manual (Sambrook et al., 4.sup.th ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2012); Current
Protocols in Molecular Biology (F. M. Ausubel, et al. eds., 2003);
Short Protocols in Molecular Biology (Ausubel et al., eds., J.
Wiley and Sons, 2002); Current Protocols in Protein Science,
(Horswill et al., 2006); Antibodies, A Laboratory Manual (Harlow
and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic
Technique and Specialized Applications (R. I. Freshney, 6.sup.th
ed., J. Wiley and Sons, 2010) for generally well understood and
commonly employed techniques and procedures for the production of
antibodies (e.g., bevacizumab), which are all incorporated herein
by reference in their entirety.
[0096] Cell Culture and Antibody Production
[0097] Generally the cells are combined (contacted) with any of the
cell culture media described herein under one or more conditions
that promote any of cell growth, maintenance and/or antibody
production. Methods of culturing a cell and producing an antibody
employ a culturing vessel (bioreactor) to contain the cell and cell
culture medium. The culturing vessel can be composed of any
material that is suitable for culturing cells, including glass,
plastic or metal. Typically, the culturing vessel will be at least
1 liter and may be 10, 100, 250, 500, 1000, 2500, 5000, 8000,
10,000 liters or more (e.g., a 12,000 liter vessel). In one aspect
the culturing vessel is capable of containing at least 2 liters, at
least 10 liters, at least 100 liters, at least 500 liters, at least
1,000 liters, at least 2,500 liters, at least 5,000 liters, at
least 7,500 liters, at least 10,000 liters, at least 12,000 liters
or more of a cell culture medium provided herein as is required for
producing manufacturing scale amounts of bevacizumab from cell
culture. Thus, the compositions and methods provided herein may
find use in a manufacturing-scale production of bevacizumab, or a
fragment thereof. 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. Culturing conditions that may be adjusted during the
culturing process include but are not limited to pH and
temperature. In some of the embodiments herein, the pH is at least
7.0, 7.15, 7.2, 7.25, 7.30, 7.35, 7.4, 7.45, or 7.50 but no more
than 8.0. The number of cells comprising a nucleic acid encoding
bevacizumab, or a fragment thereof, that can be inoculated into a
cell culture medium provided herein will be apparent to one of
skill in the art. For example, about 1.0.times.10.sup.6 to about
2.0.times.10.sup.6 cells (including any of about
1.1.times.10.sup.6, about 1.2.times.10.sup.6 about
1.3.times.10.sup.6, about 1.4.times.10.sup.6, about
1.5.times.10.sup.6, about 1.6.times.10.sup.6, about
1.7.times.10.sup.6, about 1.8.times.10.sup.6 or about
1.9.times.10.sup.6) comprising a nucleic acid encoding bevacizumab,
or a fragment thereof, can be inoculated in a medium provided
herein for initiation of a cell culture cycle. In one aspect, the
number of cells comprising a nucleic acid encoding bevacizumab, or
a fragment thereof, that can be inoculated into a cell culture
medium provided herein is from about 1.2.times.10.sup.6 to about
1.8.times.10.sup.6 cells or from about 1.3.times.10.sup.6 to about
1.7.times.10.sup.6 cells or from about 1.5.times.10.sup.6 to about
1.7.times.10.sup.6 cells.
[0098] A cell culture is generally maintained in the initial growth
phase under conditions conducive to the survival, growth and
viability (maintenance) of the cell culture. The precise conditions
will vary depending on the cell type, the organism from which the
cell was derived, and the nature and character of the expressed
antibody or fragment thereof.
[0099] The temperature of the cell culture in the initial growth
phase will be selected based primarily on the range of temperatures
at which the cell culture remains viable. For example, during the
initial growth phase, CHO cells grow well at 37.degree. C. In
general, most mammalian cells grow well within a range of about
25.degree. C. to 42.degree. C. Preferably, mammalian cells grow
well within the range of about 35.degree. C. to 40.degree. C. Those
of ordinary skill in the art will be able to select appropriate
temperature or temperatures in which to grow cells, depending on
the needs of the cells and the production requirements.
[0100] In one embodiment of the present invention, the temperature
of the initial growth phase is maintained at a single, constant
temperature. In another embodiment, the temperature of the initial
growth phase is maintained within a range of temperatures. For
example, the temperature may be steadily increased or decreased
during the initial growth phase. Alternatively, the temperature may
be increased or decreased by discrete amounts at various times
during the initial growth phase. One of ordinary skill in the art
will be able to determine whether a single or multiple temperatures
should be used, and whether the temperature should be adjusted
steadily or by discrete amounts.
[0101] The cells may be cultured during the initial growth phase
for a greater or lesser amount of time. In one variation, the cells
are cultured for a period of time sufficient to achieve a viable
cell density that is a given percentage of the maximal viable cell
density that the cells would eventually reach if allowed to grow
undisturbed. For example, the cells may be cultured for a period of
time sufficient to achieve a desired viable cell density of 1, 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95 or 99 percent of maximal viable cell density.
[0102] In another embodiment the cells are allowed to grow for a
defined period of time. For example, depending on the starting
concentration of the cell culture, the temperature at which the
cells are cultured, and the intrinsic growth rate of the cells, the
cells may be cultured for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20 or more days. In some cases, the
cells may be allowed to grow for a month or more.
[0103] The cell culture may be agitated or shaken during the
initial culture phase in order to increase oxygenation and
dispersion of nutrients to the cells. In accordance with the
present invention, one of ordinary skill in the art will understand
that it can be beneficial to control or regulate certain internal
conditions of the bioreactor during the initial growth phase,
including but not limited to pH, temperature, oxygenation, etc. For
example, pH can be controlled by supplying an appropriate amount of
acid or base and oxygenation can be controlled with sparging
devices that are well known in the art.
[0104] An initial culturing step is a growth phase, wherein batch
cell culture conditions are modified to enhance growth of
recombinant cells, to produce a seed train. The growth phase
generally refers to the period of exponential growth where cells
are generally rapidly dividing, e.g. growing. During this phase,
cells are cultured for a period of time, usually 1 to 4 days, e.g.
1, 2, 3, or 4 days, and under such conditions that cell growth is
optimal. The determination of the growth cycle for the host cell
can be determined for the particular host cell by methods known to
those skilled in the art.
