U.S. patent application number 17/842528 was filed with the patent office on 2022-09-29 for methods for improving cell viability in a production bioreactor.
The applicant listed for this patent is Genzyme Corporation. Invention is credited to Timothy Johnson, Jiuyi Lu, Jean McLarty, Yukun Ren, Neha Shah, Jason Walther, Jonathan Wang.
Application Number | 20220308042 17/842528 |
Document ID | / |
Family ID | 1000006406234 |
Filed Date | 2022-09-29 |
United States Patent
Application |
20220308042 |
Kind Code |
A1 |
Wang; Jonathan ; et
al. |
September 29, 2022 |
Methods for Improving Cell Viability In A Production BioReactor
Abstract
Provided herein are methods of predicting the effect of a
concentration of a sensitizer on cell viability in a production
bioreactor, methods of improving cell viability in a production
bioreactor, methods of predicting cell viability in a production
bioreactor, and methods for culturing a cell in a production
bioreactor.
Inventors: |
Wang; Jonathan;
(Bridgewater, NJ) ; Shah; Neha; (Bridgewater,
NJ) ; Walther; Jason; (Bridgewater, NJ) ; Lu;
Jiuyi; (Bridgewater, NJ) ; Johnson; Timothy;
(Bridgewater, NJ) ; Ren; Yukun; (Bridgewater,
NJ) ; McLarty; Jean; (Bridgewater, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Cambridge |
MA |
US |
|
|
Family ID: |
1000006406234 |
Appl. No.: |
17/842528 |
Filed: |
June 16, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16355387 |
Mar 15, 2019 |
11391725 |
|
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17842528 |
|
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62645755 |
Mar 20, 2018 |
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62644339 |
Mar 16, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/32 20130101; C12M
41/46 20130101; G01N 33/5014 20130101; C12Q 1/025 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; C12Q 1/32 20060101 C12Q001/32; C12Q 1/02 20060101
C12Q001/02; C12M 1/34 20060101 C12M001/34 |
Claims
1. A method of predicting the effect of a concentration of a
sensitizer on cell viability in a production bioreactor, the method
comprising: (a) determining cell viability under a plurality of
test conditions in a test culture vessel, wherein the plurality of
test conditions are defined by a set of parameters; and (b) based
on the cell viability in the test culture vessel under the
plurality of conditions, predicting the effect of a concentration
of a sensitizer on cell viability in a production bioreactor.
2. The method of claim 1, wherein the set of parameters comprises
shear stress, concentration of the sensitizer, and concentration of
a protectant.
3. The method of claim 1 or claim 2, wherein the plurality of
testing conditions are selected based on full factorial screening
and/or inscribed center composite design.
4. The method of any one of claims 1-3, wherein the sensitizer is
an antifoam agent.
5. The method of claim 4, wherein the antifoam is a
polydimethylsiloxane-based antifoam.
6. The method of claim 4, wherein the antifoam is simethicone.
7. The method of any one of claims 1-6, wherein the test culture
vessel has a volume of about 5 mL to about 5 L.
8. The method of claim 7, wherein the test culture vessel has a
volume of about 10 mL to about 500 mL.
9. The method of any one of claims 1-8, wherein the test culture
vessel is a baffled shake flask.
10. The method of any one of claims 1-9, wherein the production
bioreactor has a volume of about 5 L to about 20,000 L.
11. The method of claim 10, wherein the production bioreactor has a
volume of about 50 L to about 10,000 L.
12. The method of any one of claims 1-11, wherein the production
bioreactor is a perfusion bioreactor.
13. The method of any one of claims 1-11, wherein the production
bioreactor is a fed batch bioreactor.
14. The method of any one of claims 1-13, further comprising
culturing a cell in the production bioreactor using a concentration
of the sensitizer predicted to have a specific cell death rate of
less 40% per day during the culturing of the cell in the production
bioreactor.
15. The method of any one of claims 1-13, further comprising
culturing a cell in the production bioreactor using a concentration
of the sensitizer predicted to have a specific cell death rate of
less than 20% per day during the culturing of the cell in the
production bioreactor.
16. The method of any one of claims 1-13, further comprising
culturing a cell in the production bioreactor using a concentration
of the sensitizer predicted to result in a cell specific LDH
production of less than 150 nU/cell/day during the culturing of the
cell in the production bioreactor.
17. The method of any one of claims 1-13, further comprising
culturing a cell in the production bioreactor using a concentration
of the sensitizer predicted to result in a cell specific LDH
production of less than 100 nU/cell/day during the culturing of the
cell in the production bioreactor.
18. The method of any one of claims 1-13, further comprising
culturing a cell in the production bioreactor using a concentration
of the sensitizer predicted to result in an apparent growth rate of
greater than 1% per day during the culturing of the cell in the
production bioreactor.
19. The method of any one of claims 1-13, further comprising
culturing a cell in the production bioreactor using a concentration
of the sensitizer predicted to result in an apparent growth rate of
greater than 5% per day during the culturing of the cell in the
production bioreactor.
20. The method of any one of claims 14-19, wherein the cell
contains a nucleic acid encoding a recombinant therapeutic
protein.
21. The method of claim 20, further comprising collecting the
recombinant therapeutic protein.
22. The method of claim 21, further comprising purifying the
recombinant therapeutic protein.
23. The method of claim 22, further comprising formulating the
purified recombinant therapeutic protein.
24. The method of any one of claims 1-23, wherein the cell is a
bacterium, a yeast, or a mammalian cell.
25. The method of any one of claims 1-24, wherein the cell
viability is determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel.
26. The method of any one of claims 1-8 and 10-25, wherein the test
culture vessel is a baffled shake flask and the plurality of test
conditions comprise one or more of: (a) a rotary agitation of about
125 RPM to about 400 RPM; (b) a concentration of the sensitizer of
about 0 ppm to about 120 ppm; and (c) a concentration of a
protectant that is about 1 g/L to about 10 g/L.
27. The method of claim 2 or 26, wherein the protectant is a
poloxamer, a poloxamine, or a pluronic polyol.
28. The method of claim 27, wherein the poloxamer is poloxamer-188,
poloxamer-401, poloxamer-402, or poloxamer-407.
29. The method of claim 27, wherein the poloxamine is
poloxamine-904 or poloxamine-908.
30. A method of improving cell viability in a production
bioreactor, the method comprising: (a) determining cell viability
under a plurality of test conditions in a test culture vessel,
wherein the plurality of conditions are defined by a set of
parameters; (b) based on the cell viability in the test culture
vessel under the plurality of conditions, selecting a condition
that is optimal for cell viability in a production bioreactor; and
(d) culturing a cell in the production bioreactor under the
selected condition.
31. The method of claim 30, wherein the set of parameters comprises
shear stress, concentration of a sensitizer, and concentration of a
protectant.
32. The method of claim 30 or claim 31, wherein the plurality of
testing conditions are selected based on full factorial screening
and/or inscribed center composite design.
33. The method of claim 31 or 32, wherein the sensitizer is an
antifoam agent.
34. The method of claim 33, wherein the antifoam is a
polydimethylsiloxane-based antifoam.
35. The method of claim 33, wherein the antifoam is
simethicone.
36. The method of any one of claims 30-35, wherein the test culture
vessel has a volume of about 5 mL to about 5 L.
37. The method of claim 36, wherein the test culture vessel has a
volume of about 10 mL to about 500 mL.
38. The method of any one of claims 30-37, wherein the test culture
vessel is a baffled shake flask.
39. The method of any one of claims 30-38, wherein the production
bioreactor has a volume of about 5 L to about 20,000 L.
40. The method of claim 39, wherein the production bioreactor has a
volume of about 50 L to about 10,000 L.
41. The method of any one of claims 30-40, wherein the production
bioreactor is a perfusion bioreactor.
42. The method of any one of claims 30-40, wherein the production
bioreactor is a fed batch bioreactor.
43. The method of any one of claims 30-42, wherein step (c) results
in a specific cell death rate of less than 40% per day during the
culturing of the cell in the production bioreactor.
44. The method of any one of claims 30-42, wherein step (c) results
in a specific cell death rate of less than 20% per day during the
culturing of the cell in the production bioreactor.
45. The method of any one of claims 30-42, wherein step (c) results
in a cell specific LDH production of less than 150 nU/cell/day
during the culturing of the cell in the production bioreactor.
46. The method of any one of claims 30-42, wherein step (c) results
in a cell specific LDH production of less than 100 nU/cell/day
during the culturing of the cell in the production bioreactor.
47. The method of any one of claims 30-42, wherein step (c) results
in an apparent growth rate of greater than 1% per day during the
culturing of the cell in the production bioreactor.
48. The method of any one of claims 30-42, wherein step (c) results
in an apparent growth rate of greater than 5% per day during the
culturing of the cell in the production bioreactor.
49. The method of any one of claims 30-48, wherein the cell
contains a nucleic acid encoding a recombinant therapeutic
protein.
50. The method of claim 49, further comprising collecting the
recombinant therapeutic protein.
51. The method of claim 50, further comprising purifying the
recombinant therapeutic protein.
52. The method of claim 51, further comprising formulating the
purified recombinant therapeutic protein.
53. The method of any one of claims 30-52, wherein the cell is a
bacterium, a yeast, or a mammalian cell.
54. The method of any one of claims 30-53, wherein the cell
viability is determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel.
55. The method of any one of claims 30-37 and 39-54, wherein the
test culture vessel is a baffled shake flask and the plurality of
test conditions comprise one or more of: (a) a rotary agitation of
about 125 RPM to about 400 RPM; (b) a concentration of a sensitizer
of about 0 ppm to about 120 ppm; and (c) a concentration of a
protectant that is about 1 g/L to about 10 g/L.
56. The method of claim 31 or 55, wherein the protectant is a
poloxamer, a poloxamine, or a pluronic polyol.
57. The method of claim 56, wherein the poloxamer is poloxamer-188,
poloxamer-401, poloxamer-402, or poloxamer-407.
58. The method of claim 56, wherein the poloxamine is
poloxamine-904 or poloxamine-908.
59. A method for predicting cell viability in a production
bioreactor, the method comprising: (a) selecting a set of
parameters comprising at least two parameters; (b) determining cell
viability under a plurality of test conditions as defined by the
set of parameters in a test culture vessel; and (c) based on the
cell viability in the test culture vessel under the plurality of
conditions, predicting cell viability in the production
bioreactor.
60. The method of claim 59, wherein the set of parameters comprises
shear stress, concentration of a sensitizer, and concentration of a
protectant.
61. The method of claim 59 or claim 60, wherein the plurality of
test conditions are selected based on full factorial screening
and/or inscribed center composite design.
62. The method of claim 60 or 61, wherein the sensitizer is an
antifoam agent.
63. The method of claim 62, wherein the antifoam is a
polydimethylsiloxane-based antifoam.
64. The method of claim 62, wherein the antifoam is
simethicone.
65. The method of any one of claims 59-64, wherein the test culture
vessel has a volume of about 5 mL to about 5 L.
66. The method of claim 65, wherein the test culture vessel has a
volume of about 10 mL to about 500 mL.
67. The method of any one of claims 59-66, wherein the test culture
vessel is a baffled shake flask.
68. The method of any one of claims 59-67, wherein the production
bioreactor has a volume of about 5 L to about 20,000 L.
69. The method of claim 68, wherein the production bioreactor has a
volume of about 50 L to about 10,000 L.
70. The method of any one of claims 59-69, wherein the production
bioreactor is a perfusion bioreactor.
71. The method of any one of claims 59-69, wherein the production
bioreactor is a fed batch bioreactor.
72. The method of any one of claims 59-71, further comprising
culturing a cell in the production bioreactor using a condition
predicted to result in a specific cell death rate of less 40% per
day during the culturing of the cell in the production
bioreactor.
73. The method of any one of claims 59-71, further comprising
culturing a cell in the production bioreactor using a condition
predicted to result in a specific cell death rate of less than 20%
per day during the culturing of the cell in the production
bioreactor.
74. The method of any one of claims 59-71, further comprising
culturing a cell in the production bioreactor using a condition
predicted to result in a cell specific LDH production of less than
150 nU/cell/day during the culturing of the cell in the production
bioreactor.
75. The method of any one of claims 59-71, further comprising
culturing a cell in the production bioreactor using a condition
predicted to result in a cell specific LDH production of less than
100 nU/cell/day during the culturing of the cell in the production
bioreactor.
76. The method of any one of claims 59-71, further comprising
culturing a cell in the production bioreactor using a condition
predicted to result in an apparent growth rate of greater than 1%
per day during the culturing of the cell in the production
bioreactor.
77. The method of any one of claims 59-71, further comprising
culturing a cell in the production bioreactor using a condition
predicted to result in an apparent growth rate of greater than 5%
per day during the culturing of the cell in the production
bioreactor.
78. The method of any one of claims 72-77, wherein the cell
contains a nucleic acid encoding a recombinant therapeutic
protein.
79. The method of claim 78, further comprising collecting the
recombinant therapeutic protein.
80. The method of claim 79, further comprising purifying the
recombinant therapeutic protein.
81. The method of claim 80, further comprising formulating the
purified recombinant therapeutic protein.
82. The method of any one of claims 59-81, wherein the cell is a
bacterium, a yeast, or a mammalian cell.
83. The method of any one of claims 59-82, wherein the cell
viability is determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel.
84. The method of any one of claims 59-66 and 68-83, wherein the
test culture vessel is a baffled shake flask and the plurality of
test conditions comprise one or more of: (a) a rotary agitation of
about 125 RPM to about 400 RPM; (b) a concentration of the
sensitizer of about 0 ppm to about 120 ppm; and (c) a concentration
of a protectant that is about 1 g/L to about 10 g/L.
85. The method of claim 60 or 84, wherein the protectant is a
poloxamer, a poloxamine, or a pluronic polyol.
86. The method of claim 85, wherein the poloxamer is poloxamer-188,
poloxamer-401, poloxamer-402, or poloxamer-407.
87. The method of claim 85, wherein the poloxamine is
poloxamine-904 or poloxamine-908.
88. A method for culturing a cell in a production bioreactor,
comprising a culturing a cell in a liquid culture medium under one
or more of the following conditions: e) the production bioreactor
has a P/V (kW/m.sup.3) from about 1.0 to about 110 (e.g., about 3.0
to about 8.0, about 3.0 to about 7.0, from about 4.0 to about 6.0,
from about 5.0 to about 6.0, or about 6.0); f) the production
bioreactor has a volume of air per volume of liquid per minute
(VVM) (min.sup.-1) from about 0.01 to about 0.10 (e.g., from about
0.02 to about 0.08, from about 0.04 to about 0.06, or about 0.06);
g) the medium comprises a sensitizer that has a concentration of
about 0 ppm to about 5,000 ppm (e.g., about 0 ppm to about 120 ppm,
about 50 ppm to about 110 ppm, from about 80 ppm to about 100 ppm,
or about 100 ppm); and h) the liquid culture medium comprises a
protectant that has a concentration that is from about 1 g/L to
about 15 g/L (e.g., from about 2 g/L to about 10 g/L, from about 3
g/L to about 7 g/L, from about 4 g/L to about 6 g/L, or about 5
g/L).
89. The method of claim 88, wherein the sensitizer is an antifoam
agent.
90. The method of claim 89, wherein the antifoam is a
polydimethylsiloxane-based antifoam.
91. The method of claim 89, wherein the antifoam is
simethicone.
92. The method of any one of claims 88-91, wherein the protectant
is a poloxamer, a poloxamine, or a pluronic polyol.
93. The method of claim 92, wherein the poloxamer is poloxamer-188,
poloxamer-401, poloxamer-402, or poloxamer-407.
94. The method of claim 92, wherein the poloxamine is
poloxamine-904 or poloxamine-908.
95. The method of any one of claims 88-94, wherein the production
bioreactor has a volume of about 5 L to about 20,000 L.
96. The method of claim 95, wherein the production bioreactor has a
volume of about 50 L to about 10,000 L.
97. The method of any one of claims 88-96, wherein the production
bioreactor is a perfusion bioreactor.
98. The method of any one of claims 88-96, wherein the production
bioreactor is a fed batch bioreactor.
99. The method of any one of claims 88-98, wherein the method
results in a specific cell death rate of less than 40% per day
during the culturing of the cell in the production bioreactor.
100. The method of any one of claims 88-98, wherein the method
results in a specific cell death rate of less than 20% per day
during the culturing of the cell in the production bioreactor.
101. The method of any one of claims 88-98, wherein the method
results in a cell specific LDH production of less than 150
nU/cell/day during the culturing of the cell in the production
bioreactor.
102. The method of any one of claims 88-98, wherein the method
results in a cell specific LDH production of less than 100
nU/cell/day during the culturing of the cell in the production
bioreactor.
103. The method of any one of claims 88-98, wherein the method
results in an apparent growth rate of greater than 1% per day
during the culturing of the cell in the production bioreactor.
104. The method of any one of claims 88-98, wherein the method
results in an apparent growth rate of greater than 5% per day
during the culturing of the cell in the production bioreactor.
105. The method of any one of claims 88-98, wherein the cell
contains a nucleic acid encoding a recombinant therapeutic
protein.
106. The method of claim 105, further comprising collecting the
recombinant therapeutic protein.
107. The method of claim 106, further comprising purifying the
recombinant therapeutic protein.
108. The method of claim 107, further comprising formulating the
purified recombinant therapeutic protein.
109. The method of any one of claims 88-108, wherein the cell is a
bacterium, a yeast, or a mammalian cell.
110. The method of any one of claims 88-109, wherein the cell
viability is determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/355,387, filed Mar. 15, 2019, which claims
priority to U.S. Provisional Patent Application Ser. No. 62/644,339
filed Mar. 16, 2018, and U.S. Provisional Patent Application Ser.
No. 62/645,755 filed Mar. 20, 2018; entire contents of each of
these applications are herein incorporated by reference.
TECHNICAL FIELD
[0002] This disclosure is related to methods of improving cell
viability in a production bioreactor and the fields of
biotechnology and cell biology.