[0105] In the growth phase, a basal culture medium provided herein
and cells may be supplied to the culturing vessel in batch. The
culture medium in one aspect contains less than about 5% or less
than 1% or less than 0.1% serum and other proteins derived from
plants or animals (e.g., animal-derived hydrolysates and/or
plant-derived hydrolysates). In some embodiments, the basal medium
does not comprise an animal-derived or plant-derived hydrolysate.
However, serum and animal-derived proteins can be used if desired.
At a particular point in their growth, the cells may form an
inoculum to inoculate a culture medium at the start of culturing in
the production phase. Alternatively, the production phase may be
continuous with the growth phase. The cell growth phase is
generally followed by a polypeptide production phase (e.g.,
antibody production phase).
[0106] During the polypeptide production phase, the cell culture
may be maintained under a second set of culture conditions (as
compared to the growth phase) conducive to the survival and
viability of the cell culture and appropriate for expression of the
desired polypeptide (e.g., bevacizumab or fragment thereof). For
example, during the subsequent production phase, CHO cells express
recombinant polypeptides well within a range of 25.degree. C. to
35.degree. C. Multiple discrete temperature shifts may be employed
to increase cell density or viability or to increase expression of
the recombinant polypeptide. In one embodiment, a method of
increasing polypeptide production (e.g., increasing production of
bevacizumab or fragment thereof) comprises a one or more
temperature shift step during the polypeptide production phase. In
a further embodiment, a one or more temperature shift step
comprises a shift of the temperature from 37.degree. C. to
35.degree. C., from 35.degree. C. to 333.degree. C., or from
33.degree. C. to 31.degree. C. In some embodiments herein, a one or
more temperature shift step comprises a shift of temperature from
about 37.degree. C. on day 0 to 35.degree. C. on day 1 to
33.degree. C. on day 8 and to 31.degree. C. on day 10. In some
embodiments herein, a one or more temperature shift step comprises
a shift of temperature from about 37.degree. C. on day 0 to
35.degree. C. on day 3 to 33.degree. C. on day 8 and to 31.degree.
C. on day 10. In some embodiments herein, a one or more temperature
shift step comprises a shift of temperature from about 37.degree.
C. on day 0 to 34.degree. C. on day 2.5.
[0107] The cells may be maintained in the subsequent production
phase until a desired cell density or production titer is reached.
In one embodiment, the cells are maintained in the subsequent
production phase until the titer of the recombinant polypeptide
(e.g., bevacizumab or fragment thereof) reaches a maximum. In other
embodiments, the culture may be harvested prior to this point. For
example, the cells may be maintained for a period of time
sufficient to achieve a viable cell density of 1, 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99
percent of maximal viable cell density. In some cases, it may be
desirable to allow the viable cell density to reach a maximum, and
then allow the viable cell density to decline to some level before
harvesting the culture.
[0108] In certain cases, it may be beneficial or necessary to
supplement the cell culture during the subsequent production phase
with nutrients or other medium components that have been depleted
or metabolized by the cells. For example, it might be advantageous
to supplement the cell culture with nutrients or other medium
components observed to have been depleted during monitoring of the
cell culture. Alternatively or additionally, it may be beneficial
or necessary to supplement the cell culture prior to the subsequent
production phase. As non-limiting examples, it may be beneficial or
necessary to supplement the cell culture with hormones and/or other
growth factors, particular ions (such as sodium, chloride, calcium,
magnesium, and phosphate), buffers, vitamins, nucleosides or
nucleotides, trace elements (inorganic compounds usually present at
very low final concentrations), amino acids, lipids, or glucose or
other energy source. In one aspect, a basal cell culture is
supplemented with insulin and/or plant-derived hydrolysate and/or
an animal-derived hydrolysate as detailed herein.
[0109] In some embodiments herein, the methods of the invention
comprise the supplementation of an additional amount of insulin
into the cell culture during the cell production phase. For
example, an additional 15 mg/L of insulin may be added to the cell
culture on day of the production phase of the cell culture cycle.
In another example, an additional 5 mg/L of insulin may be added to
the cell culture at least three times during the production phase
of the cell culture cycle. In still another example, an additional
5 mg/L of insulin may be added to the cell culture at least six
times during the production phase of the cell culture cycle. A cell
culture cycle may be at least 3 days, 4 days, 5 days, 6 days, 7
days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14
days long. In some embodiments, the cell culture cycle is up to 20
days long. In some embodiments, the cell culture is at least 20
days long. In some embodiments, a cell may be cultured for more
than one cell culture cycle. In some of the embodiments herein, the
method further comprises the step of adding an additional amount of
animal-derived hydrolysate and plant-derived hydrolysate to the
cell culture medium provided herein (e.g., such as via a feed
medium introduced to the basal cell culture medium at a period of
time following initiation of the cell culture cycle). In some of
the embodiments herein, the additional amount of animal-derived
hydrolysate and plant-derived hydrolysate added to the cell culture
is added in an amount to provide animal-derived hydrolysate and
plant-derived hydrolysate in the cell culture medium at a
concentration selected from the concentrations listed in Table
1.
[0110] Antibody Purification
[0111] Bevacizumab or a fragment thereof preferably is recovered
from the culture medium as a secreted polypeptide, although it also
may be recovered from host cell lysates when directly expressed
without a secretory signal.
[0112] The culture medium or lysate may be centrifuged to remove
particulate cell debris. Bevacizumab or a fragment thereof
thereafter may be purified from contaminant soluble proteins and
polypeptides, with the following procedures being exemplary of
suitable purification procedures: by fractionation on
immunoaffinity or ion-exchange columns; ethanol precipitation;
reverse phase HPLC; chromatography on silica or on a
cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example,
Sephadex G-75; and protein A Sepharose columns to remove
contaminants such as IgG. A protease inhibitor such as phenyl
methyl sulfonyl fluoride (PMSF) also may be useful to inhibit
proteolytic degradation during purification. One skilled in the art
will appreciate that purification methods suitable for the antibody
or fragment thereof of interest may require modification to account
for changes in the character of the antibody or fragment thereof
upon expression in recombinant cell culture. An antibody or
fragment thereof can be generally purified using chromatographic
techniques (e.g., affinity chromatography with a low pH elution
step and ion exchange chromatography to remove process impurities).
Purified bevacizumab or a fragment thereof may be concentrated to
provide a concentrated protein composition, e.g., one with an
antibody concentration of at least 100 mg/mL or 125 mg/mL or 150
mg/mL or a concentration of about 100 mg/mL or 125 mg/mL or 150
mg/mL. It is understood that concentrated polypeptide products may
be concentrated up to levels that are permissible under the
concentration conditions, e.g., up to a concentration at which the
polypeptide is no longer soluble in solution. Non-limiting examples
of methods for producing and purifying antibodies for drug
formulations are described in Kelley, B. MAbs., 2009, 1(5):443-452,
which is incorporated herein in its entirety by reference.
IV. Pharmaceutical Formulations
[0113] Compositions comprising the cell culture medium provided
herein and one or more other component, such as a cell or a desired
antibody or fragment thereof (i.e., bevacizumab or fragment
thereof), are also provided. A mammalian cell comprising a nucleic
acid encoding bevacizumab or fragment thereof can secrete the
antibody or fragment thereof into a cell culture medium of the
invention during cell culture. Accordingly, compositions of the
invention may comprise a mammalian cell that produces bevacizumab
or fragment thereof and a cell culture medium provided herein into
which the bevacizumab or fragment thereof is secreted. Compositions
comprising bevacizumab or fragment thereof and a cell culture
medium provided herein are also contemplated. In some aspects of
the invention, a composition comprises (a) a mammalian cell
comprising a nucleic acid encoding bevacizumab or fragment thereof;
and (b) a cell culture medium as provided herein. In some aspects,
the composition comprises (a) bevacizumab or fragment thereof; and
(b) a cell culture medium as provided herein, wherein the antibody
or fragment thereof is secreted into the medium by a mammalian cell
comprising an isolated nucleic acid encoding bevacizumab or
fragment thereof. In other aspects, the composition comprises: (a)
bevacizumab or fragment thereof; and (b) a cell culture medium as
provided herein, wherein the bevacizumab or fragment thereof is
released into the medium by lysis of a mammalian cell comprising an
isolated nucleic acid encoding the bevacizumab or fragment thereof.
The mammalian cell of the composition may be any mammalian cell
detailed herein (e.g., a CHO cell) and the medium of the
composition may be any medium detailed herein, such as a medium
comprising one or more compounds as detailed in Table 1.
[0114] Compositions (e.g., pharmaceutical formulations) of
bevacizumab or a fragment thereof produced by any of the methods
described herein are prepared by mixing bevacizumab or fragment
thereof having the desired degree of purity with one or more
optional pharmaceutically acceptable carriers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), which
may be in the form of lyophilized formulations or aqueous
solutions. Pharmaceutically acceptable carriers are generally
nontoxic to recipients at the dosages and concentrations employed,
and include, but are not limited to: buffers, antioxidants,
preservatives, low molecular weight (less than about 10 residues)
polypeptides, proteins; hydrophilic polymers; amino acids;
monosaccharides, disaccharides, and other carbohydrates, chelating
agents, sugars, salt-forming counter-ions, metal complexes (e.g.
Zn-protein complexes), and/or non-ionic surfactants. In some
embodiments, the pharmaceutical formulation is administered to a
mammal such as a human. Pharmaceutical formulations of bevacizumab
or fragment thereof can be administered by any suitable means,
including parenteral, intrapulmonary, and intranasal, and, if
desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
Dosing can be by any suitable route, e.g. by injections, such as
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Accordingly,
antibody-containing formulations as provided herein may be suitable
for injection, such as subcutaneous injection into an individual
(e.g., subcutaneous injection into a human). The pharmaceutical
formulations to be used for in vivo administration are generally
sterile. Sterility may be readily accomplished, for example by
filtration through sterile filtration membranes.
[0115] In some aspects, a composition (e.g., pharmaceutical
formulation) as provided herein comprises bevacizumab or fragment
thereof at a concentration of at least 100 mg/mL, 125 mg/mL, 150
mg/mL, 200 mg/mL, or 250 mg/mL, or at a concentration of about 100
mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, or about
200 mg/mL. In other aspects, a composition (e.g., pharmaceutical
formulation) as provided herein comprises bevacizumab or fragment
thereof at a concentration of at least 1 mg/mL, 10 mg/mL, 25 mg/mL,
50 mg/mL, or 75 mg/mL, or at a concentration of about 1 mg/mL,
about 10 mg/mL, about 25 mg/mL, about 50 mg/mL, or about 75
mg/mL.
V. Articles of Manufacture and Kits
[0116] A kit for supplementing a cell culture medium with at least
two of copper, insulin and cystine are provided. The at least two
of copper, insulin and cystine may be present in an amount to
provide a concentration of the components at provided in Table 1.
The kit may contain dried constituents to be reconstituted, and may
also contain instructions for use (e.g., for use in supplementing a
medium with the kit constituents). The kit may contain the
constituents provided herein in amounts suitable to supplement a
cell culture medium for use in culturing a mammalian cell
comprising a nucleic acid encoding bevacizumab or fragment thereof.
In one aspect, a kit comprises cystine in an amount to provide from
about 0.9 mM to about 1.5 mM cystine in the cell culture medium. In
some embodiments herein, the kit further comprises insulin in an
amount to provide from about 1.4 mg/L to about 11 mg/L insulin in
the cell culture medium. In some embodiments herein, the kit
further comprises copper in an amount to provide from about 26 nM
to about 400 nM copper in the cell culture medium. In some
embodiments, a kit comprises two or more constituents selected from
the group consisting of insulin in an amount to provide from about
7.0 mg/L to about 11.0 mg/L insulin in the cell culture medium,
cystine in an amount to provide from about 0.8 mM to about 2.5 mM
cystine in the cell culture medium, and copper in an amount to
provide from about 25.0 nM to about 400.0 nM copper in the cell
culture medium.
[0117] In any of the aspects herein, the kit may further comprise
an animal-derived hydrolysate or a plant-derived hydrolysate or
both an animal-derived hydrolysate and a plant-derived hydrolysate.