BACKGROUND
[0003] Process intensification provides many advantages for the
production of recombinant therapeutic proteins. One of the
strategies for process intensification is to maintain high cell
densities to improve recombinant protein production, which further
requires high oxygen demand. Oxygen sparge in a production
bioreactor leads to foaming in the production bioreactor. Foaming
often occurs in both intensified fed batch and perfusion processes.
Thus, antifoaming agents are usually added in these processes.
However, the addition and accumulation of antifoam agents can make
cells become sensitive to shear, leading to increased cell
death.
[0004] Thus, there is a need to develop a model to characterize
acceptable cell culture conditions in a production bioreactor among
various cell culture parameters, e.g., shear, media components and
additives, and antifoam concentration.
SUMMARY
[0005] The present invention is based, e.g., on the discovery that
the presently claimed methods that utilize data obtained in a test
culture vessel (e.g., a baffled shake flask) can be used to
accurately predict the effect of a concentration of a sensitizer on
cell viability in a production bioreactor, accurately select a
condition that is optimal for cell viability in a production
bioreactor, predict cell viability in a production bioreactor, and
accurately identify a set of conditions that can be used to culture
a cell in a production bioreactor.
[0006] In one aspect, the disclosure is related to methods of
predicting the effect of a concentration of a sensitizer on cell
viability in a production bioreactor. The methods involve (a)
determining cell viability under a plurality of test conditions in
a test culture vessel, wherein the plurality of test conditions are
defined by a set of parameters; and (b) based on the cell viability
in the test culture vessel under the plurality of conditions,
predicting the effect of a concentration of a sensitizer on cell
viability in a production bioreactor.
[0007] In some embodiments, the set of parameters comprises shear
stress, concentration of the sensitizer, and concentration of a
protectant.
[0008] In some embodiments, the plurality of testing conditions are
selected based on full factorial screening and/or inscribed center
composite design.
[0009] In some embodiments, the sensitizer is an antifoam
agent.
[0010] In some embodiments, the antifoam is a
polydimethylsiloxane-based antifoam, or simethicone.
[0011] In some embodiments, the test culture vessel has a volume of
about 5 mL to about 5 L, or has a volume of about 10 mL to about
500 mL. In some embodiments, the test culture vessel is a baffled
shake flask.
[0012] In some embodiments, the production bioreactor has a volume
of about 5 L to about 20,000 L, or has a volume of about 50 L to
about 10,000 L. In some embodiments, the production bioreactor is a
perfusion bioreactor or a fed batch bioreactor.
[0013] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a concentration of the
sensitizer predicted to have a specific cell death rate of less 40%
per day during the culturing of the cell in the production
bioreactor.
[0014] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a concentration of the
sensitizer predicted to have a specific cell death rate of less
than 20% per day during the culturing of the cell in the production
bioreactor.
[0015] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a concentration of the
sensitizer predicted to result in a cell specific LDH production of
less than 150 nU/cell/day during the culturing of the cell in the
production bioreactor.
[0016] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a concentration of the
sensitizer predicted to result in a cell specific LDH production of
less than 100 nU/cell/day during the culturing of the cell in the
production bioreactor.
[0017] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a concentration of the
sensitizer predicted to result in an apparent growth rate of
greater than 1% per day during the culturing of the cell in the
production bioreactor.
[0018] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a concentration of the
sensitizer predicted to result in an apparent growth rate of
greater than 5% per day during the culturing of the cell in the
production bioreactor.
[0019] In some embodiments, the cell contains a nucleic acid
encoding a recombinant therapeutic protein.
[0020] In some embodiments, the methods further comprise collecting
the recombinant therapeutic protein. In some embodiments, the
methods further comprise purifying the recombinant therapeutic
protein. In some embodiments, the methods further comprise
formulating the purified recombinant therapeutic protein.
[0021] In some embodiments, the cell is a bacterium, a yeast, or a
mammalian cell.
[0022] In some embodiments, the cell viability is determined by
measuring a concentration of lactic acid dehydrogenase (LDH) in the
test culture vessel.
[0023] In some embodiments, the test culture vessel is a baffled
shake flask and the plurality of test conditions comprise one or
more of:
[0024] (a) a rotary agitation of about 125 RPM to about 400
RPM;
[0025] (b) a concentration of the sensitizer of about 0 ppm to
about 5000 ppm; and
[0026] (c) a concentration of a protectant that is about 1 g/L to
about 10 g/L.
[0027] In some embodiments, the protectant is a poloxamer, a
poloxamine, or a non-ionic surfactant Pluronic.RTM..
[0028] In some embodiments, the poloxamer is poloxamer-188,
poloxamer-401, poloxamer-402, or poloxamer-407.
[0029] In some embodiments, the poloxamine is poloxamine-904 or
poloxamine-908.
[0030] In another aspect, the disclosure is related to methods of
improving cell viability in a production bioreactor. The methods
involve
[0031] (a) determining cell viability under a plurality of test
conditions in a test culture vessel, wherein the plurality of
conditions are defined by a set of parameters;
[0032] (b) based on the cell viability in the test culture vessel
under the plurality of conditions, selecting a condition that is
optimal for cell viability in a production bioreactor; and
[0033] (c) culturing a cell in the production bioreactor under the
selected condition.
[0034] In some embodiments, the set of parameters comprises shear
stress, concentration of a sensitizer, and concentration of a
protectant.
[0035] In some embodiments, the plurality of testing conditions are
selected based on full factorial screening and/or inscribed center
composite design.
[0036] In some embodiments, the sensitizer is an antifoam agent. In
some embodiments, the antifoam is a polydimethylsiloxane-based
antifoam or simethicone.
[0037] In some embodiments, the test culture vessel has a volume of
about 5 mL to about 5 L.
[0038] In some embodiments, the test culture vessel has a volume of
about 10 mL to about 500 mL. In some embodiments, the test culture
vessel is a baffled shake flask.
[0039] In some embodiments, the production bioreactor has a volume
of about 5 L to about 20,000 L. In some embodiments, the production
bioreactor has a volume of about 50 L to about 10,000 L. In some
embodiments, the production bioreactor is a perfusion bioreactor or
a fed batch bioreactor.
[0040] In some embodiments, step (c) results in a specific cell
death rate of less than 40% per day during the culturing of the
cell in the production bioreactor. In some embodiments, step (c)
results in a specific cell death rate of less than 20% per day
during the culturing of the cell in the production bioreactor. In
some embodiments, step (c) results in a cell specific LDH
production of less than 150 nU/cell/day during the culturing of the
cell in the production bioreactor. In some embodiments, step (c)
results in a cell specific LDH production of less than 100
nU/cell/day during the culturing of the cell in the production
bioreactor. In some embodiments, step (c) results in an apparent
growth rate of greater than 1% per day during the culturing of the
cell in the production bioreactor. In some embodiments, step (c)
results in an apparent growth rate of greater than 5% per day
during the culturing of the cell in the production bioreactor.
[0041] In some embodiments, the cell contains a nucleic acid
encoding a recombinant therapeutic protein.
[0042] In some embodiments, the methods further comprise collecting
the recombinant therapeutic protein. In some embodiments, the
methods further comprise purifying the recombinant therapeutic
protein. In some embodiments, the methods further comprise
formulating the purified recombinant therapeutic protein.
[0043] In some embodiments, the cell is a bacterium, a yeast, or a
mammalian cell. In some embodiments, the cell viability is
determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel.
[0044] In some embodiments, the test culture vessel is a baffled
shake flask and the plurality of test conditions comprise one or
more of:
[0045] (a) a rotary agitation of about 125 RPM to about 400
RPM;
[0046] (b) a concentration of a sensitizer of about 0 ppm to about
5000 ppm; and
[0047] (c) a concentration of a protectant that is about 1 g/L to
about 10 g/L.
[0048] In some embodiments, the protectant is a poloxamer, a
poloxamine, or a non-ionic surfactant Pluronic. In some
embodiments, the poloxamer is poloxamer-188, poloxamer-401,
poloxamer-402, or poloxamer-407. In some embodiments, the
poloxamine is poloxamine-904 or poloxamine-908.
[0049] In another aspect, the disclosure is related to methods for
predicting cell viability in a production bioreactor. The methods
involve
[0050] (a) selecting a set of parameters comprising at least two
parameters;
[0051] (b) determining cell viability under a plurality of test
conditions as defined by the set of parameters in a test culture
vessel; and
[0052] (c) based on the cell viability in the test culture vessel
under the plurality of conditions, predicting cell viability in the
production bioreactor.
[0053] In some embodiments, the set of parameters comprises shear
stress, concentration of a sensitizer, and concentration of a
protectant.
[0054] In some embodiments, the plurality of test conditions are
selected based on full factorial screening and/or inscribed center
composite design.
[0055] In some embodiments, the sensitizer is an antifoam
agent.
[0056] In some embodiments, the antifoam is a
polydimethylsiloxane-based antifoam. In some embodiments, the
antifoam is simethicone.
[0057] In some embodiments, the test culture vessel has a volume of
about 5 mL to about 5 L.
[0058] In some embodiments, the test culture vessel has a volume of
about 10 mL to about 500 mL.
[0059] In some embodiments, the test culture vessel is a baffled
shake flask.
[0060] In some embodiments, the production bioreactor has a volume
of about 5 L to about 20,000 L, or has a volume of about 50 L to
about 10,000 L.
[0061] In some embodiments, the production bioreactor is a
perfusion bioreactor or a fed batch bioreactor.
[0062] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a condition predicted to
result in a specific cell death rate of less 40% per day during the
culturing of the cell in the production bioreactor.
[0063] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a condition predicted to
result in a specific cell death rate of less than 20% per day
during the culturing of the cell in the production bioreactor.
[0064] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a condition predicted to
result in a cell specific LDH production of less than 150
nU/cell/day during the culturing of the cell in the production
bioreactor.
[0065] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a condition predicted to
result in a cell specific LDH production of less than 100
nU/cell/day during the culturing of the cell in the production
bioreactor.
[0066] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a condition predicted to
result in an apparent growth rate of greater than 1% per day during
the culturing of the cell in the production bioreactor.
[0067] In some embodiments, the methods further comprise culturing
a cell in the production bioreactor using a condition predicted to
result in an apparent growth rate of greater than 5% per day during
the culturing of the cell in the production bioreactor.
[0068] In some embodiments, the cell contains a nucleic acid
encoding a recombinant therapeutic protein.
[0069] In some embodiments, the methods further comprise collecting
the recombinant therapeutic protein. In some embodiments, the
methods further comprise purifying the recombinant therapeutic
protein. In some embodiments, the methods further comprise
formulating the purified recombinant therapeutic protein.
[0070] In some embodiments, the cell is a bacterium, a yeast, or a
mammalian cell.
[0071] In some embodiments, the cell viability is determined by
measuring a concentration of lactic acid dehydrogenase (LDH) in the
test culture vessel.
[0072] In some embodiments, the test culture vessel is a baffled
shake flask and the plurality of test conditions comprise one or
more of:
[0073] (a) a rotary agitation of about 125 RPM to about 400
RPM;
[0074] (b) a concentration of the sensitizer of about 0 ppm to
about 5000 ppm; and
[0075] (c) a concentration of a protectant that is about 1 g/L to
about 10 g/L.
[0076] In some embodiments, the protectant is a poloxamer, a
poloxamine, or a non-ionic surfactant Pluronic. In some
embodiments, the poloxamer is poloxamer-188, poloxamer-401,
poloxamer-402, or poloxamer-407. In some embodiments, the
poloxamine is poloxamine-904 or poloxamine-908.
[0077] In another aspect, the disclosure also provides methods for
culturing a cell in a production bioreactor, comprising a culturing
a cell in a liquid culture medium under one or more of the
following conditions: [0078] a) the production bioreactor has a PN
(W/m.sup.3) from about 1.0 to about 110.0 (e.g., from about 10.0 to
about 100.0, from about 20.0 to about 80.0, from about 40.0 to
about 60.0, or about 6.0); [0079] b) the production bioreactor has
a volume of air per volume of liquid per minute (VVM) (min.sup.-1)
from about 0.01 to about 0.10 (e.g., from about 0.02 to about 0.08,
from about 0.04 to about 0.06, or about 0.06); [0080] c) the medium
comprises a sensitizer that has a concentration of about 0 ppm to
about 5000 ppm (e.g., from about 50 ppm to about 4000 ppm, from
about 1000 ppm to about 2000 ppm, or about 100 ppm); and [0081] d)
the liquid culture medium comprises a protectant that has a
concentration that is from about 1 g/L to about 15 g/L (e.g., from
about 2 g/L to about 10 g/L, from about 3 g/L to about 7 g/L, from
about 4 g/L to about 6 g/L, or about 5 g/L).
[0082] In some embodiments, the sensitizer is an antifoam
agent.
[0083] In some embodiments, the antifoam is a
polydimethylsiloxane-based antifoam. In some embodiments, the
antifoam is simethicone.
[0084] In some embodiments, the protectant is a poloxamer, a
poloxamine, or a non-ionic surfactant Pluronic.RTM..
[0085] In some embodiments, the poloxamer is poloxamer-188,
poloxamer-401, poloxamer-402, or poloxamer-407. In some
embodiments, the poloxamine is poloxamine-904 or
poloxamine-908.
[0086] In some embodiments, the production bioreactor has a volume
of about 5 L to about 20,000 L. In some embodiments, the production
bioreactor has a volume of about 50 L to about 10,000 L. In some
embodiments, the production bioreactor is a perfusion bioreactor or
a fed batch bioreactor.
[0087] In some embodiments, the methods result in a specific cell
death rate of less than 40% per day during the culturing of the
cell in the production bioreactor. In some embodiments, the methods
result in a specific cell death rate of less than 20% per day
during the culturing of the cell in the production bioreactor. In
some embodiments, the methods result in a cell specific LDH
production of less than 150 nU/cell/day during the culturing of the
cell in the production bioreactor. In some embodiments, the methods
result in a cell specific LDH production of less than 100
nU/cell/day during the culturing of the cell in the production
bioreactor. In some embodiments, the methods result in an apparent
growth rate of greater than 1% per day during the culturing of the
cell in the production bioreactor. In some embodiments, the methods
result in an apparent growth rate of greater than 5% per day during
the culturing of the cell in the production bioreactor.
[0088] In some embodiments, the cell contains a nucleic acid
encoding a recombinant therapeutic protein.
[0089] In some embodiments, the methods further comprise collecting
the recombinant therapeutic protein. In some embodiments, the
methods further comprise purifying the recombinant therapeutic
protein. In some embodiments, the methods further comprise
formulating the purified recombinant therapeutic protein.
[0090] In some embodiments, the cell is a bacterium, a yeast, or a
mammalian cell. In some embodiments, the cell viability is
determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel.
[0091] As used herein, the word "a" before a noun represents one or
more of the particular noun. For example, the phrase "a cell"
represents "one or more cells."
[0092] The term "protectant" means an agent that decreases a cell's
sensitivity (e.g., a mammalian cell's sensitivity) to shear stress
during culturing in a vessel (e.g., a test culture vessel or a
production bioreactor). A non-limiting examples of protectants
include, e.g., poloxamers (e.g., poloxamer-188, poloxamer-401,
poloxamer-402, and poloxamer-407), poloxamines (e.g.,
poloxamine-904 or poloxaminer-908), and non-ionic surfactant
Pluronic.RTM.. Additional examples of protectants are described
herein and are known in the art.
[0093] The term "sensitizer" means an agent that is added to and/or
is present in a liquid culture medium of a culture of cells (e.g.,
a mammalian cell culture) that increases the cells' sensitivity to
shear stress during culturing in a vessel (e.g., a test culture
vessel or a production bioreactor). Non-limiting examples of
sensitizers include antifoam (e.g., a polydimethylsiloxane-based
antifoam or simethicone). Additional examples of sensitizers are
described herein and are known in the art.
[0094] The term "mammalian cell" means any cell from or derived
from any mammal (e.g., a human, a hamster, a mouse, a green monkey,
a rat, a pig, a cow, or a rabbit). For example, a mammalian cell
can be an immortalized cell. In some embodiments, the mammalian
cell is a differentiated cell. In some embodiments, the mammalian
cell is an undifferentiated cell. Non-limiting examples of
mammalian cells are described herein. Additional examples of
mammalian cells are known in the art.
[0095] The term "culturing" or "cell culturing" means the
maintenance or proliferation of a mammalian cell under a controlled
set of physical conditions.
[0096] The term "culture of cells" or "cell culture" means a liquid
culture medium containing a plurality of cells that is maintained
or proliferated under a controlled set of physical conditions.
[0097] The term "liquid culture medium" means a fluid that contains
sufficient nutrients to allow a cell (e.g., a mammalian cell) to
grow or proliferate in vitro. For example, a liquid culture medium
can contain one or more of: amino acids (e.g., 20 amino acids), a
purine (e.g., hypoxanthine), a pyrimidine (e.g., thymidine),
choline, inositol, thiamine, folic acid, biotin, calcium,
niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin,
pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron,
copper, zinc, and sodium bicarbonate. In some embodiments, a liquid
culture medium can contain serum from a mammal. In some
embodiments, a liquid culture medium does not contain serum or
another extract from a mammal (a defined liquid culture medium). In
some embodiments, a liquid culture medium can contain trace metals,
a mammalian growth hormone, and/or a mammalian growth factor.
Another example of liquid culture medium is minimal medium (e.g., a
medium containing only inorganic salts, a carbon source, and
water). Non-limiting examples of liquid culture medium are
described herein. Additional examples of liquid culture medium are
known in the art and are commercially available. A liquid culture
medium can contain any density of mammalian cells. For example, as
used herein, a volume of liquid culture medium removed from a
bioreactor can be substantially free of mammalian cells.
[0098] The term "animal-derived component free liquid culture
medium" means a liquid culture medium that does not contain any
components (e.g., proteins or serum) derived from a mammal.
[0099] The term "serum-free liquid culture medium" means a liquid
culture medium that does not contain a mammalian serum.
[0100] The term "serum-containing liquid culture medium" means a
liquid culture medium that contains a mammalian serum.
[0101] The term "chemically-defined liquid culture medium" is a
term of art and means a liquid culture medium in which all of the
chemical components are known. For example, a chemically-defined
liquid culture medium does not contain fetal bovine serum, bovine
serum albumin, or human serum albumin, as these preparations
typically contain a complex mix of albumins and lipids.