In some of the embodiments herein, the kit further comprises a
plant-derived hydrolysate in an amount to provide from about 1.4
g/L to about 6.2 g/L plant-derived hydrolysate in the cell culture
medium. In some of the embodiments herein, the kit further
comprises an animal-derived hydrolysate in an amount to provide
from about 5.6 g/L to about 38.0 g/L animal-derived hydrolysate in
the cell culture medium.
[0118] In another aspect of the invention, an article of
manufacture is provided comprising a container which holds the cell
culture medium of the invention and optionally provides
instructions for its use. Suitable containers include, for example,
bottles and bags. The container may be formed from a variety of
materials such as glass or plastic. The container holds the cell
culture medium and the label on, or associated with, the container
may indicate directions for use (e.g., for use in culturing cells).
The article of manufacture may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents and package inserts with instructions for
use.
[0119] An article of manufacture comprising a container which holds
bevacizumab, or a fragment thereof produced by a method detailed
herein and optionally provides instructions for its use is also
provided.
EMBODIMENTS
[0120] Various embodiments and aspects of the invention are
detailed herein and throughout. Embodiments include, without
limitation, the following:
[0121] Method 1: A method of producing bevacizumab, or a fragment
thereof, comprising the step of culturing a mammalian cell
comprising a nucleic acid encoding bevacizumab or fragment thereof
in a cell culture medium, wherein the cell culture medium comprises
two or more components selected from the group consisting of
copper, insulin, and cystine, and wherein the cell produces
bevacizumab, or a fragment thereof.
[0122] Method 2: A method of culturing a mammalian cell comprising
a nucleic acid encoding bevacizumab, or a fragment thereof, the
method comprising the step of contacting the mammalian cell with a
cell culture medium comprising two or more components selected from
the group consisting of copper, insulin and cystine.
[0123] Method 3: A method of enhancing the amount of bevacizumab,
or a fragment thereof, produced from a mammalian cell comprising a
nucleic acid encoding bevacizumab, or a fragment thereof, the
method comprising the step of culturing the mammalian cell in a
cell culture medium comprising at least two of insulin, copper and
cystine, wherein the amount of bevacizumab, or a fragment thereof,
produced from the mammalian cell is enhanced relative to culturing
the mammalian cell in a cell culture medium without at least two of
insulin, copper and cystine.
[0124] Method 4: A method of culturing a mammalian cell comprising
a nucleic acid encoding bevacizumab, or a fragment thereof, in a
cell culture medium comprising at least two of insulin, copper and
cystine, wherein the amount of bevacizumab, or a fragment thereof,
produced from the mammalian cell is enhanced relative to culturing
the mammalian cell in a cell culture medium without at least two of
insulin, copper and cystine.
[0125] For any of methods 1-4, the method may comprise any one or
more of the following features (i)-(xviii) or sub-feature thereof
or any combination of feature or sub feature: [0126] (i) the cell
culture medium comprises copper and insulin [0127] (ii) the cell
culture medium comprises copper and cystine [0128] (iii) the cell
culture medium comprises insulin and cystine [0129] (iv) the cell
culture medium comprises copper, insulin, and cystine [0130] (v)
the cell culture medium (including a medium containing any of
features (i)-(iv)) further comprises a plant-derived hydrolysate,
an animal-derived hydrolysate or both a plant-derived hydrolysate
and an animal-derived hydrolysate. [0131] (vi) the cell culture
medium (including a medium containing any one or more or all of
features (i)-(v)) comprises insulin at a concentration of any one
of: a. from about 1.0 mg/L to about 100.0 mg/L b. from about 10.0
mg/L to about 100.0 mg/L c. from about 10.0 mg/L to about 50.0 mg/L
d. from about 10.0 mg/L to about 35.0 mg/L e. from about 10.0 mg/L
to about 25.0 mg/L f. from about 5.0 mg/L to about 80.0 mg/L g.
from about 5.0 mg/L to about 60.0 mg/L h. from about 5.0 mg/L to
about 50.0 mg/L i. from about 5.0 mg/L to about 40.0 mg/L j. from
about 5.0 mg/L to about 25.0 mg/L k. from about 10.0 mg/L to about
25.0 mg/L l. from about 10.0 mg/L to about 40.0 mg/L m. from about
15.0 mg/L to about 20.0 mg/L n. from about 5.0 mg/L to about 15.0
mg/L o. from about 6.0 mg/L to about 12.0 mg/L p. from about 7.0
mg/L to about 11.0 mg/L q. from about 8.0 mg/L to about 10.0 mg/L
r. about any one of 5.0 mg/L, 6.0 mg/L, 7.0 mg/L, 8.0 mg/L, 9.0
mg/L, 10.0 mg/L, 11.0 mg/L, 12.0 mg/L, 13.0 mg/L, 14.0 mg/L, 15.0
mg/L, 16.0 mg/L, 17.0 mg/L, 18.0 mg/L, 19.0 mg/L, 20.0 mg/L, 21.0
mg/L, 22.0 mg/L, 23.0 mg/L, 24.0 mg/L, 25.0 mg/L, 26.0 mg/L, 27.0
mg/L, 28.0 mg/L, 29.0 mg/L or 30.0 mg/L or 31.0 mg/L or 32 mg/L or
33 mg/L or 34 mg/L or 35 mg/L or 36 mg/L or 37 mg/L or 38 mg/L or
39 mg/L or 40 mg/L s. about any one of 7 mg/L, 8.0 mg/L, 9.0 mg/L,
10.0 mg/L or 11.0 mg/L t. about 25 mg/L [0132] (vii) the cell
culture medium (including a medium containing any one or more or
all of features (i)-(vi)) comprises copper at a concentration of
any one of: a. from about 69 nM to about 1,000 nM b. from about
69.0 nM to about 400.0 nM c. from about 80 nM to about 400 nM. d.
from about 100 nM to about 400 nM e. from about 125 nM to about 400
nM f. from about 150 nM to about 400 nM g. from about 200 nM to
about 400 nM h. from about 250 nM to about 400 nM i. from about 300
nM to about 400 nM j. from about 325 nM to about 375 nM k. from
about 325 nM to about 350 nM l. about any one of 100 nM, 125 nM,
150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350
nM, 375 nM or 400 nM. m. about any one of 330 nM, 335 nM, 340 nM,
345 nM or 350 nM n. about 335 nM, 336 nM, 337 nM, 338 nM, 339 nM or
400 nM o. about 339 nM [0133] (viii) the cell culture medium
(including a medium containing any one or more or all of features
(i)-(vii)) comprises cystine at a concentration of any one of: a.
from about 0.7 mM to about 2.0 mM b. from about 0.8 mM to about 2.5
mM c. from about 0.8 mM to about 2.0 mM d. from about 0.8 mM to
about 1.75 mM e. from about 0.8 mM to about 1.6 mM f. from about
1.0 mM to about 2.0 mM g. from about 1.0 mM to about 1.6 mM h. from
about 1.2 mM to about 1.4 mM i. about any one of 0.8 mM or 0.9 mM
or 1.0 mM or 1.1 mM or 1.2 mM or 1.3 mM or 1.4 mM or 1.5 mM j.
about any one of 1.1 mM, 1.3 mM or 1.5 mM [0134] (ix) the cell
culture medium (including a medium containing any one or more or
all of features (i)-(viii)) comprises an animal-derived hydrolysate
at a concentration of any one of: a. from about 6.0 g/L to about
20.0 g/L b. from about 5.6 g/L to about 38.0 g/L c. from about 7.0
g/L to about 25.0 g/L d. from about 7.0 g/L to about 20.0 g/L e.
from about 7.0 g/L to about 15.0 g/L f. from about 8.0 g/L to about
12.0 g/L g. from about 9.0 g/L to about 11.0 g/L h. from about 7.0
g/L to about 11.0 g/L i. about any one of 5 g/L, 10 g/L, 15 g/L, 20
g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, 45 g/L or 50 g/L j. about any
one of 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, or 12 g/L
k. about 10 g/L l. about 13 g/L [0135] (x) the cell culture medium
(including a medium containing any one or more or all of features
(i)-(ix)) comprises a plant-derived hydrolysate at a concentration
of any one of: a. from about 1.0 g/L to about 10.0 g/L b. from
about 1.4 g/L to about 11.0 g/L c. from about 1.4 g/L to about 6.2
g/L d. from about 1.5 g/L to about 5.5 g/L e. from about 1.5 g/L to
about 4.5 g/L f. from about 1.5 g/L to about 3.5 g/L g. from about
2.0 g/L to about 3.0 g/L h. from about 1.5 g/L to about 2.5 g/L i.
from about 1.75 g/L to about 2.75 g/L j. from about 2.25 g/L to
about 2.75 g/L k. about any one of 1.75 g/L, 2.0 g/L, 2.25 g/L, 2.5
g/L, 3.0 g/L, 3.25, 3.5 g/L, 3.75 g/L, or 4.0 g/L l. about any one
of 2.0 g/L, 2.25 g/L, 2.5 g/L or 3.0 g/L m. about 2.5 g/L n. about
3.1 g/L [0136] (xi) the cell culture medium (including a medium
containing any one or more or all of features (i)-(x)) comprises
both an animal-derived hydrolysate and a plant-derived hydrolysate,
and wherein the animal-derived hydrolysate is present in a greater
amount than the plant-derived hydrolysate [0137] (xii) the cell
culture medium (including a medium containing any one or more or
all of features (i)-(xi)) comprises insulin and the method further
comprises the step of adding an additional amount of insulin to the
cell culture medium, wherein the additional amount of insulin may:
(a) be added to the cell culture medium once or at least three
times or at least six times during the cell culture cycle and (b)
may be added in an amount to provide insulin in the cell culture
medium at a concentration of any one of: a. from about 1.0 mg/L to
about 100.0 mg/L b. from about 10.0 mg/L to about 100.0 mg/L c.
from about 10.0 mg/L to about 50.0 mg/L d. from about 10.0 mg/L to
about 35.0 mg/L e. from about 10.0 mg/L to about 25.0 mg/L f. from
about 5.0 mg/L to about 80.0 mg/L g. from about 5.0 mg/L to about
60.0 mg/L h. from about 5.0 mg/L to about 50.0 mg/L i. from about
5.0 mg/L to about 40.0 mg/L j. from about 5.0 mg/L to about 25.0
mg/L k. from about 10.0 mg/L to about 25.0 mg/L l. from about 10.0
mg/L to about 40.0 mg/L m. from about 15.0 mg/L to about 20.0 mg/L
n. from about 5.0 mg/L to about 15.0 mg/L o. from about 6.0 mg/L to
about 12.0 mg/L p. from about 7.0 mg/L to about 11.0 mg/L q. from
about 8.0 mg/L to about 10.0 mg/L r. about any one of 5.0 mg/L, 6.0
mg/L, 7.0 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0
mg/L, 13.0 mg/L, 14.0 mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0
mg/L, 19.0 mg/L, 20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0
mg/L, 25.0 mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0
mg/L or 31.0 mg/L or 32 mg/L or 33 mg/L or 34 mg/L or 35 mg/L or 36
mg/L or 37 mg/L or 38 mg/L or 39 mg/L or 40 mg/L s. about any one
of 7 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L or 11.0 mg/L t. about 25
mg/L u. about 15 mg/L [0138] (xiii) the method further comprises
the step of adding cysteine to the cell culture medium (including a
medium containing any one or more or all of features (i)-(xii)),
which cysteine may be added to the cell culture medium (a) as a
component of a batch feed that is added to a basal medium that does
not comprise cysteine and/or (b) may be added in an amount to
provide cysteine in the cell culture medium at a concentration of
from about 0.5 to about 5.0 mM or from about 0.5 to about 2.0 mM or
from about 0.5 to about 2.0 mM (such as at a concentration of 0.8
mM) or from about 7.0 to about 8.0 mM (such as at a concentration
of about 7.5 mM) [0139] (xiv) the method further comprises the step
of adding cystine to the cell culture medium (including a medium
containing any one or more or all of features (i)-(xii)), which
cystine may be added to the cell culture medium as a component of a
batch feed that is added to a basal medium and which may be added
in an amount to provide cystine in the cell culture medium at a
concentration of from about 0.1 to about 1.5 mM (such as at a
concentration of 0.2 mM) [0140] (xv) the cell is cultured (e.g., in
any cell culture media including those having any one or more or
all of features (i)-(xiv)) at a temperature ranging from about
28.degree. C. to about 37.degree. C. or from about 31.degree. C. to
about 35.degree. C. [0141] (xvi) the cell is cultured (e.g., in any
cell culture media including those having any one or more or all of
features (i)-(xiv)) at a first temperature of about 35.degree. C.