[0102] The term "protein-free liquid culture medium" means a liquid
culture medium that does not contain any protein (e.g., any
detectable protein).
[0103] The term "perfusion bioreactor" means a bioreactor
containing a plurality of cells (e.g., mammalian cells) in a first
liquid culture medium, wherein the culturing of the cells present
in the bioreactor includes periodic or continuous removal of the
first liquid culture medium and at the same time or shortly
thereafter adding substantially the same volume of a second liquid
culture medium to the bioreactor. In some examples, the second
liquid culture medium can be the same as the first liquid culture
medium. In some examples, there is an incremental change (e.g.,
increase or decrease) in the volume of the first liquid culture
medium removed and added over incremental periods (e.g., an about
24-hour period, a period of between about 1 minute and about
24-hours, or a period of greater than 24 hours) during the
culturing period (e.g., the culture medium refeed rate on a daily
basis). The fraction of media removed and replaced each day can
vary depending on the particular cells being cultured, the initial
seeding density, and the cell density at a particular time. "RV" or
"reactor volume" means the volume of the culture medium present at
the beginning of the culturing process (e.g., the total volume of
the culture medium present after seeding).
[0104] The term "fed-batch bioreactor" is a term of art and means a
bioreactor containing a plurality of cells (e.g., mammalian cells)
in a first liquid culture medium, wherein the culturing of the
cells present in the bioreactor includes the periodic or continuous
addition of a second liquid culture medium to the first liquid
culture medium without substantial or significant removal of the
first liquid culture medium or second liquid culture medium from
the cell culture. The second liquid culture medium can be the same
as the first liquid culture medium. In some examples of fed-batch
culture, the second liquid culture medium is a concentrated form of
the first liquid culture medium. In some examples of fed-batch
culture, the second liquid culture medium is added as a dry powder.
In some examples of fed-batch culture, the second liquid culture
medium is completely different formulation as compared to the first
liquid culture medium.
[0105] The term "concentrated fed-batch bioreactor) is a term of
art and means a bioreactor containing a plurality of cells (e.g.,
mammalian cells) in a first liquid culture medium, wherein the
culturing of the cells present in the bioreactor includes the
periodic or continuous addition of a second liquid culture medium
to the first liquid culture medium with substantial or significant
removal of the first liquid culture medium or second liquid culture
medium from the cell culture while without removing product from
the bioreactor. The second liquid culture medium can be the same as
the first liquid culture medium. In some examples of concentrated
fed-batch culture, the second liquid culture medium is a
concentrated form of the first liquid culture medium. In some
examples of concentrated fed-batch culture, the second liquid
culture medium is added as a dry powder. In some examples of
concentrated fed-batch culture, the second liquid culture medium is
completely different formulation of the first liquid culture
medium.
[0106] "Rotary agitation" is a term well-known in the art and
refers to the movement of a shake flask in a generally circular
fashion, e.g., clock-wise or counter-clockwise, in order to, e.g.,
increase the dissolved 02 concentration in a liquid culture medium
contained therein. Agitation can be performed using any art-known
method, e.g., an instrument that moves the shake flask in a
circular or ellipsoidal motion, such as a rotary shaker. Exemplary
devices that can be used to perform rotary agitation are described
herein. Additional examples of such devices are also known in the
art and are commercially available.
[0107] The term "shake flask" is meant a vessel (e.g., a sterile
vessel) that can hold a volume of liquid culture medium that has at
least one gas permeable surface (e.g., an end that has at a
gas-permeable element, e.g., a membrane, which may also act as a
sterile barrier) and/or at least one vent cap, and at least a
portion of its shape is approximately frustoconical. For example, a
shake flask can be a baffled shake flask or an Erlenmeyer flask, or
any art-recognized modified version thereof.
[0108] The term "production bioreactor" is a term of art and means
a large-scale bioreactor (e.g., having an internal volume over 5 L,
1,000 L, 5,000 L, 10,000 L, 20,000 L, 50,000 L, or 100,000 L). For
example, a production bioreactor can be a perfusion bioreactor.
[0109] The term "test culture vessel" is a term of art and means a
small-scale bioreactor (e.g., relative to a production bioreactor)
having a volume of about 2 mL to about 500 mL. In some examples,
the height and width ratio of the test culture vessel corresponds
to about the height and width ratio of the production bioreactor in
the methods described herein. In some embodiments, the test culture
vessel is a shake flask (e.g., a baffled shake flask). Additional
examples of a test culture vessel are described herein and are
known in the art.
[0110] The term "batch culturing" is a term of art and means a
vessel (e.g., bioreactor) containing a plurality of cells (e.g.,
mammalian cells) in a liquid culture medium, wherein the culturing
of the cells present in the vessel (e.g., bioreactor) does not
include the addition of a substantial or significant amount of
fresh liquid culture medium to the cell culture and does not
include the removal of a substantial or significant amount of
liquid culture medium from the cell culture during culturing.
[0111] The term "fed-batch culturing" is a term of art and means a
vessel (e.g., a production bioreactor) including a plurality of
cells (e.g., mammalian cells) in a liquid culture medium, wherein
the culturing of the cells present in the vessel (e.g., production
bioreactor) includes the periodic or continuous addition of fresh
liquid culture medium to the vessel without substantial or
significant removal of liquid culture medium from the vessel during
culturing. The fresh liquid culture medium can be the same as the
liquid culture medium present in the vessel at the start of the
culturing. In some examples of fed-batch culturing, the fresh
liquid culture medium is a concentrated form of the liquid culture
medium present in the vessel at the start of culturing. In some
examples of fed-batch culture, the fresh culture medium is added as
a dry powder. In some examples of fed-batch culturing, the fresh
culture medium is a completely different formulation compared to
the liquid culture medium present in the vessel at the start of
culturing.
[0112] The methods described herein provide several advantages. For
example, it can align different scales, predict .DELTA.parameters,
and optimize existing design space. It can also increase
predictability/alignment of performance across scales, create more
representative small scale models, increase process robustness,
improve shear tolerance for process intensification, and provide
guidance on sensitizer and protectant addition across different
shear profiles (FIG. 12).
[0113] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can also be used. The materials, methods, and examples are
illustrative only and not intended to be limiting. All
publications, patent applications, patents, sequences, database
entries, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control.
[0114] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
DESCRIPTION OF DRAWINGS
[0115] FIG. 1 is a diagram showing how bubbles in a cell culture
medium can cause cell death.
[0116] FIG. 2 is a diagram illustrating addition of antifoam (an
exemplary sensitizer) can move the operating conditions out of the
acceptable operating space.
[0117] FIG. 3 is a diagram showing the relationship between
specific lactate dehydrogenase (LDH) production and cell death
rate.
[0118] FIG. 4 is a diagram showing the relationship between
protectants, sensitizers, and rotary agitation, and the impact of
protectants, sensitizers, and agitation on specific LDH production
in a cell culture.
[0119] FIG. 5 is a diagram showing that the specific LDH production
is a consequence of sensitizer concentration, protectant
concentration, and rotary agitation (shear stress).
[0120] FIG. 6 shows full factorial design for the three selected
parameters: sensitizer concentration, protectant concentration, and
rotary agitation (shear stress). The top panel shows the full
factorial design (2.sup.3), where the response is specific LDH
production (death rate) and the factors are shear, concentration of
protectant, and concentration of sensitizer.
[0121] The middle panel shows that failure resulted in an extreme
value, there is a large range between point of failure versus the
rest of the data, the point of failure is difficult to model, and
the extreme value is excluded (inoperable region). The bottom panel
shows that centerpoints are poorly predicted (indication of
curvature, response is likely non-linear, and increasing RPM
doesn't scale linearly).
[0122] FIG. 7 is a diagram showing full factorial design combined
with inscribed center composite design for the selected parameters
of sensitizer concentration, protectant concentration, and rotary
agitation (shear stress).
[0123] FIG. 8 is a diagram showing the advantages of the full
factorial design combined with inscribed center composite
design.
[0124] FIG. 9 is a diagram illustrating that the methods described
herein can be used to identify region of poor performance (light
shading) and region of operation failure (dark shading).
[0125] FIG. 10 is a diagram illustrating methods of normalizing
shear stress across scale via specific LDH production and methods
of predicting effects of parameter changes (.DELTA.parameter).
[0126] FIG. 11 is a diagram illustrating how the models described
herein can be used to optimize operating conditions of a production
bioreactor. The data show that the operating condition (rectangle
with filled with open squares) currently works, but can be further
optimized (light shaded rectangle to the right of the large arrow)
(e.g., increase concentration range of sensitizer tolerance in
process and improve resilience to vessel foam out).
[0127] FIG. 12 lists the capabilities and utilities of the models
described herein. The model is able to align different scales,
predict changes in parameters, and optimize existing design spaces.
The model also provides for an increase in the
predictability/alignment of performance across scales (e.g., create
more representative small scale models and augment understanding of
shear across scale on top of traditional methodologies). The model
also provide for increased process robustness (e.g., improved shear
tolerance for process intensification and improved guidance on
concentration of sensitizer and concentration of protectant
addition across different shear profiles).
[0128] FIG. 13A is a graph that compares the observed specific LDH
production values in the tested benchtop vessels against the
predicted specific LDH production values.
[0129] FIG. 13B depicts data from the experiment described in
Example 2.
[0130] FIG. 13C lists the observed specific LDH production values
with the predicted values.
[0131] FIG. 14 compares the observed effects of antifoam agents on
specific LDH production and the predicted effects of antifoam
agents on specific LDH production.
[0132] FIG. 15A is a graph that compares the observed specific LDH
production values in the tested production bioreactors with the
predicted values.
[0133] FIG. 15B lists the observed specific LDH production values
in the tested production bioreactors with the predicted values.
DETAILED DESCRIPTION
[0134] This disclosure relates to methods of optimizing process
intensification for production of proteins or polypeptides. In one
aspect, the disclosure is related to a small scale test culture
vessel (e.g., baffled shake flask) model that can characterize an
operation space as defined by various operation parameters, e.g.,
protectant (e.g., poloxamer-188) concentration, sensitizer (e.g.,
antifoam), shear stress (e.g., agitation rate or oxygen sparging
rate), media components and additives, pH, and time etc.
Particularly, the methods described herein can be used to predict
the effect of these operation parameters on cell culture growth and
viability in a production bioreactor. Orthogonal approaches have
been used to correlate shear stress between small scale and larger
scale models, and the predicted operation space has been validated
by the data at the benchtop scale and at the production scale.
[0135] Thus, provided herein are methods of predicting the effect
of a concentration of a sensitizer on cell viability in a
production bioreactor, methods of improving cell viability in a
production bioreactor, methods for predicting cell viability in a
production bioreactor, and methods for culturing a cell in a
production bioreactor. Non-limiting aspects of these methods are
described herein, and may be combined without limitation.
Process Intensification
[0136] Process intensification refers to improvements in
manufacturing and processing by optimizing existing operation
schemes so that the manufacturing process can be more precise and
more efficient. These improvements can bring various advantages,
e.g., increasing biomass, improving volumetric productivity,
optimizing facility capacity utilization, reducing cost of goods,
maintaining or improving product quality consistency, reducing
facility footprint, and/or reducing unit operations. At the
molecular level, process intensification can also involve enhancing
mixing, improving reaction kinetics, and/or increasing yields and
specificity.
[0137] The present disclosure is related to process intensification
for the production of proteins or polypeptides (e.g., recombinant
therapeutic proteins, recombinant proteins, or recombinant
polypeptides). One strategy to increase recombinant therapeutic
protein or polypeptide production is to increase cell densities.
High cell densities usually require high oxygen consumption, and
the required oxygen sparge often leads to foaming issues (i.e.,
bubbles). As shown in FIG. 1, bubbles can lead to cell death, e.g.,
through cell-bubble contact, cell bubble attachment, or lethal
force from bubble rupture. Formula 1 shows the relationship between
cell death rate, volumetric aeration, and bubble size.
Death .times. Rate .varies. ( Volumetric .times. Aeration ) (
Bubble .times. Size ) 3 ( Formula .times. 1 ) ##EQU00001##
Thus, antifoaming agents are often added to the cell culture
process. However, the addition and/or accumulation of antifoam
agents can cause a negative impact on cell culture performance
(e.g., mammalian cell culture performance). In some cases, the
addition and accumulation of antifoam agents can lead to increased
cell death rate, particularly at the production scale (production
bioreactor scale). Therefore, it is important to assess the effects
of operation parameters or changes in these operation parameters
(e.g., antifoam agents or some other parameters) at the production
scale (production bioreactor scale).
Sensitizers, Agitation, and Protectants
[0138] Because it is prohibitively expensive to test the effects of
various operation parameters at the production scale (production
bioreactor scale), pilot experiments are usually performed in test
culture vessels (e.g., shake flasks, laboratory fermenters, or
pilot fermenters) first. These test culture vessels can be operated
to test the effects of various parameters, e.g., agitation,
sensitizer concentration, protectant concentration, temperature,
pH, nutrient composition, biomass loading and feeding strategy at a
small scale, and correlate the observed effects with effects at the
production scale (production bioreactor scale). In many traditional
methods, power/volume (P/V), volumetric mass transfer coefficient
(kLa), tip speed, mixing time, computational fluid dynamics (CFD)
modelling are often used to correlate the results in test culture
vessels with the effects at the production scale (production
bioreactor scale).
[0139] Among these various parameters, sensitizer parameters (e.g.,
concentration of sensitizer) are especially difficult to scale-up.
In some embodiments of any of the methods described herein, a
sensitizer can be an antifoam agent (e.g., simethicone, simethicone
emulsion, or a polydimethylsiloxane (PDMS)-based oil). A
description of the antifoam agents can be found, e.g., in Bre{hacek
over (v)}evi et al. "Mechanism of antifoaming action of
simethicone," J. Appl. Toxicol. 14(3):207-211, 1994; and Bergeron,
et al. "Polydimethylsiloxane (PDMS)-based antifoams," Colloids and
Surfaces A: Physicochemical and Engineering Aspects
122(1-3):103-120, 1997; both of which are incorporated herein by
reference in its entirety.
[0140] Additional examples of sensitizers include, e.g., organic
oil-based sensitizers, silicone oil-based sensitizers, and mixed
liquid-solid mixture/emulsified sensitizers (e.g., fumed silica,
oil combination antifoams). A sensitizer can be a silicone foam
control agent, e.g., XIAMETER.RTM. ACP-0100 Antifoam Compound,
XIAMETER.RTM. ACP-1500 Antifoam Compound, XIAMETER.RTM. AFE-1510
Antifoam Emulsion, XIAMETER.RTM. AFE-1520 Antifoam Emulsion,
XIAMETER.RTM. AFE-1530 Antifoam Emulsion, XIAMETER.RTM. ACP-1400
Antifoam Compound, XIAMETER.RTM. AFE-0700 Antifoam Emulsion,
XIAMETER.RTM. ACP-1920 Powdered Antifoam, and XIAMETER.RTM. PMX-200
Silicone Fluid. Additional examples of sensitizers include Dow
Corning.RTM. antifoam C emulsion, Antifoam 204 (Sigma Aldrich),
Antifoam A (Sigma Aldrich), Antifoam B emulsion (Sigma Aldrich),
Antifoam C emulsion (Sigma Aldrich), Antifoam Y-30 Emulsion (Sigma
Aldrich), Antifoam 0-30 (Sigma Aldrich), and Antifoam SE-15 (Sigma
Aldrich). A non-limiting example of an organic, non-silicone
polypropylene-based polyether sensitizer is antifoam 204. A
non-limiting example of a simethicone emulsion is Antifoam EX-Cell.
Non-limiting examples of mixed solid-liquid antifoams include
silicone oil (PDMS), compound A, modified CA (MCA), emulsion A
(EA), and analogue of EA (AEA) described in Marinova et al.,
Langmuir 17:2426-2436, 2001. Non-limiting examples of
polydimethylsiloxane (PMSA)-based antifoams are described in
Bergeron et al., Colloids and Surfaces A: Physiochemical and
Engineering Aspects 122:103-120, 1997.
[0141] Additional examples of sensitizers are known in the art.
See, e.g., Rashmika et al., Biotechnol. Prog. 34(1):262-270,
2018.
[0142] Some other parameters, for example, baffling, flask size,
agitation, also have an impact on the cell's sensitivity to shear.
Because of the interactions with other parameters, these sensitizer
parameters (e.g., concentration of an antifoam agent) often have
non-linear relation in the scale-up process, and are geometrically
un-related across different scales. Therefore, traditional models
are usually powerless in terms of predicting the effects of the
sensitizer parameters at the production scale.
[0143] What makes the prediction even more challenging is that the
sensitizer parameter can change in the entire production period.
For example, during the production process (e.g., the culturing of
a cell in a production bioreactor), the cell density increases, and
a higher oxygen sparge is required. Therefore, more antifoam agents
are required and are added to the production bioreactor. The
increased concentration of antifoam agents make the cells even more
sensitive to shear stress, and moves the operating condition
quickly out of acceptable operating space (FIG. 2).
[0144] In addition, protectants can protect cells from the
increased shear stress and are often added to the liquid culture
medium. The increased protectant concentration may be necessary to
mitigate the negative impacts of sensitizers, but the interaction
of protectants with other parameters makes it difficult to
determine the exact amount of protectant that is needed. Some
common protectants include, e.g., poloxamer, poloxamine, and/or
non-ionic surfactant Pluronic.RTM.. In some embodiments, the
protectant is a poloxamer (e.g., poloxamer-188, poloxamer-401,
poloxamer-402, or poloxamer-407). In some other embodiments, the
protectant is a poloxamine (e.g., poloxamine-904 or
poloxamine-908). In some additional embodiments, the protectant is
a non-ionic surfactant Pluronic.RTM. (e.g., L-61, L-121, P-103,
P-123, P-84, P-104, P-65, P-105, L-35, F-127, F-38, F-68, or F-108)
or a reverse non-ionic surfactant Pluronic.RTM. (e.g., 25 R-2, 25
R-4, 25 R-5, 25 R-8, 10 R-5, or 10 R-8). A detailed description of
exemplary protectants can be found, e.g., in Moghimi et al.,
"Poloxamers and poloxamines in nanoparticle engineering and
experimental medicine," Trends in Biotechnology 18(10):412-420,
2000; Murhammer et al., "Structural features of nonionic polyglycol
polymer molecules responsible for the protective effect in sparged
animal cell bioreactors," Biotechnology Progress 6(2):142-148,
1990; Murhammer, "Pluronic.RTM. polyols, cell protection,"
Encyclopedia of Bioprocess Technology (1999); each of which is
incorporated herein by reference in its entirety.
Modeling Based on Factorial Design and Center Composite Design
[0145] The disclosure provides modeling methods to capture the
effects (e.g., cell viability, cell death rate, recombinant
therapeutic protein production, or yield) of various operation
parameters, e.g., protectants (media component/additive, e.g.,
Pluronic.RTM.), sensitizers (e.g., antifoam agents), and shear
stress (agitation) for the purposes of optimizing reactors at any
scale based on the results from test culture vessel models.
[0146] In one aspect, the methods described herein can be used to
predict cell viability in a production bioreactor. The methods
involve selecting a set of parameters comprising at least two
(e.g., three, four, five, or six) parameters; determining cell
viability under a plurality of test conditions as defined by the
set of parameters in a test culture vessel (e.g., any of the test
culture vessels described herein); and based on the cell viability
in the test culture vessel under the plurality of conditions,
predicting cell viability in the production bioreactor. Based on
the prediction, the conditions with the improved cell viability can
be identified. Also provided herein are methods of predicting the
effect of a concentration of a sensitizer on cell viability in a
production bioreactor. The methods involve determining cell
viability under a plurality of test conditions in a test culture
vessel, where the plurality of test conditions are defined by a set
of parameters; and based on the cell viability in the test culture
vessel under the plurality of conditions, predicting the effect of
a concentration of a sensitizer on cell viability in a production
bioreactor.
[0147] The test culture vessel can be a spin tube (e.g. baffled
spin tube), a flask (e.g., a shake flask having a volume of about 3
L) (e.g., baffled shake flask.), an automated laboratory fermenter
(Ambr 15 and Amber 250 from Sartorius), a laboratory fermenter, or
a pilot fermenter. In some embodiments, the test culture vessel can
have a volume of less than 5 L, less than 3 L, less than 1 L, less
than 500 ml, or less than 100 ml (e.g., less than 1 L, or from 50
ml to 500 ml). In some embodiments, the test culture vessel has a
volume (an interior volume) of about 5 mL to about 5 L (e.g., about
10 mL to about 500 mL, or about 2.5 L to about 3.5 L). For example,
a test culture vessel can have a volume (an interior volume) of
about 5 mL to about 5 L, about 5 mL to about 4.5 L, about 5 mL to
about 4.0 L, about 5 mL to about 3.5 L, about 5 mL to about 3.0 L,
about 5 mL to about 2.5 L, about 5 mL to about 2 L, about 5 mL to
about 1.5 L, about 5 mL to about 1 L, about 5 mL to about 900 mL,
about 5 mL to about 800 mL, about 5 mL to about 700 mL, about 5 mL
to about 600 mL, about 5 mL to about 500 mL, about 5 mL to about
450 mL, about 5 mL to about 400 mL, about 5 mL to about 350 mL,
about 5 mL to about 300 mL, about 5 mL to about 250 mL, about 5 mL
to about 200 mL, about 5 mL to about 150 mL, about 5 mL to about
100 mL, about 5 mL to about 80 mL, about 5 mL to about 60 mL, about
5 mL to about 50 mL, about 5 mL to about 40 mL, about 5 mL to about
30 mL, about 5 mL to about 25 mL, about 5 mL to about 20 mL, about
5 mL to about 15 mL, about 5 mL to about 10 mL, about 10 mL to
about 5 L, about 10 mL to about 4.5 L, about 10 mL to about 4.0 L,
about 10 mL to about 3.5 L, about 10 mL to about 3.0 L, about 10 mL
to about 2.5 L, about 10 mL to about 2 L, about 10 mL to about 1.5
L, about 10 mL to about 1 L, about 10 mL to about 900 mL, about 10
mL to about 800 mL, about 10 mL to about 700 mL, about 10 mL to
about 600 mL, about 10 mL to about 500 mL, about 10 mL to about 450
mL, about 10 mL to about 400 mL, about 10 mL to about 350 mL, about
10 mL to about 300 mL, about 10 mL to about 250 mL, about 10 mL to
about 200 mL, about 10 mL to about 150 mL, about 10 mL to about 100
mL, about 10 mL to about 80 mL, about 10 mL to about 60 mL, about
10 mL to about 50 mL, about 10 mL to about 40 mL, about 10 mL to
about 30 mL, about 10 mL to about 25 mL, about 10 mL to about 20
mL, about 10 mL to about 15 mL, about 15 mL to about 5 L, about 15
mL to about 4.5 L, about 15 mL to about 4.0 L, about 15 mL to about
3.5 L, about 15 mL to about 3.0 L, about 15 mL to about 2.5 L,
about 15 mL to about 2 L, about 15 mL to about 1.5 L, about 15 mL
to about 1 L, about 15 mL to about 900 mL, about 15 mL to about 800
mL, about 15 mL to about 700 mL, about 15 mL to about 600 mL, about
15 mL to about 500 mL, about 15 mL to about 450 mL, about 15 mL to
about 400 mL, about 15 mL to about 350 mL, about 15 mL to about 300
mL, about 15 mL to about 250 mL, about 15 mL to about 200 mL, about
15 mL to about 150 mL, about 15 mL to about 100 mL, about 15 mL to
about 80 mL, about 15 mL to about 60 mL, about 15 mL to about 50
mL, about 15 mL to about 40 mL, about 15 mL to about 30 mL, about
15 mL to about 25 mL, about 15 mL to about 20 mL, about 20 mL to
about 5 L, about 20 mL to about 4.5 L, about 20 mL to about 4.0 L,
about 20 mL to about 3.5 L, about 20 mL to about 3.0 L, about 20 mL
to about 2.5 L, about 20 mL to about 2 L, about 20 mL to about 1.5
L, about 20 mL to about 1 L, about 20 mL to about 900 mL, about 20
mL to about 800 mL, about 20 mL to about 700 mL, about 20 mL to
about 600 mL, about 20 mL to about 500 mL, about 20 mL to about 450
mL, about 20 mL to about 400 mL, about 20 mL to about 350 mL, about
20 mL to about 300 mL, about 20 mL to about 250 mL, about 20 mL to
about 200 mL, about 20 mL to about 150 mL, about 20 mL to about 100
mL, about 20 mL to about 80 mL, about 20 mL to about 60 mL, about
20 mL to about 50 mL, about 20 mL to about 40 mL, about 20 mL to
about 30 mL, about 20 mL to about 25 mL, about 25 mL to about 5 L,
about 25 mL to about 4.5 L, about 25 mL to about 4.0 L, about 25 mL
to about 3.5 L, about 25 mL to about 3.0 L, about 25 mL to about
2.5 L, about 25 mL to about 2 L, about 25 mL to about 1.5 L, about
25 mL to about 1 L, about 25 mL to about 900 mL, about 25 mL to
about 800 mL, about 25 mL to about 700 mL, about 25 mL to about 600
mL, about 25 mL to about 500 mL, about 25 mL to about 450 mL, about
25 mL to about 400 mL, about 25 mL to about 350 mL, about 25 mL to
about 300 mL, about 25 mL to about 250 mL, about 25 mL to about 200
mL, about 25 mL to about 150 mL, about 25 mL to about 100 mL, about
25 mL to about 80 mL, about 25 mL to about 60 mL, about 25 mL to
about 50 mL, about 25 mL to about 40 mL, about 25 mL to about 30
mL, about 30 mL to about 5 L, about 30 mL to about 4.5 L, about 30
mL to about 4.0 L, about 30 mL to about 3.5 L, about 30 mL to about
3.0 L, about 30 mL to about 2.5 L, about 30 mL to about 2 L, about
30 mL to about 1.5 L, about 30 mL to about 1 L, about 30 mL to
about 900 mL, about 30 mL to about 800 mL, about 30 mL to about 700
mL, about 30 mL to about 600 mL, about 30 mL to about 500 mL, about
30 mL to about 450 mL, about 30 mL to about 400 mL, about 30 mL to
about 350 mL, about 30 mL to about 300 mL, about 30 mL to about 250
mL, about 30 mL to about 200 mL, about 30 mL to about 150 mL, about
30 mL to about 100 mL, about 30 mL to about 80 mL, about 30 mL to
about 60 mL, about 30 mL to about 50 mL, about 30 mL to about 40
mL, about 40 mL to about 5 L, about 40 mL to about 4.5 L, about 40
mL to about 4.0 L, about 40 mL to about 3.5 L, about 40 mL to about
3.0 L, about 40 mL to about 2.5 L, about 40 mL to about 2 L, about
40 mL to about 1.5 L, about 40 mL to about 1 L, about 40 mL to
about 900 mL, about 40 mL to about 800 mL, about 40 mL to about 700
mL, about 40 mL to about 600 mL, about 40 mL to about 500 mL, about
40 mL to about 450 mL, about 40 mL to about 400 mL, about 40 mL to
about 350 mL, about 40 mL to about 300 mL, about 40 mL to about 250
mL, about 40 mL to about 200 mL, about 40 mL to about 150 mL, about
40 mL to about 100 mL, about 40 mL to about 80 mL, about 40 mL to
about 60 mL, about 40 mL to about 50 mL, about 50 mL to about 5 L,
about 50 mL to about 4.5 L, about 50 mL to about 4.0 L, about 50 mL
to about 3.5 L, about 50 mL to about 3.0 L, about 50 mL to about
2.5 L, about 50 mL to about 2 L, about 50 mL to about 1.5 L, about
50 mL to about 1 L, about 50 mL to about 900 mL, about 50 mL to
about 800 mL, about 50 mL to about 700 mL, about 50 mL to about 600
mL, about 50 mL to about 500 mL, about 50 mL to about 450 mL, about
50 mL to about 400 mL, about 50 mL to about 350 mL, about 50 mL to
about 300 mL, about 50 mL to about 250 mL, about 50 mL to about 200
mL, about 50 mL to about 150 mL, about 50 mL to about 100 mL, about
50 mL to about 80 mL, about 50 mL to about 60 mL, about 60 mL to
about 5 L, about 60 mL to about 4.5 L, about 60 mL to about 4.0 L,
about 60 mL to about 3.5 L, about 60 mL to about 3.0 L, about 60 mL
to about 2.5 L, about 60 mL to about 2 L, about 60 mL to about 1.5
L, about 60 mL to about 1 L, about 60 mL to about 900 mL, about 60
mL to about 800 mL, about 60 mL to about 700 mL, about 60 mL to
about 600 mL, about 60 mL to about 500 mL, about 60 mL to about 450
mL, about 60 mL to about 400 mL, about 60 mL to about 350 mL, about
60 mL to about 300 mL, about 60 mL to about 250 mL, about 60 mL to
about 200 mL, about 60 mL to about 150 mL, about 60 mL to about 100
mL, about 60 mL to about 80 mL, about 80 mL to about 5 L, about 80
mL to about 4.5 L, about 80 mL to about 4.0 L, about 80 mL to about
3.5 L, about 80 mL to about 3.0 L, about 80 mL to about 2.5 L,
about 80 mL to about 2 L, about 80 mL to about 1.5 L, about 80 mL
to about 1 L, about 80 mL to about 900 mL, about 80 mL to about 800
mL, about 80 mL to about 700 mL, about 80 mL to about 600 mL, about
80 mL to about 500 mL, about 80 mL to about 450 mL, about 80 mL to
about 400 mL, about 80 mL to about 350 mL, about 80 mL to about 300
mL, about 80 mL to about 250 mL, about 80 mL to about 200 mL, about
80 mL to about 150 mL, about 80 mL to about 100 mL, about 100 mL to
about 5 L, about 100 mL to about 4.5 L, about 100 mL to about 4.0
L, about 100 mL to about 3.5 L, about 100 mL to about 3.0 L, about
100 mL to about 2.5 L, about 100 mL to about 2 L, about 100 mL to
about 1.5 L, about 100 mL to about 1 L, about 100 mL to about 900
mL, about 100 mL to about 800 mL, about 100 mL to about 700 mL,
about 100 mL to about 600 mL, about 100 mL to about 500 mL, about
100 mL to about 450 mL, about 100 mL to about 400 mL, about 100 mL
to about 350 mL, about 100 mL to about 300 mL, about 100 mL to
about 250 mL, about 100 mL to about 200 mL, about 100 mL to about
150 mL, about 150 mL to about 5 L, about 150 mL to about 4.5 L,
about 150 mL to about 4.0 L, about 150 mL to about 3.5 L, about 150
mL to about 3.0 L, about 150 mL to about 2.5 L, about 150 mL to
about 2 L, about 150 mL to about 1.5 L, about 150 mL to about 1 L,
about 150 mL to about 900 mL, about 150 mL to about 800 mL, about
150 mL to about 700 mL, about 150 mL to about 600 mL, about 150 mL
to about 500 mL, about 150 mL to about 450 mL, about 150 mL to
about 400 mL, about 150 mL to about 350 mL, about 150 mL to about
300 mL, about 150 mL to about 250 mL, about 150 mL to about 200 mL,
about 200 mL to about 5 L, about 200 mL to about 4.5 L, about 200
mL to about 4.0 L, about 200 mL to about 3.5 L, about 200 mL to
about 3.0 L, about 200 mL to about 2.5 L, about 200 mL to about 2
L, about 200 mL to about 1.5 L, about 200 mL to about 1 L, about
200 mL to about 900 mL, about 200 mL to about 800 mL, about 200 mL
to about 700 mL, about 200 mL to about 600 mL, about 200 mL to
about 500 mL, about 200 mL to about 450 mL, about 200 mL to about
400 mL, about 200 mL to about 350 mL, about 200 mL to about 300 mL,
about 200 mL to about 250 mL, about 250 mL to about 5 L, about 250
mL to about 4.5 L, about 250 mL to about 4.0 L, about 250 mL to
about 3.5 L, about 250 mL to about 3.0 L, about 250 mL to about 2.5
L, about 250 mL to about 2 L, about 250 mL to about 1.5 L, about
250 mL to about 1 L, about 250 mL to about 900 mL, about 250 mL to
about 800 mL, about 250 mL to about 700 mL, about 250 mL to about
600 mL, about 250 mL to about 500 mL, about 250 mL to about 450 mL,
about 250 mL to about 400 mL, about 250 mL to about 350 mL, about
250 mL to about 300 mL, about 300 mL to about 5 L, about 300 mL to
about 4.5 L, about 300 mL to about 4.0 L, about 300 mL to about 3.5
L, about 300 mL to about 3.0 L, about 300 mL to about 2.5 L, about
300 mL to about 2 L, about 300 mL to about 1.5 L, about 300 mL to
about 1 L, about 300 mL to about 900 mL, about 300 mL to about 800
mL, about 300 mL to about 700 mL, about 300 mL to about 600 mL,
about 300 mL to about 500 mL, about 300 mL to about 450 mL, about
300 mL to about 400 mL, about 300 mL to about 350 mL, about 350 mL
to about 5 L, about 350 mL to about 4.5 L, about 350 mL to about
4.0 L, about 350 mL to about 3.5 L, about 350 mL to about 3.0 L,
about 350 mL to about 2.5 L, about 350 mL to about 2 L, about 350
mL to about 1.5 L, about 350 mL to about 1 L, about 350 mL to about
900 mL, about 350 mL to about 800 mL, about 350 mL to about 700 mL,
about 350 mL to about 600 mL, about 350 mL to about 500 mL, about
350 mL to about 450 mL, about 350 mL to about 400 mL, about 400 mL
to about 5 L, about 400 mL to about 4.5 L, about 400 mL to about
4.0 L, about 400 mL to about 3.5 L, about 400 mL to about 3.0 L,
about 400 mL to about 2.5 L, about 400 mL to about 2 L, about 400
mL to about 1.5 L, about 400 mL to about 1 L, about 400 mL to about
900 mL, about 400 mL to about 800 mL, about 400 mL to about 700 mL,
about 400 mL to about 600 mL, about 400 mL to about 500 mL, about
400 mL to about 450 mL, about 450 mL to about 5 L, about 450 mL to
about 4.5 L, about 450 mL to about 4.0 L, about 450 mL to about 3.5
L, about 450 mL to about 3.0 L, about 450 mL to about 2.5 L, about
450 mL to about 2 L, about 450 mL to about 1.5 L, about 450 mL to
about 1 L, about 450 mL to about 900 mL, about 450 mL to about 800
mL, about 450 mL to about 700 mL, about 450 mL to about 600 mL,
about 450 mL to about 500 mL, about 500 mL to about 5 L, about 500
mL to about 4.5 L, about 500 mL to about 4.0 L, about 500 mL to
about 3.5 L, about 500 mL to about 3.0 L, about 500 mL to about 2.5
L, about 500 mL to about 2 L, about 500 mL to about 1.5 L, about
500 mL to about 1 L, about 500 mL to about 900 mL, about 500 mL to
about 800 mL, about 500 mL to about 700 mL, about 500 mL to about
600 mL, about 600 mL to about 5 L, about 600 mL to about 4.5 L,
about 600 mL to about 4.0 L, about 600 mL to about 3.5 L, about 600
mL to about 3.0 L, about 600 mL to about 2.5 L, about 600 mL to
about 2 L, about 600 mL to about 1.5 L, about 600 mL to about 1 L,
about 600 mL to about 900 mL, about 600 mL to about 800 mL, about
600 mL to about 700 mL, about 700 mL to about 5 L, about 700 mL to
about 4.5 L, about 700 mL to about 4.0 L, about 700 mL to about 3.5
L, about 700 mL to about 3.0 L, about 700 mL to about 2.5 L, about
700 mL to about 2 L, about 700 mL to about 1.5 L, about 700 mL to
about 1 L, about 700 mL to about 900 mL, about 700 mL to about 800
mL, about 800 mL to about 5 L, about 800 mL to about 4.5 L, about
800 mL to about 4.0 L, about 800 mL to about 3.5 L, about 800 mL to
about 3.0 L, about 800 mL to about 2.5 L, about 800 mL to about 2
L, about 800 mL to about 1.5 L, about 800 mL to about 1 L, about
800 mL to about 900 mL, about 900 mL to about 5 L, about 900 mL to
about 4.5 L, about 900 mL to about 4.0 L, about 900 mL to about 3.5
L, about 900 mL to about 3.0 L, about 900 mL to about 2.5 L, about
900 mL to about 2 L, about 900 mL to about 1.5 L, about 900 mL to
about 1 L, about 1 L to about 5 L, about 1 L to about 4.5 L, about
1 L to about 4.0 L, about 1 L to about 3.5 L, about 1 L to about
3.0 L, about 1 L to about 2.5 L, about 1 L to about 2 L, about 1 L
to about 1.5 L, about 1.5 L to about 5 L, about 1.5 L to about 4.5
L, about 1.5 L to about 4.0 L, about 1.5 L to about 3.5 L, about
1.5 L to about 3.0 L, about 1.5 L to about 2.5 L, about 1.5 L to
about 2 L, about 2 L to about 5 L, about 2 L to about 4.5 L, about
2 L to about 4.0 L, about 2 L to about 3.5 L, about 2 L to about
3.0 L, about 2 L to about 2.5 L, about 2.5 L to about 5 L, about
2.5 L to about 4.5 L, about 2.5 L to about 4.0 L, about 2.5 L to
about 3.5 L, about 2.5 L to about 3.0 L, about 3.0 L to about 5 L,
about 3.0 L to about 4.5 L, about 3.0 L to about 4.0 L, about 3.0 L
to about 3.5 L, about 3.5 L to about 5 L, about 3.5 L to about 4.5
L, about 3.5 L to about 4.0 L, about 4.0 L to about 5 L, about 4.0
L to about 4.5 L, or about 4.5 L to about 5.0 L. In some
embodiments, the ratio of the height to the width of the test
culture vessel is about the same as the ratio of the height to the
width of the production bioreactor.