for a first period of time, is cultured at a second temperature of
about 33.degree. C. for a second period of time, and is cultured at
a third temperature of about 31.degree. C. for a third period of
time [0142] (xvii) bevacizumab, or a fragment thereof, is secreted
into the cell culture medium (including a medium containing any one
or more or all of features (i)-(xvi)) [0143] (xviii) the method
further comprises the step of recovering the bevacizumab, or a
fragment thereof, from the cell culture (including a medium
containing any one or more or all of features (i)-(xvi))
[0144] Also provided herein is bevacizumab, or fragment thereof,
produced by any method provided herein, including without
limitation any of methods 1-4, which method may further comprise
any one or more or all of the features (i)-(xviii) or sub-feature
thereof or any combination of the foregoing.
[0145] Also provided is a composition comprising: (i) bevacizumab,
or a fragment thereof, produced by any method provided herein,
including without limitation any of methods 1-4, which method may
further comprise any one or more or all of the features (i)-(xviii)
or sub-feature thereof or any combination of the foregoing and (ii)
a pharmaceutically acceptable carrier.
[0146] A kit for supplementing a cell culture medium for use in
culturing a mammalian cell comprising a nucleic acid encoding
bevacizumab, or a fragment thereof, is also provided, the kit
comprising at least two of components (i)-(iii): [0147] (i) insulin
in an amount to provide a concentration of any one of: a. from
about 1.0 mg/L to about 100.0 mg/L b. from about 10.0 mg/L to about
100.0 mg/L c. from about 10.0 mg/L to about 50.0 mg/L d. from about
10.0 mg/L to about 35.0 mg/L e. from about 10.0 mg/L to about 25.0
mg/L f. from about 5.0 mg/L to about 80.0 mg/L g. from about 5.0
mg/L to about 60.0 mg/L h. from about 5.0 mg/L to about 50.0 mg/L
i. from about 5.0 mg/L to about 40.0 mg/L j. from about 5.0 mg/L to
about 25.0 mg/L k. from about 10.0 mg/L to about 25.0 mg/L l. from
about 10.0 mg/L to about 40.0 mg/L m. from about 15.0 mg/L to about
20.0 mg/L n. from about 5.0 mg/L to about 15.0 mg/L o. from about
6.0 mg/L to about 12.0 mg/L p. from about 7.0 mg/L to about 11.0
mg/L q. from about 8.0 mg/L to about 10.0 mg/L r. about any one of
5.0 mg/L, 6.0 mg/L, 7.0 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L, 11.0
mg/L, 12.0 mg/L, 13.0 mg/L, 14.0 mg/L, 15.0 mg/L, 16.0 mg/L, 17.0
mg/L, 18.0 mg/L, 19.0 mg/L, 20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0
mg/L, 24.0 mg/L, 25.0 mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0
mg/L or 30.0 mg/L or 31.0 mg/L or 32 mg/L or 33 mg/L or 34 mg/L or
35 mg/L or 36 mg/L or 37 mg/L or 38 mg/L or 39 mg/L or 40 mg/L s.
about any one of 7 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L or 11.0
mg/L. t. about 25 mg/L [0148] (ii) cystine in an amount to provide
a concentration of any one of: a. from about 0.7 mM to about 2.0 mM
b. from about 0.8 mM to about 2.5 mM c. from about 0.8 mM to about
2.0 mM d. from about 0.8 mM to about 1.75 mM e. from about 0.8 mM
to about 1.6 mM f. from about 1.0 mM to about 2.0 mM g. from about
1.0 mM to about 1.6 mM h. from about 1.2 mM to about 1.4 mM i.
about any one of 0.8 mM or 0.9 mM or 1.0 mM or 1.1 mM or 1.2 mM or
1.3 mM or 1.4 mM or 1.5 mM j. about any one of 1.1 mM, 1.3 mM or
1.5 mM [0149] (iii) copper at a concentration of any one of: a.
from about 69.0 nM to about 1,000.0 nM b. from about 69.0 nM to
about 400.0 nM c. from about 80 nM to about 400 nM. d. from about
100 nM to about 400 nM e. from about 125 nM to about 400 nM f. from
about 150 nM to about 400 nM g. from about 200 nM to about 400 nM
h. from about 250 nM to about 400 nM i. from about 300 nM to about
400 nM j. from about 325 nM to about 375 nM k. from about 325 nM to
about 350 nM l. about any one of 100 nM, 125 nM, 150 nM, 175 nM,
200 nM, 225 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM or
400 nM. m. about any one of 330 nM, 335 nM, 340 nM, 345 nM or 350
nM n. about 335 nM, 336 nM, 337 nM, 338 nM, 339 nM or 400 nM o.
about 339 nM
[0150] The kit may further comprise other components, including any
one or more of: [0151] i. an animal-derived hydrolysate, such as in
an amount to provide concentration of any one of: a. from about 6.0
g/L to about 20.0 g/L b. from about 5.6 g/L to about 38.0 g/L c.
from about 7.0 g/L to about 25.0 g/L d. from about 7.0 g/L to about
20.0 g/L e. from about 7.0 g/L to about 15.0 g/L f. from about 8.0
g/L to about 12.0 g/L g. from about 9.0 g/L to about 11.0 g/L h.
from about 7.0 g/L to about 11.0 g/L i. about any one of 5 g/L, 10
g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L, 40 g/L, 45 g/L or 50
g/L j. about any one of 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L,
11 g/L, or 12 g/L k. about 10 g/L l. about 13 g/L [0152] ii.
plant-derived hydrolysate, such as in an amount to provide a
concentration of any one of: a. from about 1.0 g/L to about 10.0
g/L b. from about 1.4 g/L to about 11.0 g/L c. from about 1.4 g/L
to about 6.2 g/L d. from about 1.5 g/L to about 5.5 g/L e. from
about 1.5 g/L to about 4.5 g/L f. from about 1.5 g/L to about 3.5
g/L g. from about 2.0 g/L to about 3.0 g/L h. from about 1.5 g/L to
about 2.5 g/L i. from about 1.75 g/L to about 2.75 g/L j. from
about 2.25 g/L to about 2.75 g/L k. about any one of 1.75 g/L, 2.0
g/L, 2.25 g/L, 2.5 g/L, 3.0 g/L, 3.25, 3.5 g/L, 3.75 g/L, or 4.0
g/L l. about any one of 2.0 g/L, 2.25 g/L, 2.5 g/L or 3.0 g/L m.