[0148] In some embodiments, the volume of liquid culture medium
disposed in the test culture vessel (during culturing) can range
from about 0.5 mL to about 1.5 L, about 0.5 mL to about 1.4 L,
about 0.5 mL to about 1.3 L, about 0.5 mL to about 1.2 L, about 0.5
mL to about 1.1 L, about 0.5 mL to about 1.0 L, about 0.5 mL to
about 900 mL, about 0.5 mL to about 800 mL, about 0.5 mL to about
700 mL, about 0.5 mL to about 600 mL, about 0.5 mL to about 500 mL,
about 0.5 mL to about 400 mL, about 0.5 mL to about 300 mL, about
0.5 mL to about 250 mL, about 0.5 mL to about 200 mL, about 0.5 mL
to about 150 mL, about 0.5 mL to about 100 mL, about 0.5 mL to
about 80 mL, about 0.5 mL to about 60 mL, about 0.5 mL to about 50
mL, about 0.5 mL to about 40 mL, about 0.5 mL to about 30 mL, about
0.5 mL to about 25 mL, about 0.5 mL to about 20 mL, about 0.5 mL to
about 10 mL, about 0.5 mL to about 5 mL, about 0.5 mL to about 2.5
mL, about 2.5 mL to about 1.5 L, about 2.5 mL to about 1.4 L, about
2.5 mL to about 1.3 L, about 0.5 mL to about 1.2 L, about 0.5 mL to
about 1.1 L, about 0.5 mL to about 1.0 L, about 2.5 mL to about 900
mL, about 2.5 mL to about 800 mL, about 2.5 mL to about 700 mL,
about 2.5 mL to about 600 mL, about 2.5 mL to about 500 mL, about
2.5 mL to about 400 mL, about 2.5 mL to about 300 mL, about 2.5 mL
to about 250 mL, about 2.5 mL to about 200 mL, about 2.5 mL to
about 150 mL, about 2.5 mL to about 100 mL, about 2.5 mL to about
80 mL, about 2.5 mL to about 60 mL, about 2.5 mL to about 50 mL,
about 2.5 mL to about 40 mL, about 2.5 mL to about 30 mL, about 2.5
mL to about 25 mL, about 2.5 mL to about 20 mL, about 2.5 mL to
about 10 mL, about 2.5 mL to about 5 mL, about 5 mL to about 1.5 L,
about 5 mL to about 1.4 L, about 5 mL to about 1.3 L, about 5 mL to
about 1.2 L, about 5 mL to about 1.1 L, about 5 mL to about 1.0 L,
about 5 mL to about 900 mL, about 5 mL to about 800 mL, about 5 mL
to about 700 mL, about 5 mL to about 600 mL, about 5 mL to about
500 mL, about 5 mL to about 400 mL, about 5 mL to about 300 mL,
about 5 mL to about 250 mL, about 5 mL to about 200 mL, about 5 mL
to about 150 mL, about 5 mL to about 100 mL, about 5 mL to about 80
mL, about 5 mL to about 60 mL, about 5 mL to about 50 mL, about 5
mL to about 40 mL, about 5 mL to about 30 mL, about 5 mL to about
25 mL, about 5 mL to about 20 mL, about 5 mL to about 10 mL, about
10 mL to about 1.5 L, about 10 mL to about 1.4 L, about 10 mL to
about 1.3 L, about 10 mL to about 1.2 L, about 10 mL to about 1.1
L, about 10 mL to about 1.0 L, about 10 mL to about 900 mL, about
10 mL to about 800 mL, about 10 mL to about 700 mL, about 10 mL to
about 600 mL, about 10 mL to about 500 mL, about 10 mL to about 400
mL, about 10 mL to about 300 mL, about 10 mL to about 250 mL, about
10 mL to about 200 mL, about 10 mL to about 150 mL, about 10 mL to
about 100 mL, about 10 mL to about 80 mL, about 10 mL to about 60
mL, about 10 mL to about 50 mL, about 10 mL to about 40 mL, about
10 mL to about 30 mL, about 10 mL to about 25 mL, about 10 mL to
about 20 mL, about 20 mL to about 1.5 L, about 20 mL to about 1.4
L, about 20 mL to about 1.3 L, about 20 mL to about 1.2 L, about 20
mL to about 1.1 L, about 20 mL to about 1.0 L, about 20 mL to about
900 mL, about 20 mL to about 800 mL, about 20 mL to about 700 mL,
about 20 mL to about 600 mL, about 20 mL to about 500 mL, about 20
mL to about 400 mL, about 20 mL to about 300 mL, about 20 mL to
about 250 mL, about 20 mL to about 200 mL, about 20 mL to about 150
mL, about 20 mL to about 100 mL, about 20 mL to about 80 mL, about
20 mL to about 60 mL, about 20 mL to about 50 mL, about 20 mL to
about 40 mL, about 20 mL to about 30 mL, about 20 mL to about 25
mL, about 25 mL to about 1.5 L, about 25 mL to about 1.4 L, about
25 mL to about 1.3 L, about 25 mL to about 1.2 L, about 25 mL to
about 1.1 L, about 25 mL to about 1.0 L, about 25 mL to about 900
mL, about 25 mL to about 800 mL, about 25 mL to about 700 mL, about
25 mL to about 600 mL, about 25 mL to about 500 mL, about 25 mL to
about 400 mL, about 25 mL to about 300 mL, about 25 mL to about 250
mL, about 25 mL to about 200 mL, about 25 mL to about 150 mL, about
25 mL to about 100 mL, about 25 mL to about 80 mL, about 25 mL to
about 60 mL, about 25 mL to about 50 mL, about 25 mL to about 40
mL, about 25 mL to about 30 mL, about 30 mL to about 1.5 L, about
30 mL to about 1.4 L, about 30 mL to about 1.3 L, about 30 mL to
about 1.2 L, about 30 mL to about 1.1 L, about 30 mL to about 1.0
L, about 30 mL to about 900 mL, about 30 mL to about 800 mL, about
30 mL to about 700 mL, about 30 mL to about 600 mL, about 30 mL to
about 500 mL, about 30 mL to about 400 mL, about 30 mL to about 300
mL, about 30 mL to about 250 mL, about 30 mL to about 200 mL, about
30 mL to about 150 mL, about 30 mL to about 100 mL, about 30 mL to
about 80 mL, about 30 mL to about 60 mL, about 30 mL to about 50
mL, about 30 mL to about 40 mL, about 40 mL to about 1.5 L, about
40 mL to about 1.4 L, about 40 mL to about 1.3 L, about 40 mL to
about 1.2 L, about 40 mL to about 1.1 L, about 40 mL to about 1.0
L, about 40 mL to about 900 mL, about 40 mL to about 800 mL, about
40 mL to about 700 mL, about 40 mL to about 600 mL, about 40 mL to
about 500 mL, about 40 mL to about 400 mL, about 40 mL to about 300
mL, about 40 mL to about 250 mL, about 40 mL to about 200 mL, about
40 mL to about 150 mL, about 40 mL to about 100 mL, about 40 mL to
about 80 mL, about 40 mL to about 60 mL, about 40 mL to about 50
mL, about 50 mL to about 1.5 L, about 50 mL to about 1.4 L, about
50 mL to about 1.3 L, about 50 mL to about 1.2 L, about 50 mL to
about 1.1 L, about 50 mL to about 1.0 L, about 50 mL to about 900
mL, about 50 mL to about 800 mL, about 50 mL to about 700 mL, about
50 mL to about 600 mL, about 50 mL to about 500 mL, about 50 mL to
about 400 mL, about 50 mL to about 300 mL, about 50 mL to about 250
mL, about 50 mL to about 200 mL, about 50 mL to about 150 mL, about
50 mL to about 100 mL, about 50 mL to about 80 mL, about 50 mL to
about 60 mL, about 60 mL to about 1.5 L, about 60 mL to about 1.4
L, about 60 mL to about 1.3 L, about 60 mL to about 1.2 L, about 60
mL to about 1.1 L, about 60 mL to about 1.0 L, about 60 mL to about
900 mL, about 60 mL to about 800 mL, about 60 mL to about 700 mL,
about 60 mL to about 600 mL, about 60 mL to about 500 mL, about 60
mL to about 400 mL, about 60 mL to about 300 mL, about 60 mL to
about 250 mL, about 60 mL to about 200 mL, about 60 mL to about 150
mL, about 60 mL to about 100 mL, about 60 mL to about 80 mL, about
80 mL to about 1.5 L, about 80 mL to about 1.4 L, about 80 mL to
about 1.3 L, about 80 mL to about 1.2 L, about 80 mL to about 1.1
L, about 80 mL to about 1.0 L, about 80 mL to about 900 mL, about
80 mL to about 800 mL, about 80 mL to about 700 mL, about 80 mL to
about 600 mL, about 80 mL to about 500 mL, about 80 mL to about 400
mL, about 80 mL to about 300 mL, about 80 mL to about 250 mL, about
80 mL to about 200 mL, about 80 mL to about 150 mL, about 80 mL to
about 100 mL, about 100 mL to about 1.5 L, about 100 mL to about
1.4 L, about 100 mL to about 1.3 L, about 100 mL to about 1.2 L,
about 100 mL to about 1.1 L, about 100 mL to about 1.0 L, about 100
mL to about 900 mL, about 100 mL to about 800 mL, about 100 mL to
about 700 mL, about 100 mL to about 600 mL, about 100 mL to about
500 mL, about 100 mL to about 400 mL, about 100 mL to about 300 mL,
about 100 mL to about 250 mL, about 100 mL to about 200 mL, about
100 mL to about 150 mL, about 150 mL to about 1.5 L, about 150 mL
to about 1.4 L, about 150 mL to about 1.3 L, about 150 mL to about
1.2 L, about 150 mL to about 1.1 L, about 150 mL to about 1.0 L,
about 150 mL to about 900 mL, about 150 mL to about 800 mL, about
150 mL to about 700 mL, about 150 mL to about 600 mL, about 150 mL
to about 500 mL, about 150 mL to about 400 mL, about 150 mL to
about 300 mL, about 150 mL to about 250 mL, about 150 mL to about
200 mL, about 200 mL to about 1.5 L, about 200 mL to about 1.4 L,
about 200 mL to about 1.3 L, about 200 mL to about 1.2 L, about 200
mL to about 1.1 L, about 200 mL to about 1.0 L, about 200 mL to
about 900 mL, about 200 mL to about 800 mL, about 200 mL to about
700 mL, about 200 mL to about 600 mL, about 200 mL to about 500 mL,
about 200 mL to about 400 mL, about 200 mL to about 300 mL, about
200 mL to about 250 mL, about 250 mL to about 1.5 L, about 250 mL
to about 1.4 L, about 250 mL to about 1.3 L, about 250 mL to about
1.2 L, about 250 mL to about 1.1 L, about 250 mL to about 1.0 L,
about 250 mL to about 900 mL, about 250 mL to about 800 mL, about
250 mL to about 700 mL, about 250 mL to about 600 mL, about 250 mL
to about 500 mL, about 250 mL to about 400 mL, about 250 mL to
about 300 mL, about 300 mL to about 1.5 L, about 300 mL to about
1.4 L, about 300 mL to about 1.3 L, about 300 mL to about 1.2 L,
about 300 mL to about 1.1 L, about 300 mL to about 1.0 L, about 300
mL to about 900 mL, about 300 mL to about 800 mL, about 300 mL to
about 700 mL, about 300 mL to about 600 mL, about 300 mL to about
500 mL, about 300 mL to about 400 mL, about 400 mL to about 1.5 L,
about 400 mL to about 1.4 L, about 400 mL to about 1.3 L, about 400
mL to about 1.2 L, about 400 mL to about 1.1 L, about 400 mL to
about 1.0 L, about 400 mL to about 900 mL, about 400 mL to about
800 mL, about 400 mL to about 700 mL, about 400 mL to about 600 mL,
about 400 mL to about 500 mL, about 500 mL to about 1.5 L, about
500 mL to about 1.4 L, about 500 mL to about 1.3 L, about 500 mL to
about 1.2 L, about 500 mL to about 1.1 L, about 500 mL to about 1.0
L, about 500 mL to about 900 mL, about 500 mL to about 800 mL,
about 500 mL to about 700 mL, about 500 mL to about 600 mL, about
600 mL to about 1.5 L, about 600 mL to about 1.4 L, about 600 mL to
about 1.3 L, about 600 mL to about 1.2 L, about 600 mL to about 1.1
L, about 600 mL to about 1.0 L, about 600 mL to about 900 mL, about
600 mL to about 800 mL, about 600 mL to about 700 mL, about 700 mL
to about 1.5 L, about 700 mL to about 1.4 L, about 700 mL to about
1.3 L, about 700 mL to about 1.2 L, about 700 mL to about 1.1 L,
about 700 mL to about 1.0 L, about 700 mL to about 900 mL, about
700 mL to about 800 mL, about 800 mL to about 1.5 L, about 800 mL
to about 1.4 L, about 800 mL to about 1.3 L, about 800 mL to about
1.2 L, about 800 mL to about 1.1 L, about 800 mL to about 1.0 L,
about 800 mL to about 900 mL, about 900 mL to about 1.5 L, about
900 mL to about 1.4 L, about 900 mL to about 1.3 L, about 900 mL to
about 1.2 L, about 900 mL to about 1.1 L, about 900 mL to about 1.0
L, about 1.0 L to about 1.5 L, about 1.0 L to about 1.4 L, about
1.0 L to about 1.3 L, about 1.0 L to about 1.2 L, about 1.0 L to
about 1.1 L, about 1.1 L to about 1.5 L, about 1.1 L to about 1.4
L, about 1.1 L to about 1.3 L, about 1.1 L to about 1.2 L, about
1.2 L to about 1.5 L, about 1.2 L to about 1.4 L, about 1.2 L to
about 1.3 L, about 1.3 L to about 1.5 L, about 1.3 L to about 1.4
L, or about 1.4 L to about 1.5 L.
[0149] In some embodiments, the test culture vessel is a shake
flask, e.g., a baffled shake flask. A shake flask can have a
volume, for example, of about 250 mL to about 300 mL, about 300 mL
to about 400 mL, or about 400 mL to about 500 mL. The shake flask
can include at least one gas permeable surface (e.g., at least one
surface having a gas permeable membrane which may also act as a
sterile barrier) and/or at least one vented cap. A shake flask may
have on its outer surface a structure that allows the shake flask
to be stably placed in a tissue culture incubator (e.g., a rotary
incubator).
[0150] The interior surface of a shake flask may have at least one
coating (e.g., at least one coating of gelatin, collagen,
poly-L-ornithine, polystyrene, and laminin). Exemplary shake flasks
that can be used in any of the methods described herein can be
purchased from Corning Inc. (Tewsbury, Mass.), Presens (Brondby,
Denmark), Nalge-Nunc International (Rochester, N.Y.). Exemplary
shake flasks include baffled shake flasks or Erlenmeyer flasks, or
any art-recognized modified versions thereof. Additional examples
of shake flasks (e.g., different shapes and dimensions of shake
flasks) and interior surface coatings of shake flasks are known in
the art and can be used in the present methods.
[0151] In some embodiments, the test culture vessel can have a
rotary agitation of about 125 RPM to about 400 RPM (e.g., about 150
RPM to about 350 RPM, or about 200 RPM to about 300 RPM). For
example, the test culture vessel can have a rotary agitation of
about 125 RPM to about 400 RPM, about 125 RPM to about 380 RPM,
about 125 RPM to about 360 RPM, about 125 RPM to about 340 RPM,
about 125 RPM to about 320 RPM, about 125 RPM to about 300 RPM,
about 125 RPM to about 280 RPM, about 125 RPM to about 260 RPM,
about 125 RPM to about 240 RPM, about 125 RPM to about 220 RPM,
about 125 RPM to about 200 RPM, about 125 RPM to about 180 RPM,
about 125 RPM to about 160 RPM, about 125 RPM to about 140 RPM,
about 140 RPM to about 400 RPM, about 140 RPM to about 380 RPM,
about 140 RPM to about 360 RPM, about 140 RPM to about 340 RPM,
about 140 RPM to about 320 RPM, about 140 RPM to about 300 RPM,
about 140 RPM to about 280 RPM, about 140 RPM to about 260 RPM,
about 140 RPM to about 240 RPM, about 140 RPM to about 220 RPM,
about 140 RPM to about 200 RPM, about 140 RPM to about 180 RPM,
about 140 RPM to about 160 RPM, about 160 RPM to about 400 RPM,
about 160 RPM to about 380 RPM, about 160 RPM to about 360 RPM,
about 160 RPM to about 340 RPM, about 160 RPM to about 320 RPM,
about 160 RPM to about 300 RPM, about 160 RPM to about 280 RPM,
about 160 RPM to about 260 RPM, about 160 RPM to about 240 RPM,
about 160 RPM to about 220 RPM, about 160 RPM to about 200 RPM,
about 160 RPM to about 180 RPM, about 180 RPM to about 400 RPM,
about 180 RPM to about 380 RPM, about 180 RPM to about 360 RPM,
about 180 RPM to about 340 RPM, about 180 RPM to about 320 RPM,
about 180 RPM to about 300 RPM, about 180 RPM to about 280 RPM,
about 180 RPM to about 260 RPM, about 180 RPM to about 240 RPM,
about 180 RPM to about 220 RPM, about 180 RPM to about 200 RPM,
about 200 RPM to about 400 RPM, about 200 RPM to about 380 RPM,
about 200 RPM to about 360 RPM, about 200 RPM to about 340 RPM,
about 200 RPM to about 320 RPM, about 200 RPM to about 300 RPM,
about 200 RPM to about 280 RPM, about 200 RPM to about 260 RPM,
about 200 RPM to about 240 RPM, about 200 RPM to about 220 RPM,
about 220 RPM to about 400 RPM, about 220 RPM to about 380 RPM,
about 220 RPM to about 360 RPM, about 220 RPM to about 340 RPM,
about 220 RPM to about 320 RPM, about 220 RPM to about 300 RPM,
about 220 RPM to about 280 RPM, about 220 RPM to about 260 RPM,
about 220 RPM to about 240 RPM, about 240 RPM to about 400 RPM,
about 240 RPM to about 380 RPM, about 240 RPM to about 360 RPM,
about 240 RPM to about 340 RPM, about 240 RPM to about 320 RPM,
about 240 RPM to about 300 RPM, about 240 RPM to about 280 RPM,
about 240 RPM to about 260 RPM, about 260 RPM to about 400 RPM,
about 260 RPM to about 380 RPM, about 260 RPM to about 360 RPM,
about 260 RPM to about 340 RPM, about 260 RPM to about 320 RPM,
about 260 RPM to about 300 RPM, about 260 RPM to about 280 RPM,
about 280 RPM to about 400 RPM, about 280 RPM to about 380 RPM,
about 280 RPM to about 360 RPM, about 280 RPM to about 340 RPM,
about 280 RPM to about 320 RPM, about 280 RPM to about 300 RPM,
about 300 RPM to about 400 RPM, about 300 RPM to about 380 RPM,
about 300 RPM to about 360 RPM, about 300 RPM to about 340 RPM,
about 300 RPM to about 320 RPM, about 320 RPM to about 400 RPM,
about 320 RPM to about 380 RPM, about 320 RPM to about 360 RPM,
about 320 RPM to about 340 RPM, about 340 RPM to about 400 RPM,
about 340 RPM to about 380 RPM, about 340 RPM to about 360 RPM,
about 360 RPM to about 400 RPM, about 360 RPM to about 380 RPM, or
about 380 RPM to about 400 RPM.
[0152] In some embodiments, rotary agitation of the test culture
vessel can be performed using a rotary incubator with a throw
(orbit) diameter of between about 25 mm to about 50 mm. In some
embodiments, the test culture vessel can have a concentration of a
sensitizer of about 0 ppm to about 5,000 ppm (e.g., about 0 ppm to
about 120 ppm, about 30 ppm to about 120 ppm, or about 50 ppm to
about 100 ppm). In some embodiments, the test culture vessel can
have a concentration of a sensitizer (e.g., an antifoam, e.g., any
of the antifoams described herein) of 0 ppm to about 200 ppm, 0 ppm
to about 190 ppm, 0 ppm to about 180 ppm, 0 ppm to about 170 ppm, 0
ppm to about 160 ppm, 0 ppm to about 150 ppm, 0 ppm to about 140
ppm, 0 ppm to about 130 ppm, 0 ppm to about 120 ppm, 0 ppm to about
110 ppm, 0 ppm to about 100 ppm, 0 ppm, to about 90 ppm, 0 ppm to
about 80 ppm, 0 ppm to about 70 ppm, 0 ppm to about 60 ppm, 0 ppm
to about 50 ppm, 0 ppm to about 40 ppm, 0 ppm to about 30 ppm, 0
ppm to about 20 ppm, 0 ppm to about 10 ppm, about 10 ppm to about
200 ppm, about 10 ppm to about 190 ppm, about 10 ppm to about 180
ppm, about 10 ppm to about 170 ppm, about 10 ppm to about 160 ppm,
about 10 ppm to about 150 ppm, about 10 ppm to about 140 ppm, about
10 ppm to about 130 ppm, about 10 ppm to about 120 ppm, about 10
ppm to about 110 ppm, about 10 ppm to about 100 ppm, about 10 ppm
to about 90 ppm, about 10 ppm to about 80 ppm, about 10 ppm to
about 70 ppm, about 10 ppm to about 60 ppm, about 10 ppm to about
50 ppm, about 10 ppm to about 40 ppm, about 10 ppm to about 30 ppm,
about 10 ppm to about 20 ppm, about 20 ppm to about 200 ppm, about
20 ppm to about 190 ppm, about 20 ppm to about 180 ppm, about 20
ppm to about 170 ppm, about 20 ppm to about 160 ppm, about 20 ppm
to about 150 ppm, about 20 ppm to about 140 ppm, about 20 ppm to
about 130 ppm, about 20 ppm to about 120 ppm, about 20 ppm to about
110 ppm, about 20 ppm to about 100 ppm, about 20 ppm to about 90
ppm, about 20 ppm to about 80 ppm, about 20 ppm to about 70 ppm,
about 20 ppm to about 60 ppm, about 20 ppm to about 50 ppm, about
20 ppm to about 40 ppm, about 20 ppm to about 30 ppm, about 30 ppm
to about 200 ppm, about 30 ppm to about 190 ppm, about 30 ppm to
about 180 ppm, about 30 ppm to about 170 ppm, about 30 ppm to about
160 ppm, about 30 ppm to about 150 ppm, about 30 ppm to about 140
ppm, about 30 ppm to about 130 ppm, about 30 ppm to about 120 ppm,
about 30 ppm to about 110 ppm, about 30 ppm to about 100 ppm, about
30 ppm to about 90 ppm, about 30 ppm to about 80 ppm, about 30 ppm
to about 70 ppm, about 30 ppm to about 60 ppm, about 30 ppm to
about 50 ppm, about 30 ppm to about 40 ppm, about 40 ppm to about
200 ppm, about 40 ppm to about 190 ppm, about 40 ppm to about 180
ppm, about 40 ppm to about 170 ppm, about 40 ppm to about 160 ppm,
about 40 ppm to about 150 ppm, about 40 ppm to about 140 ppm, about
40 ppm to about 130 ppm, about 40 ppm to about 120 ppm, about 40
ppm to about 110 ppm, about 40 ppm to about 100 ppm, about 40 ppm
to about 90 ppm, about 40 ppm to about 80 ppm, about 40 ppm to
about 70 ppm, about 40 ppm to about 60 ppm, about 40 ppm to about
50 ppm, about 50 ppm to about 200 ppm, about 50 ppm to about 190
ppm, about 50 ppm to about 180 ppm, about 50 ppm to about 170 ppm,
about 50 ppm to about 160 ppm, about 50 ppm to about 150 ppm, about
50 ppm to about 140 ppm, about 50 ppm to about 130 ppm, about 50
ppm to about 120 ppm, about 50 ppm to about 110 ppm, about 50 ppm
to about 100 ppm, about 50 ppm to about 90 ppm, about 50 ppm to
about 80 ppm, about 50 ppm to about 70 ppm, about 50 ppm to about
60 ppm, about 60 ppm to about 200 ppm, about 60 ppm to about 190
ppm, about 60 ppm to about 180 ppm, about 60 ppm to about 170 ppm,
about 60 ppm to about 160 ppm, about 60 ppm to about 150 ppm, about
60 ppm to about 140 ppm, about 60 ppm to about 130 ppm, about 60
ppm to about 120 ppm, about 60 ppm to about 110 ppm, about 60 ppm
to about 100 ppm, about 60 ppm to about 90 ppm, about 60 ppm to
about 80 ppm, about 60 ppm to about 70 ppm, about 70 ppm to about
200 ppm, about 70 ppm to about 190 ppm, about 70 ppm to about 180
ppm, about 70 ppm to about 170 ppm, about 70 ppm to about 160 ppm,
about 70 ppm to about 150 ppm, about 70 ppm to about 140 ppm, about
70 ppm to about 130 ppm, about 70 ppm to about 120 ppm, about 70
ppm to about 110 ppm, about 70 ppm to about 100 ppm, about 70 ppm
to about 90 ppm, about 70 ppm to about 80 ppm, about 80 ppm to
about 200 ppm, about 80 ppm to about 190 ppm, about 80 ppm to about
180 ppm, about 80 ppm to about 170 ppm, about 80 ppm to about 160
ppm, about 80 ppm to about 150 ppm, about 80 ppm to about 140 ppm,
about 80 ppm to about 130 ppm, about 80 ppm to about 120 ppm, about
80 ppm to about 110 ppm, about 80 ppm to about 100 ppm, about 80
ppm to about 90 ppm, about 90 ppm to about 200 ppm, about 90 ppm to
about 190 ppm, about 90 ppm to about 180 ppm, about 90 ppm to about
170 ppm, about 90 ppm to about 160 ppm, about 90 ppm to about 150
ppm, about 90 ppm to about 140 ppm, about 90 ppm to about 130 ppm,
about 90 ppm to about 120 ppm, about 90 ppm to about 110 ppm, about
90 ppm to about 100 ppm, about 100 ppm to about 200 ppm, about 100
ppm to about 190 ppm, about 100 ppm to about 180 ppm, about 100 ppm
to about 170 ppm, about 100 ppm to about 160 ppm, about 100 ppm to
about 150 ppm, about 100 ppm to about 140 ppm, about 100 ppm to
about 130 ppm, about 100 ppm to about 120 ppm, about 100 ppm to
about 110 ppm, about 110 ppm to about 200 ppm, about 110 ppm to
about 190 ppm, about 110 ppm to about 180 ppm, about 110 ppm to
about 170 ppm, about 110 ppm to about 160 ppm, about 110 ppm to
about 150 ppm, about 110 ppm to about 140 ppm, about 110 ppm to
about 130 ppm, about 110 ppm to about 120 ppm, about 120 ppm to
about 200 ppm, about 120 ppm to about 190 ppm, about 120 ppm to
about 180 ppm, about 120 ppm to about 170 ppm, about 120 ppm to
about 160 ppm, about 120 ppm to about 150 ppm, about 120 ppm to
about 140 ppm, about 120 ppm to about 130 ppm, about 130 ppm to
about 200 ppm, about 130 ppm to about 190 ppm, about 130 ppm to
about 180 ppm, about 130 ppm to about 170 ppm, about 130 ppm to
about 160 ppm, about 130 ppm to about 150 ppm, about 130 ppm to
about 140 ppm, about 140 ppm to about 200 ppm, about 140 ppm to
about 190 ppm, about 140 ppm to about 180 ppm, about 140 ppm to
about 170 ppm, about 140 ppm to about 160 ppm, about 140 ppm to
about 150 ppm, about 150 ppm to about 200 ppm, about 150 ppm to
about 190 ppm, about 150 ppm to about 180 ppm, about 150 ppm to
about 170 ppm, about 150 ppm to about 160 ppm, about 160 ppm to
about 200 ppm, about 160 ppm to about 190 ppm, about 160 ppm to
about 180 ppm, about 160 ppm to about 170 ppm, about 170 ppm to
about 200 ppm, about 170 ppm to about 190 ppm, about 170 ppm to
about 180 ppm, about 180 ppm to about 200 ppm, about 180 ppm to
about 190 ppm, or about 190 ppm to about 200 ppm (where the
sensitizer is present in a liquid culture medium disposed in the
test culture vessel, e.g., for a period of time during the
culturing).
[0153] In some embodiments, the test culture vessel can have a
concentration of a sensitizer (e.g., an antifoam, e.g., any of the
antifoams described herein) of 0 ppm to about 5,000 ppm, 0 ppm to
about 4,500 ppm, 0 ppm to about 4,000 ppm, 0 ppm to about 3,500
ppm, 0 ppm to about 3,000 ppm, 0 ppm to about 2,500 ppm, 0 ppm to
about 2,000 ppm, 0 ppm to about 1,500 ppm, 0 ppm to about 1,000
ppm, 0 ppm to about 800 ppm, 0 ppm to about 600 ppm, 0 ppm to about
500 ppm, 0 ppm to about 450 ppm, 0 ppm to about 400 ppm, 0 ppm to
about 350 ppm, 0 ppm to about 300 ppm, 0 ppm to about 250 ppm, 0
ppm to about 200 ppm, 0 ppm to about 150 ppm, 0 ppm to about 140
ppm, 0 ppm to about 120 ppm, 0 ppm to about 100 ppm, 0 ppm to about
80 ppm, 0 ppm to about 60 ppm, 0 ppm to about 50 ppm, 0 ppm to
about 40 ppm, 0 ppm to about 30 ppm, 0 ppm to about 20 ppm, 0 ppm
to about 10 ppm, about 10 ppm to about 5,000 ppm, about 10 ppm to
about 4,500 ppm, about 10 ppm to about 4,000 ppm, about 10 ppm to
about 3,500 ppm, about 10 ppm to about 3,000 ppm, about 10 ppm to
about 2,500 ppm, about 10 ppm to about 2,000 ppm, about 10 ppm to
about 1,500 ppm, about 10 ppm to about 1,000 ppm, about 10 ppm to
about 800 ppm, about 10 ppm to about 600 ppm, about 10 ppm to about
500 ppm, about 10 ppm to about 450 ppm, about 10 ppm to about 400
ppm, about 10 ppm to about 350 ppm, about 10 ppm to about 300 ppm,
about 10 ppm to about 250 ppm, about 10 ppm to about 200 ppm, about
10 ppm to about 150 ppm, about 10 ppm to about 140 ppm, about 10
ppm to about 120 ppm, about 10 ppm to about 100 ppm, about 10 ppm
to about 80 ppm, about 10 ppm to about 60 ppm, about 10 ppm to
about 50 ppm, about 10 ppm to about 40 ppm, about 10 ppm to about
30 ppm, about 10 ppm to about 20 ppm, about 20 ppm to about 5,000
ppm, about 20 ppm to about 4,500 ppm, about 20 ppm to about 4,000
ppm, about 20 ppm to about 3,500 ppm, about 20 ppm to about 3,000
ppm, about 20 ppm to about 2,500 ppm, about 20 ppm to about 2,000
ppm, about 20 ppm to about 1,500 ppm, about 20 ppm to about 1,000
ppm, about 20 ppm to about 800 ppm, about 20 ppm to about 600 ppm,
about 20 ppm to about 500 ppm, about 20 ppm to about 450 ppm, about
20 ppm to about 400 ppm, about 20 ppm to about 350 ppm, about 20
ppm to about 300 ppm, about 20 ppm to about 250 ppm, about 20 ppm
to about 200 ppm, about 20 ppm to about 150 ppm, about 20 ppm to
about 140 ppm, about 20 ppm to about 120 ppm, about 20 ppm to about
100 ppm, about 20 ppm to about 80 ppm, about 20 ppm to about 60
ppm, about 20 ppm to about 50 ppm, about 20 ppm to about 40 ppm,
about 20 ppm to about 30 ppm, about 30 ppm to about 5,000 ppm,
about 30 ppm to about 4,500 ppm, about 30 ppm to about 4,000 ppm,
about 30 ppm to about 3,500 ppm, about 30 ppm to about 3,000 ppm,
about 30 ppm to about 2,500 ppm, about 30 ppm to about 2,000 ppm,
about 30 ppm to about 1,500 ppm, about 30 ppm to about 1,000 ppm,
about 30 ppm to about 800 ppm, about 30 ppm to about 600 ppm, about
30 ppm to about 500 ppm, about 30 ppm to about 450 ppm, about 30
ppm to about 400 ppm, about 30 ppm to about 350 ppm, about 30 ppm
to about 300 ppm, about 30 ppm to about 250 ppm, about 30 ppm to
about 200 ppm, about 30 ppm to about 150 ppm, about 30 ppm to about
140 ppm, about 30 ppm to about 120 ppm, about 30 ppm to about 100
ppm, about 30 ppm to about 80 ppm, about 30 ppm to about 60 ppm,
about 30 ppm to about 50 ppm, about 30 ppm to about 40 ppm, about
40 ppm to about 5,000 ppm, about 40 ppm to about 4,500 ppm, about
40 ppm to about 4,000 ppm, about 40 ppm to about 3,500 ppm, about
40 ppm to about 3,000 ppm, about 40 ppm to about 2,500 ppm, about
40 ppm to about 2,000 ppm, about 40 ppm to about 1,500 ppm, about
40 ppm to about 1,000 ppm, about 40 ppm to about 800 ppm, about 40
ppm to about 600 ppm, about 40 ppm to about 500 ppm, about 40 ppm
to about 450 ppm, about 40 ppm to about 400 ppm, about 40 ppm to
about 350 ppm, about 40 ppm to about 300 ppm, about 40 ppm to about
250 ppm, about 40 ppm to about 200 ppm, about 40 ppm to about 150
ppm, about 40 ppm to about 140 ppm, about 40 ppm to about 120 ppm,
about 40 ppm to about 100 ppm, about 40 ppm to about 80 ppm, about
40 ppm to about 60 ppm, about 40 ppm to about 50 ppm, about 50 ppm
to about 5,000 ppm, about 50 ppm to about 4,500 ppm, about 50 ppm
to about 4,000 ppm, about 50 ppm to about 3,500 ppm, about 50 ppm
to about 3,000 ppm, about 50 ppm to about 2,500 ppm, about 50 ppm
to about 2,000 ppm, about 50 ppm to about 1,500 ppm, about 50 ppm
to about 1,000 ppm, about 50 ppm to about 800 ppm, about 50 ppm to
about 600 ppm, about 50 ppm to about 500 ppm, about 50 ppm to about
450 ppm, about 50 ppm to about 400 ppm, about 50 ppm to about 350
ppm, about 50 ppm to about 300 ppm, about 50 ppm to about 250 ppm,
about 50 ppm to about 200 ppm, about 50 ppm to about 150 ppm, about