about 2.5 g/L n. about 3.1 g/L
[0153] A cell culture medium for use in culturing a mammalian cell
comprising a nucleic acid encoding bevacizumab, or a fragment
thereof, is also provided, the cell culture medium comprising at
least two of components (i)-(iii): [0154] (i) insulin in an amount
to provide a concentration of any one of: a. from about 1.0 mg/L to
about 100.0 mg/L b. from about 10.0 mg/L to about 100.0 mg/L c.
from about 10.0 mg/L to about 50.0 mg/L d. from about 10.0 mg/L to
about 35.0 mg/L e. from about 10.0 mg/L to about 25.0 mg/L f. from
about 5.0 mg/L to about 80.0 mg/L g. from about 5.0 mg/L to about
60.0 mg/L h. from about 5.0 mg/L to about 50.0 mg/L i. from about
5.0 mg/L to about 40.0 mg/L j. from about 5.0 mg/L to about 25.0
mg/L k. from about 10.0 mg/L to about 25.0 mg/L l. from about 10.0
mg/L to about 40.0 mg/L m. from about 15.0 mg/L to about 20.0 mg/L
n. from about 5.0 mg/L to about 15.0 mg/L o. from about 6.0 mg/L to
about 12.0 mg/L p. from about 7.0 mg/L to about 11.0 mg/L q. from
about 8.0 mg/L to about 10.0 mg/L r. about any one of 5.0 mg/L, 6.0
mg/L, 7.0 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L, 11.0 mg/L, 12.0
mg/L, 13.0 mg/L, 14.0 mg/L, 15.0 mg/L, 16.0 mg/L, 17.0 mg/L, 18.0
mg/L, 19.0 mg/L, 20.0 mg/L, 21.0 mg/L, 22.0 mg/L, 23.0 mg/L, 24.0
mg/L, 25.0 mg/L, 26.0 mg/L, 27.0 mg/L, 28.0 mg/L, 29.0 mg/L or 30.0
mg/L or 31.0 mg/L or 32 mg/L or 33 mg/L or 34 mg/L or 35 mg/L or 36
mg/L or 37 mg/L or 38 mg/L or 39 mg/L or 40 mg/L s. about any one
of 7 mg/L, 8.0 mg/L, 9.0 mg/L, 10.0 mg/L or 11.0 mg/L t. about 25
mg/L [0155] (ii) cystine in an amount to provide a concentration of
any one of: a. from about 0.7 mM to about 2.0 mM b. from about 0.8
mM to about 2.5 mM c. from about 0.8 mM to about 2.0 mM d. from
about 0.8 mM to about 1.75 mM e. from about 0.8 mM to about 1.6 mM
f. from about 1.0 mM to about 2.0 mM g. from about 1.0 mM to about
1.6 mM h. from about 1.2 mM to about 1.4 mM i. about any one of 0.8
mM or 0.9 mM or 1.0 mM or 1.1 mM or 1.2 mM or 1.3 mM or 1.4 mM or
1.5 mM j. about any one of 1.1 mM, 1.3 mM or 1.5 mM [0156] (iii)
copper at a concentration of any one of: a. from about 69.0 nM to
about 1,000.0 nM b. from about 69.0 nM to about 400.0 nM c. from
about 80 nM to about 400 nM. d. from about 100 nM to about 400 nM
e. from about 125 nM to about 400 nM f. from about 150 nM to about
400 nM g. from about 200 nM to about 400 nM h. from about 250 nM to
about 400 nM i. from about 300 nM to about 400 nM j. from about 325
nM to about 375 nM k. from about 325 nM to about 350 nM l. about
any one of 100 nM, 125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM,
275 nM, 300 nM, 325 nM, 350 nM, 375 nM or 400 nM. m. about any one
of 330 nM, 335 nM, 340 nM, 345 nM or 350 nM n. about 335 nM, 336
nM, 337 nM, 338 nM, 339 nM or 400 nM o. about 339 nM
[0157] The cell culture medium may comprise other components,
including any one or more of: [0158] 1) an animal-derived
hydrolysate, such as in an amount to provide concentration of any
one of: a. from about 6.0 g/L to about 20.0 g/L b. from about 5.6
g/L to about 25.0 g/L c. from about 7.0 g/L to about 25.0 g/L d.
from about 7.0 g/L to about 20.0 g/L e. from about 7.0 g/L to about
15.0 g/L f. from about 8.0 g/L to about 12.0 g/L g. from about 9.0
g/L to about 11.0 g/L h. from about 7.0 g/L to about 11.0 g/L i.
about any one of 5 g/L, 10 g/L, 15 g/L, 20 g/L or 25 g/L j. about
any one of 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, or 12
g/L k. about 10 g/L l. about 13 g/L [0159] 2) plant-derived
hydrolysate, such as in an amount to provide a concentration of any
one of: a. from about 1.0 g/L to about 10.0 g/L b. from about 1.4
g/L to about 11.0 g/L c. from about 1.4 g/L to about 6.2 g/L d.
from about 1.5 g/L to about 5.5 g/L e. from about 1.5 g/L to about
4.5 g/L f. from about 1.5 g/L to about 3.5 g/L g. from about 2.0
g/L to about 3.0 g/L h. from about 1.5 g/L to about 2.5 g/L i. from
about 1.75 g/L to about 2.75 g/L j. from about 2.25 g/L to about
2.75 g/L k. about any one of 1.75 g/L, 2.0 g/L, 2.25 g/L, 2.5 g/L,
3.0 g/L, 3.25, 3.5 g/L, 3.75 g/L, or 4.0 g/L l. about any one of
2.0 g/L, 2.25 g/L, 2.5 g/L or 3.0 g/L m. about 2.5 g/L n. about 3.1
g/L
[0160] The cell culture medium may further be supplemented with
additional cell culture medium components, where the additional
cell culture medium components may comprise, e.g., insulin and/or
cysteine, such as insulin in an amount to provide any concentration
of insulin provided herein, including the concentrations listed in
the present embodiments (such as 15 mg/L) and/or cysteine in an
amount to provide any concentration of cysteine provided herein
(such as 0.8 mM), including the concentrations listed in the
present embodiments.