50 ppm to about 140 ppm, about 50 ppm to about 120 ppm, about 50
ppm to about 100 ppm, about 50 ppm to about 80 ppm, about 50 ppm to
about 60 ppm, about 60 ppm to about 5,000 ppm, about 60 ppm to
about 4,500 ppm, about 60 ppm to about 4,000 ppm, about 60 ppm to
about 3,500 ppm, about 60 ppm to about 3,000 ppm, about 60 ppm to
about 2,500 ppm, about 60 ppm to about 2,000 ppm, about 60 ppm to
about 1,500 ppm, about 60 ppm to about 1,000 ppm, about 60 ppm to
about 800 ppm, about 60 ppm to about 600 ppm, about 60 ppm to about
500 ppm, about 60 ppm to about 450 ppm, about 60 ppm to about 400
ppm, about 60 ppm to about 350 ppm, about 60 ppm to about 300 ppm,
about 60 ppm to about 250 ppm, about 60 ppm to about 200 ppm, about
60 ppm to about 150 ppm, about 60 ppm to about 140 ppm, about 60
ppm to about 120 ppm, about 60 ppm to about 100 ppm, about 60 ppm
to about 80 ppm, about 80 ppm to about 5,000 ppm, about 80 ppm to
about 4,500 ppm, about 80 ppm to about 4,000 ppm, about 80 ppm to
about 3,500 ppm, about 80 ppm to about 3,000 ppm, about 80 ppm to
about 2,500 ppm, about 80 ppm to about 2,000 ppm, about 80 ppm to
about 1,500 ppm, about 80 ppm to about 1,000 ppm, about 80 ppm to
about 800 ppm, about 80 ppm to about 600 ppm, about 80 ppm to about
500 ppm, about 80 ppm to about 450 ppm, about 80 ppm to about 400
ppm, about 80 ppm to about 350 ppm, about 80 ppm to about 300 ppm,
about 80 ppm to about 250 ppm, about 80 ppm to about 200 ppm, about
80 ppm to about 150 ppm, about 80 ppm to about 140 ppm, about 80
ppm to about 120 ppm, about 80 ppm to about 100 ppm, about 100 ppm
to about 5,000 ppm, about 100 ppm to about 4,500 ppm, about 100 ppm
to about 4,000 ppm, about 100 ppm to about 3,500 ppm, about 100 ppm
to about 3,000 ppm, about 100 ppm to about 2,500 ppm, about 100 ppm
to about 2,000 ppm, about 100 ppm to about 1,500 ppm, about 100 ppm
to about 1,000 ppm, about 100 ppm to about 800 ppm, about 100 ppm
to about 600 ppm, about 100 ppm to about 500 ppm, about 100 ppm to
about 450 ppm, about 100 ppm to about 400 ppm, about 100 ppm to
about 350 ppm, about 100 ppm to about 300 ppm, about 100 ppm to
about 250 ppm, about 100 ppm to about 200 ppm, about 100 ppm to
about 150 ppm, about 100 ppm to about 140 ppm, about 100 ppm to
about 120 ppm, about 120 ppm to about 5,000 ppm, about 120 ppm to
about 4,500 ppm, about 120 ppm to about 4,000 ppm, about 120 ppm to
about 3,500 ppm, about 120 ppm to about 3,000 ppm, about 120 ppm to
about 2,500 ppm, about 120 ppm to about 2,000 ppm, about 120 ppm to
about 1,500 ppm, about 120 ppm to about 1,000 ppm, about 120 ppm to
about 800 ppm, about 120 ppm to about 600 ppm, about 120 ppm to
about 500 ppm, about 120 ppm to about 450 ppm, about 120 ppm to
about 400 ppm, about 120 ppm to about 350 ppm, about 120 ppm to
about 300 ppm, about 120 ppm to about 250 ppm, about 120 ppm to
about 200 ppm, about 120 ppm to about 150 ppm, about 120 ppm to
about 140 ppm, about 140 ppm to about 5,000 ppm, about 140 ppm to
about 4,500 ppm, about 140 ppm to about 4,000 ppm, about 140 ppm to
about 3,500 ppm, about 140 ppm to about 3,000 ppm, about 140 ppm to
about 2,500 ppm, about 140 ppm to about 2,000 ppm, about 140 ppm to
about 1,500 ppm, about 140 ppm to about 1,000 ppm, about 140 ppm to
about 800 ppm, about 140 ppm to about 600 ppm, about 140 ppm to
about 500 ppm, about 140 ppm to about 450 ppm, about 140 ppm to
about 400 ppm, about 140 ppm to about 350 ppm, about 140 ppm to
about 300 ppm, about 140 ppm to about 250 ppm, about 140 ppm to
about 200 ppm, about 140 ppm to about 150 ppm, about 150 ppm to
about 5,000 ppm, about 150 ppm to about 4,500 ppm, about 150 ppm to
about 4,000 ppm, about 150 ppm to about 3,500 ppm, about 150 ppm to
about 3,000 ppm, about 150 ppm to about 2,500 ppm, about 150 ppm to
about 2,000 ppm, about 150 ppm to about 1,500 ppm, about 150 ppm to
about 1,000 ppm, about 150 ppm to about 800 ppm, about 150 ppm to
about 600 ppm, about 150 ppm to about 500 ppm, about 150 ppm to
about 450 ppm, about 150 ppm to about 400 ppm, about 150 ppm to
about 350 ppm, about 150 ppm to about 300 ppm, about 150 ppm to
about 250 ppm, about 150 ppm to about 200 ppm, about 200 ppm to
about 5,000 ppm, about 200 ppm to about 4,500 ppm, about 200 ppm to
about 4,000 ppm, about 200 ppm to about 3,500 ppm, about 200 ppm to
about 3,000 ppm, about 200 ppm to about 2,500 ppm, about 200 ppm to
about 2,000 ppm, about 200 ppm to about 1,500 ppm, about 200 ppm to
about 1,000 ppm, about 200 ppm to about 800 ppm, about 200 ppm to
about 600 ppm, about 200 ppm to about 500 ppm, about 200 ppm to
about 450 ppm, about 200 ppm to about 400 ppm, about 200 ppm to
about 350 ppm, about 200 ppm to about 300 ppm, about 200 ppm to
about 250 ppm, about 250 ppm to about 5,000 ppm, about 250 ppm to
about 4,500 ppm, about 250 ppm to about 4,000 ppm, about 250 ppm to
about 3,500 ppm, about 250 ppm to about 3,000 ppm, about 250 ppm to
about 2,500 ppm, about 250 ppm to about 2,000 ppm, about 250 ppm to
about 1,500 ppm, about 250 ppm to about 1,000 ppm, about 250 ppm to
about 800 ppm, about 250 ppm to about 600 ppm, about 250 ppm to
about 500 ppm, about 250 ppm to about 450 ppm, about 250 ppm to
about 400 ppm, about 250 ppm to about 350 ppm, about 250 ppm to
about 300 ppm, about 300 ppm to about 5,000 ppm, about 300 ppm to
about 4,500 ppm, about 300 ppm to about 4,000 ppm, about 300 ppm to
about 3,500 ppm, about 300 ppm to about 3,000 ppm, about 300 ppm to
about 2,500 ppm, about 300 ppm to about 2,000 ppm, about 300 ppm to
about 1,500 ppm, about 300 ppm to about 1,000 ppm, about 300 ppm to
about 800 ppm, about 300 ppm to about 600 ppm, about 300 ppm to
about 500 ppm, about 300 ppm to about 450 ppm, about 300 ppm to
about 400 ppm, about 300 ppm to about 350 ppm, about 350 ppm to
about 5,000 ppm, about 350 ppm to about 4,500 ppm, about 350 ppm to
about 4,000 ppm, about 350 ppm to about 3,500 ppm, about 350 ppm to
about 3,000 ppm, about 350 ppm to about 2,500 ppm, about 350 ppm to
about 2,000 ppm, about 350 ppm to about 1,500 ppm, about 350 ppm to
about 1,000 ppm, about 350 ppm to about 800 ppm, about 350 ppm to
about 600 ppm, about 350 ppm to about 500 ppm, about 350 ppm to
about 450 ppm, about 350 ppm to about 400 ppm, about 400 ppm to
about 5,000 ppm, about 400 ppm to about 4,500 ppm, about 400 ppm to
about 4,000 ppm, about 400 ppm to about 3,500 ppm, about 400 ppm to
about 3,000 ppm, about 400 ppm to about 2,500 ppm, about 400 ppm to
about 2,000 ppm, about 400 ppm to about 1,500 ppm, about 400 ppm to
about 1,000 ppm, about 400 ppm to about 800 ppm, about 400 ppm to
about 600 ppm, about 400 ppm to about 500 ppm, about 400 ppm to
about 450 ppm, about 450 ppm to about 5,000 ppm, about 450 ppm to
about 4,500 ppm, about 450 ppm to about 4,000 ppm, about 450 ppm to
about 3,500 ppm, about 450 ppm to about 3,000 ppm, about 450 ppm to
about 2,500 ppm, about 450 ppm to about 2,000 ppm, about 450 ppm to
about 1,500 ppm, about 450 ppm to about 1,000 ppm, about 450 ppm to
about 800 ppm, about 450 ppm to about 600 ppm, about 450 ppm to
about 500 ppm, about 500 ppm to about 5,000 ppm, about 500 ppm to
about 4,500 ppm, about 500 ppm to about 4,000 ppm, about 500 ppm to
about 3,500 ppm, about 500 ppm to about 3,000 ppm, about 500 ppm to
about 2,500 ppm, about 500 ppm to about 2,000 ppm, about 500 ppm to
about 1,500 ppm, about 500 ppm to about 1,000 ppm, about 500 ppm to
about 800 ppm, about 500 ppm to about 600 ppm, about 600 ppm to
about 5,000 ppm, about 600 ppm to about 4,500 ppm, about 600 ppm to
about 4,000 ppm, about 600 ppm to about 3,500 ppm, about 600 ppm to
about 3,000 ppm, about 600 ppm to about 2,500 ppm, about 600 ppm to
about 2,000 ppm, about 600 ppm to about 1,500 ppm, about 600 ppm to
about 1,000 ppm, about 600 ppm to about 800 ppm, about 800 ppm to
about 5,000 ppm, about 800 ppm to about 4,500 ppm, about 800 ppm to
about 4,000 ppm, about 800 ppm to about 3,500 ppm, about 800 ppm to
about 3,000 ppm, about 800 ppm to about 2,500 ppm, about 800 ppm to
about 2,000 ppm, about 800 ppm to about 1,500 ppm, about 800 ppm to
about 1,000 ppm, about 1,000 ppm to about 5,000 ppm, about 1,000
ppm to about 4,500 ppm, about 1,000 ppm to about 4,000 ppm, about
1,000 ppm to about 3,500 ppm, about 1,000 ppm to about 3,000 ppm,
about 1,000 ppm to about 2,500 ppm, about 1,000 ppm to about 2,000
ppm, about 1,000 ppm to about 1,500 ppm, about 1,500 ppm to about
5,000 ppm, about 1,500 ppm to about 4,500 ppm, about 1,500 ppm to
about 4,000 ppm, about 1,500 ppm to about 3,500 ppm, about 1,500
ppm to about 3,000 ppm, about 1,500 ppm to about 2,500 ppm, about
1,500 ppm to about 2,000 ppm, about 2,000 ppm to about 5,000 ppm,
about 2,000 ppm to about 4,500 ppm, about 2,000 ppm to about 4,000
ppm, about 2,000 ppm to about 3,500 ppm, about 2,000 ppm to about
3,000 ppm, about 2,000 ppm to about 2,500 ppm, about 2,500 ppm to
about 5,000 ppm, about 2,500 ppm to about 4,500 ppm, about 2,500
ppm to about 4,000 ppm, about 2,500 ppm to about 3,500 ppm, about
2,500 ppm to about 3,000 ppm, about 3,000 ppm to about 5,000 ppm,
about 3,000 ppm to about 4,500 ppm, about 3,000 ppm to about 4,000
ppm, about 3,000 ppm to about 3,500 ppm, about 3,500 ppm to about
5,000 ppm, about 3,500 ppm to about 4,500 ppm, about 3,500 ppm to
about 4,000 ppm, about 4,000 ppm to about 5,000 ppm, about 4,000
ppm to about 4,500 ppm, or about 4,500 ppm to about 5,000 ppm
(where the sensitizer is present in a liquid culture medium
disposed in the test culture vessel, e.g., for a period of time
during the culturing).
[0154] In some embodiments, the test culture vessel can have a
concentration of a protectant that is about 1 g/L to about 10 g/L
(e.g., from about 2 g/L to about 8 g/L, from about 3 g/L to about 7
g/L, from about 4 g/L to about 6 g/L, or about 5 g/L). In some
embodiments, the test culture vessel can have a concentration of a
protectant (e.g., any of the protectants described herein or known
in the art) of about 1.0 g/L to about 10.0 g/L, about 1.0 g/L to
about 9.5 g/L, about 1.0 g/L to about 9.0 g/L, about 1.0 g/L to
about 8.5 g/L, about 1.0 g/L to about 8.0 g/L, about 1.0 g/L to
about 7.5 g/L, about 1.0 g/L to about 7.0 g/L, about 1.0 g/L to
about 6.5 g/L, about 1.0 g/L to about 6.0 g/L, about 1.0 g/L to
about 5.5 g/L, about 1.0 g/L to about 5.0 g/L, about 1.0 g/L to
about 4.5 g/L, about 1.0 g/L to about 4.0 g/L, about 1.0 g/L to
about 3.5 g/L, about 1.0 g/L to about 3.0 g/L, about 1.0 g/L to
about 2.5 g/L, about 1.0 g/L to about 2.0 g/L, about 1.0 g/L to
about 1.5 g/L, about 1.5 g/L to about 10.0 g/L, about 1.5 g/L to
about 9.5 g/L, about 1.5 g/L to about 9.0 g/L, about 1.5 g/L to
about 8.5 g/L, about 1.5 g/L to about 8.0 g/L, about 1.5 g/L to
about 7.5 g/L, about 1.5 g/L to about 7.0 g/L, about 1.5 g/L to
about 6.5 g/L, about 1.5 g/L to about 6.0 g/L, about 1.5 g/L to
about 5.5 g/L, about 1.5 g/L to about 5.0 g/L, about 1.5 g/L to
about 4.5 g/L, about 1.5 g/L to about 4.0 g/L, about 1.5 g/L to
about 3.5 g/L, about 1.5 g/L to about 3.0 g/L, about 1.5 g/L to
about 2.5 g/L, about 1.5 g/L to about 2.0 g/L, about 2.0 g/L to
about 10.0 g/L, about 2.0 g/L to about 9.5 g/L, about 2.0 g/L to
about 9.0 g/L, about 2.0 g/L to about 8.5 g/L, about 2.0 g/L to
about 8.0 g/L, about 2.0 g/L to about 7.5 g/L, about 2.0 g/L to
about 7.0 g/L, about 2.0 g/L to about 6.5 g/L, about 2.0 g/L to
about 6.0 g/L, about 2.0 g/L to about 5.5 g/L, about 2.0 g/L to
about 5.0 g/L, about 2.0 g/L to about 4.5 g/L, about 2.0 g/L to
about 4.0 g/L, about 2.0 g/L to about 3.5 g/L, about 2.0 g/L to
about 3.0 g/L, about 2.0 g/L to about 2.5 g/L, about 2.5 g/L to
about 10.0 g/L, about 2.5 g/L to about 9.5 g/L, about 2.5 g/L to
about 9.0 g/L, about 2.5 g/L to about 8.5 g/L, about 2.5 g/L to
about 8.0 g/L, about 2.5 g/L to about 7.5 g/L, about 2.5 g/L to
about 7.0 g/L, about 2.5 g/L to about 6.5 g/L, about 2.5 g/L to
about 6.0 g/L, about 2.5 g/L to about 5.5 g/L, about 2.5 g/L to
about 5.0 g/L, about 2.5 g/L to about 4.5 g/L, about 2.5 g/L to
about 4.0 g/L, about 2.5 g/L to about 3.5 g/L, about 2.5 g/L to
about 3.0 g/L, about 3.0 g/L to about 10.0 g/L, about 3.0 g/L to
about 9.5 g/L, about 3.0 g/L to about 9.0 g/L, about 3.0 g/L to
about 8.5 g/L, about 3.0 g/L to about 8.0 g/L, about 3.0 g/L to
about 7.5 g/L, about 3.0 g/L to about 7.0 g/L, about 3.0 g/L to
about 6.5 g/L, about 3.0 g/L to about 6.0 g/L, about 3.0 g/L to
about 5.5 g/L, about 3.0 g/L to about 5.0 g/L, about 3.0 g/L to
about 4.5 g/L, about 3.0 g/L to about 4.0 g/L, about 3.0 g/L to
about 3.5 g/L, about 3.5 g/L to about 10.0 g/L, about 3.5 g/L to
about 9.5 g/L, about 3.5 g/L to about 9.0 g/L, about 3.5 g/L to
about 8.5 g/L, about 3.5 g/L to about 8.0 g/L, about 3.5 g/L to
about 7.5 g/L, about 3.5 g/L to about 7.0 g/L, about 3.5 g/L to
about 6.5 g/L, about 3.5 g/L to about 6.0 g/L, about 3.5 g/L to
about 5.5 g/L, about 3.5 g/L to about 5.0 g/L, about 3.5 g/L to
about 4.5 g/L, about 3.5 g/L to about 4.0 g/L, about 4.0 g/L to
about 10.0 g/L, about 4.0 g/L to about 9.5 g/L, about 4.0 g/L to
about 9.0 g/L, about 4.0 g/L to about 8.5 g/L, about 4.0 g/L to
about 8.0 g/L, about 4.0 g/L to about 7.5 g/L, about 4.0 g/L to
about 7.0 g/L, about 4.0 g/L to about 6.5 g/L, about 4.0 g/L to
about 6.0 g/L, about 4.0 g/L to about 5.5 g/L, about 4.0 g/L to
about 5.0 g/L, about 4.0 g/L to about 4.5 g/L, about 4.5 g/L to
about 10.0 g/L, about 4.5 g/L to about 9.5 g/L, about 4.5 g/L to
about 9.0 g/L, about 4.5 g/L to about 8.5 g/L, about 4.5 g/L to
about 8.0 g/L, about 4.5 g/L to about 7.5 g/L, about 4.5 g/L to
about 7.0 g/L, about 4.5 g/L to about 6.5 g/L, about 4.5 g/L to
about 6.0 g/L, about 4.5 g/L to about 5.5 g/L, about 4.5 g/L to
about 5.0 g/L, about 5.0 g/L to about 10.0 g/L, about 5.0 g/L to
about 9.5 g/L, about 5.0 g/L to about 9.0 g/L, about 5.0 g/L to
about 8.5 g/L, about 5.0 g/L to about 8.0 g/L, about 5.0 g/L to
about 7.5 g/L, about 5.0 g/L to about 7.0 g/L, about 5.0 g/L to
about 6.5 g/L, about 5.0 g/L to about 6.0 g/L, about 5.0 g/L to
about 5.5 g/L, about 5.5 g/L to about 10.0 g/L, about 5.5 g/L to
about 9.5 g/L, about 5.5 g/L to about 9.0 g/L, about 5.5 g/L to
about 8.5 g/L, about 5.5 g/L to about 8.0 g/L, about 5.5 g/L to
about 7.5 g/L, about 5.5 g/L to about 7.0 g/L, about 5.5 g/L to
about 6.5 g/L, about 5.5 g/L to about 6.0 g/L, about 6.0 g/L to
about 10.0 g/L, about 6.0 g/L to about 9.5 g/L, about 6.0 g/L to
about 9.0 g/L, about 6.0 g/L to about 8.5 g/L, about 6.0 g/L to
about 8.0 g/L, about 6.0 g/L to about 7.5 g/L, about 6.0 g/L to
about 7.0 g/L, about 6.0 g/L to about 6.5 g/L, about 6.5 g/L to
about 10.0 g/L, about 6.5 g/L to about 9.5 g/L, about 6.5 g/L to
about 9.0 g/L, about 6.5 g/L to about 8.5 g/L, about 6.5 g/L to
about 8.0 g/L, about 6.5 g/L to about 7.5 g/L, about 6.5 g/L to
about 7.0 g/L, about 7.0 g/L to about 10.0 g/L, about 7.0 g/L to
about 9.5 g/L, about 7.0 g/L to about 9.0 g/L, about 7.0 g/L to
about 8.5 g/L, about 7.0 g/L to about 8.0 g/L, about 7.0 g/L to
about 7.5 g/L, about 7.5 g/L to about 10.0 g/L, about 7.5 g/L to
about 9.5 g/L, about 7.5 g/L to about 9.0 g/L, about 7.5 g/L to
about 8.5 g/L, about 7.5 g/L to about 8.0 g/L, about 8.0 g/L to
about 10.0 g/L, about 8.0 g/L to about 9.5 g/L, about 8.0 g/L to
about 9.0 g/L, about 8.0 g/L to about 8.5 g/L, about 8.5 g/L to
about 10.0 g/L, about 8.5 g/L to about 9.5 g/L, about 8.5 g/L to
about 9.0 g/L, about 9.0 g/L to about 10.0 g/L, about 9.0 g/L to
about 9.5 g/L, or about 9.5 g/L to about 10.0 g/L (where the
protectant is present in a liquid culture medium disposed in the
test culture vessel, e.g., for a period of time during the
culturing).