[0161] The cell culture medium may further be supplemented with
additional cell culture medium components, where the additional
cell culture medium components may comprise, e.g., insulin and/or
cysteine and/or cystine, such as insulin in an amount to provide
any concentration of insulin provided herein, including the
concentrations listed in the present embodiments (such as 15 mg/L)
and/or cysteine in an amount to provide any concentration of
cysteine provided herein (such as 0.8 mM), including the
concentrations listed in the present embodiments and/or cystine in
an amount to provide any concentration of cystine provided herein
(such as 0.2 mM), including the concentrations listed in the
present embodiments.
[0162] Also provided herein is a composition comprising (a) a
mammalian cell comprising a nucleic acid encoding bevacizumab, or a
fragment thereof; and (b) a cell culture medium detailed herein,
including without limitation a cell culture medium provided in the
present embodiments. Further provided is a composition comprising:
(a) bevacizumab, or a fragment thereof; and (b) a cell culture
medium detailed herein, including without limitation a cell culture
medium provided in the present embodiments.
[0163] The following Examples are provided to illustrate but not to
limit the invention.
EXAMPLES
Example 1
Impact of Cell Culture Medium Components on Amount of Bevacizumab
Produced by a Mammalian Cell Line
[0164] Chinese hamster ovary (CHO) cells producing bevacizumab were
cultured in cell culture media containing various amounts of
insulin, and the impact of insulin on the amount of bevacizumab
produced was assessed. Production of bevacizumab was initiated in
cell culture by inoculating cells in basal medium containing 339 nM
copper, 1% animal hydrolysate and 0.25% plant hydrolysate and a
batch feed medium was added on day 3 over a 14 day cell culture
cycle in a bioreactor. The basal cell culture medium of a control
cell culture was supplemented with less than 10 mg/L of insulin and
no additional insulin was given during the cell culture cycle. In
two representative experimental cases (case 1 and case 2), the
insulin level in the basal cell culture media was 10 mg/L.
Additional insulin was added three to six times during the cell
culture cycle so as to provide cell culture case 1 and cell culture
case 2 with a final amount of 25 mg/L and 40 mg/L insulin at the
end of the 14 day cell culture cycle, respectively (Table A). The
cells were cultured at 37.degree. C. on day 1 and the temperature
was subsequently shifted down to 35.degree. C. on day 1, 33.degree.
C. on day 8, and 31.degree. C. on day 10. Titer improvement was
quantified by percent increase over the control. Total addition of
insulin during the cell culture cycle from less than 10 mg/L to 25
mg/L or 40 mg/L led to a titer improvement of about 16% or about
18% as compared with the control, respectively (Table A).
TABLE-US-00003 TABLE A Exemplary insulin addition protocol and
results Insulin addition Insulin addition case 1 case 2 Total
insulin 25 mg/L 40 mg/L concentration added during a 14 day
duration Titer 16% 18% improvement percent increase
[0165] CHO cells producing bevacizumab were cultured in cell
culture media containing different amounts of hydrolysate from
different sources and the impact of the specific hydrolysate on the
amount of bevacizumab produced was assessed. In two representative
protocols, production of bevacizumab was initiated in cell culture
by inoculating cells in basal cell culture medium containing 339 nM
copper and a batch feed medium was added on day 3 over a 14 day
cell culture cycle in a bioreactor. The basal cell culture medium
for Protocol 1 had 1% animal-derived hydrolysate without
plant-derived hydrolysate while Protocol 2 had basal cell culture
medium supplemented with 0.75% animal-derived hydrolysate in
combination with 0.25% plant-derived hydrolysate (Table B).
Analysis of the antibody titer produced by the two protocols
demonstrated that Protocol 2 provided a 27% increase in the amount
of bevacizumab produced from the cells as compared to the amount of
bevacizumab produced by cells cultured using Protocol 1 (Table
B).
TABLE-US-00004 TABLE B Exemplary experimental protocols Protocol 1
Protocol 2 Porcine peptone 1% 0.75% Plant peptone None 0.25% Titer
improvement percent increase Not Applicable 27%
Example 2
Impact of Cell Culture Medium Components on Amount of Bevacizumab
Produced by a Mammalian Cell Line
[0166] CHO cells producing bevacizumab were cultured in basal media
with either the amino acid cysteine in the monomer form (Cys,
cysteine) or in the dimer form (Cys-Cys, cystine) (Table C).
Production of bevacizumab was initiated in cell culture by
inoculating cells in basal medium containing 339 nM copper, 1%
animal hydrolysate and 0.25% plant hydrolysate and a batch feed was
added on day 3 over a 14 day cell culture cycle in a bioreactor.
Insulin levels in the basal media were at a concentration of either
less than 10 mg/L or 10 mg/L. Two of the experimental cell cultures
(Cysteine+Insulin and Cystine+Insulin) had additional insulin added
three times or six times during the cell culture cycle so as to
provide a final amount of 25 mg/L or 40 mg/L (Table C). The cells
were cultured at 37.degree. C. on day 1 the temperature was
subsequently shifted down to 35.degree. C. on day 1, 33.degree. C.
on day 8, and 31.degree. C. on day 10. Titer improvement was
quantified by percent increase over the control. Replacement of
cysteine (Cys) by cystine (Cys-Cys) improved titer by 11% over the
control. Impact of insulin addition was also observed in basal
media made using cystine (Cys-Cys).
TABLE-US-00005 TABLE C Summary of protocols and results Cysteine
Cysteine + Cystine + (Control) Cystine Insulin Insulin Cysteine
(Cys) 2.6 mM 0 mM 2.6 mM 0 mM concentration in basal media Cystine
0 mM 1.3 mM 0 mM 1.3 mM (Cys-Cys) concentration in basal media
Total insulin Less than Less than 25 mg/L 40 mg/L concentration 10
mg/L 10 mg/L added during a 14 day duration Titer Not 11% 11% 14%
improvement Applicable percent increase
* * * * *