The production bioreactor can be a perfusion bioreactor, a fed
batch bioreactor, or a concentrated fed-batch bioreactor. In some
embodiments, the production bioreactor can have a volume of about 5
L to about 20,000 L (e.g., about 50 L to about 10,000 L). In some
embodiments, the production bioreactor can have a volume of more
than 5 L, more than 10 L, more than 50 L, more than 100 L, more
than 1000 L, more than 2000 L, more than 5000 L, more than 20000 L,
more than 100000 L, or more than 150000 L (e.g., more than 1000 L).
In some embodiments, the production bioreactor can have a volume of
about 5 L to about 20,000 L, about 5 L to about 18,000 L, about 5 L
to about 16,000 L, about 5 L to about 14,000 L, about 5 L to about
12,000 L, about 5 L to about 10,000 L, about 5 L to about 9,500 L,
about 5 L to about 9,000 L, about 5 L to about 8,500 L, about 5 L
to about 8,000 L, about 5 L to about 7,500 L, about 5 L to about
7,000 L, about 5 L to about 6,500 L, about 5 L to about 6,000 L,
about 5 L to about 5,500 L, about 5 L to about 5,000 L, about 5 L
to about 4,500 L, about 5 L to about 4,000 L, about 5 L to about
3,500 L, about 5 L to about 3,000 L, about 5 L to about 2,500 L,
about 5 L to about 2,000 L, about 5 L to about 1,500 L, about 5 L
to about 1,000 L, about 5 L to about 900 L, about 5 L to about 800
L, about 5 L to about 700 L, about 5 L to about 600 L, about 5 L to
about 500 L, about 5 L to about 400 L, about 5 L to about 300 L,
about 5 L to about 250 L, about 5 L to about 200 L, about 5 L to
about 150 L, about 5 L to about 100 L, about 5 L to about 80 L,
about 5 L to about 60 L, about 5 L to about 50 L, about 5 L to
about 25 L, about 5 L to about 10 L, about 10 L to about 20,000 L,
about 10 L to about 18,000 L, about 10 L to about 16,000 L, about
10 L to about 14,000 L, about 10 L to about 12,000 L, about 10 L to
about 10,000 L, about 10 L to about 9,500 L, about 10 L to about
9,000 L, about 10 L to about 8,500 L, about 10 L to about 8,000 L,
about 10 L to about 7,500 L, about 10 L to about 7,000 L, about 10
L to about 6,500 L, about 10 L to about 6,000 L, about 10 L to
about 5,500 L, about 10 L to about 5,000 L, about 10 L to about
4,500 L, about 10 L to about 4,000 L, about 10 L to about 3,500 L,
about 10 L to about 3,000 L, about 10 L to about 2,500 L, about 10
L to about 2,000 L, about 10 L to about 1,500 L, about 10 L to
about 1,000 L, about 10 L to about 900 L, about 10 L to about 800
L, about 10 L to about 700 L, about 10 L to about 600 L, about 10 L
to about 500 L, about 10 L to about 400 L, about 10 L to about 300
L, about 10 L to about 250 L, about 10 L to about 200 L, about 10 L
to about 150 L, about 10 L to about 100 L, about 10 L to about 80
L, about 10 L to about 60 L, about 10 L to about 50 L, about 10 L
to about 25 L, about 25 L to about 20,000 L, about 25 L to about
18,000 L, about 25 L to about 16,000 L, about 25 L to about 14,000
L, about 25 L to about 12,000 L, about 25 L to about 10,000 L,
about 25 L to about 9,500 L, about 25 L to about 9,000 L, about 25
L to about 8,500 L, about 25 L to about 8,000 L, about 25 L to
about 7,500 L, about 25 L to about 7,000 L, about 25 L to about
6,500 L, about 25 L to about 6,000 L, about 25 L to about 5,500 L,
about 25 L to about 5,000 L, about 25 L to about 4,500 L, about 25
L to about 4,000 L, about 25 L to about 3,500 L, about 25 L to
about 3,000 L, about 25 L to about 2,500 L, about 25 L to about
2,000 L, about 25 L to about 1,500 L, about 25 L to about 1,000 L,
about 25 L to about 900 L, about 25 L to about 800 L, about 25 L to
about 700 L, about 25 L to about 600 L, about 25 L to about 500 L,
about 25 L to about 400 L, about 25 L to about 300 L, about 25 L to
about 250 L, about 25 L to about 200 L, about 25 L to about 150 L,
about 25 L to about 100 L, about 25 L to about 80 L, about 25 L to
about 60 L, about 25 L to about 50 L, about 50 L to about 20,000 L,
about 50 L to about 18,000 L, about 50 L to about 16,000 L, about
50 L to about 14,000 L, about 50 L to about 12,000 L, about 50 L to
about 10,000 L, about 50 L to about 9,500 L, about 50 L to about
9,000 L, about 50 L to about 8,500 L, about 50 L to about 8,000 L,
about 50 L to about 7,500 L, about 50 L to about 7,000 L, about 50
L to about 6,500 L, about 50 L to about 6,000 L, about 50 L to
about 5,500 L, about 50 L to about 5,000 L, about 50 L to about
4,500 L, about 50 L to about 4,000 L, about 50 L to about 3,500 L,
about 50 L to about 3,000 L, about 50 L to about 2,500 L, about 50
L to about 2,000 L, about 50 L to about 1,500 L, about 50 L to
about 1,000 L, about 50 L to about 900 L, about 50 L to about 800
L, about 50 L to about 700 L, about 50 L to about 600 L, about 50 L
to about 500 L, about 50 L to about 400 L, about 50 L to about 300
L, about 50 L to about 250 L, about 50 L to about 200 L, about 50 L
to about 150 L, about 50 L to about 100 L, about 50 L to about 80
L, about 50 L to about 60 L, about 60 L to about 20,000 L, about 60
L to about 18,000 L, about 60 L to about 16,000 L, about 60 L to
about 14,000 L, about 60 L to about 12,000 L, about 60 L to about
10,000 L, about 60 L to about 9,500 L, about 60 L to about 9,000 L,
about 60 L to about 8,500 L, about 60 L to about 8,000 L, about 60
L to about 7,500 L, about 60 L to about 7,000 L, about 60 L to
about 6,500 L, about 60 L to about 6,000 L, about 60 L to about
5,500 L, about 60 L to about 5,000 L, about 60 L to about 4,500 L,
about 60 L to about 4,000 L, about 60 L to about 3,500 L, about 60
L to about 3,000 L, about 60 L to about 2,500 L, about 60 L to
about 2,000 L, about 60 L to about 1,500 L, about 60 L to about
1,000 L, about 60 L to about 900 L, about 60 L to about 800 L,
about 60 L to about 700 L, about 60 L to about 600 L, about 60 L to
about 500 L, about 60 L to about 400 L, about 60 L to about 300 L,
about 60 L to about 250 L, about 60 L to about 200 L, about 60 L to
about 150 L, about 60 L to about 100 L, about 60 L to about 80 L,
about 80 L to about 20,000 L, about 80 L to about 18,000 L, about
80 L to about 16,000 L, about 80 L to about 14,000 L, about 80 L to
about 12,000 L, about 80 L to about 10,000 L, about 80 L to about
9,500 L, about 80 L to about 9,000 L, about 80 L to about 8,500 L,
about 80 L to about 8,000 L, about 80 L to about 7,500 L, about 80
L to about 7,000 L, about 80 L to about 6,500 L, about 80 L to
about 6,000 L, about 80 L to about 5,500 L, about 80 L to about
5,000 L, about 80 L to about 4,500 L, about 80 L to about 4,000 L,
about 80 L to about 3,500 L, about 80 L to about 3,000 L, about 80
L to about 2,500 L, about 80 L to about 2,000 L, about 80 L to
about 1,500 L, about 80 L to about 1,000 L, about 80 L to about 900
L, about 80 L to about 800 L, about 80 L to about 700 L, about 80 L
to about 600 L, about 80 L to about 500 L, about 80 L to about 400
L, about 80 L to about 300 L, about 80 L to about 250 L, about 80 L
to about 200 L, about 80 L to about 150 L, about 80 L to about 100
L, about 100 L to about 20,000 L, about 100 L to about 18,000 L,
about 100 L to about 16,000 L, about 100 L to about 14,000 L, about
100 L to about 12,000 L, about 100 L to about 10,000 L, about 100 L
to about 9,500 L, about 100 L to about 9,000 L, about 100 L to
about 8,500 L, about 100 L to about 8,000 L, about 100 L to about
7,500 L, about 100 L to about 7,000 L, about 100 L to about 6,500
L, about 100 L to about 6,000 L, about 100 L to about 5,500 L,
about 100 L to about 5,000 L, about 100 L to about 4,500 L, about
100 L to about 4,000 L, about 100 L to about 3,500 L, about 100 L
to about 3,000 L, about 100 L to about 2,500 L, about 100 L to
about 2,000 L, about 100 L to about 1,500 L, about 100 L to about
1,000 L, about 100 L to about 900 L, about 100 L to about 800 L,
about 100 L to about 700 L, about 100 L to about 600 L, about 100 L
to about 500 L, about 100 L to about 400 L, about 100 L to about
300 L, about 100 L to about 250 L, about 100 L to about 200 L,
about 100 L to about 150 L, about 150 L to about 20,000 L, about
150 L to about 18,000 L, about 150 L to about 16,000 L, about 150 L
to about 14,000 L, about 150 L to about 12,000 L, about 150 L to
about 10,000 L, about 150 L to about 9,500 L, about 150 L to about
9,000 L, about 150 L to about 8,500 L, about 150 L to about 8,000
L, about 150 L to about 7,500 L, about 150 L to about 7,000 L,
about 150 L to about 6,500 L, about 150 L to about 6,000 L, about
150 L to about 5,500 L, about 150 L to about 5,000 L, about 150 L
to about 4,500 L, about 150 L to about 4,000 L, about 150 L to
about 3,500 L, about 150 L to about 3,000 L, about 150 L to about
2,500 L, about 150 L to about 2,000 L, about 150 L to about 1,500
L, about 150 L to about 1,000 L, about 150 L to about 900 L, about
150 L to about 800 L, about 150 L to about 700 L, about 150 L to
about 600 L, about 150 L to about 500 L, about 150 L to about 400
L, about 150 L to about 300 L, about 150 L to about 250 L, about
150 L to about 200 L, about 200 L to about 20,000 L, about 200 L to
about 18,000 L, about 200 L to about 16,000 L, about 200 L to about
14,000 L, about 200 L to about 12,000 L, about 200 L to about
10,000 L, about 200 L to about 9,500 L, about 200 L to about 9,000
L, about 200 L to about 8,500 L, about 200 L to about 8,000 L,
about 200 L to about 7,500 L, about 200 L to about 7,000 L, about
200 L to about 6,500 L, about 200 L to about 6,000 L, about 200 L
to about 5,500 L, about 200 L to about 5,000 L, about 200 L to
about 4,500 L, about 200 L to about 4,000 L, about 200 L to about
3,500 L, about 200 L to about 3,000 L, about 200 L to about 2,500
L, about 200 L to about 2,000 L, about 200 L to about 1,500 L,
about 200 L to about 1,000 L, about 200 L to about 900 L, about 200
L to about 800 L, about 200 L to about 700 L, about 200 L to about
600 L, about 200 L to about 500 L, about 200 L to about 400 L,
about 200 L to about 300 L, about 200 L to about 250 L, about 250 L
to about 20,000 L, about 250 L to about 18,000 L, about 250 L to
about 16,000 L, about 250 L to about 14,000 L, about 250 L to about
12,000 L, about 250 L to about 10,000 L, about 250 L to about 9,500
L, about 250 L to about 9,000 L, about 250 L to about 8,500 L,
about 250 L to about 8,000 L, about 250 L to about 7,500 L, about
250 L to about 7,000 L, about 250 L to about 6,500 L, about 250 L
to about 6,000 L, about 250 L to about 5,500 L, about 250 L to
about 5,000 L, about 250 L to about 4,500 L, about 250 L to about
4,000 L, about 250 L to about 3,500 L, about 250 L to about 3,000
L, about 250 L to about 2,500 L, about 250 L to about 2,000 L,
about 250 L to about 1,500 L, about 250 L to about 1,000 L, about
250 L to about 900 L, about 250 L to about 800 L, about 250 L to
about 700 L, about 250 L to about 600 L, about 250 L to about 500
L, about 250 L to about 400 L, about 250 L to about 300 L, about
300 L to about 20,000 L, about 300 L to about 18,000 L, about 300 L
to about 16,000 L, about 300 L to about 14,000 L, about 300 L to
about 12,000 L, about 300 L to about 10,000 L, about 300 L to about
9,500 L, about 300 L to about 9,000 L, about 300 L to about 8,500
L, about 300 L to about 8,000 L, about 300 L to about 7,500 L,
about 300 L to about 7,000 L, about 300 L to about 6,500 L, about
300 L to about 6,000 L, about 300 L to about 5,500 L, about 300 L
to about 5,000 L, about 300 L to about 4,500 L, about 300 L to
about 4,000 L, about 300 L to about 3,500 L, about 300 L to about
3,000 L, about 300 L to about 2,500 L, about 300 L to about 2,000
L, about 300 L to about 1,500 L, about 300 L to about 1,000 L,
about 300 L to about 900 L, about 300 L to about 800 L, about 300 L
to about 700 L, about 300 L to about 600 L, about 300 L to about
500 L, about 300 L to about 400 L, about 400 L to about 20,000 L,
about 400 L to about 18,000 L, about 400 L to about 16,000 L, about
400 L to about 14,000 L, about 400 L to about 12,000 L, about 400 L
to about 10,000 L, about 400 L to about 9,500 L, about 400 L to
about 9,000 L, about 400 L to about 8,500 L, about 400 L to about
8,000 L, about 400 L to about 7,500 L, about 400 L to about 7,000
L, about 400 L to about 6,500 L, about 400 L to about 6,000 L,
about 400 L to about 5,500 L, about 400 L to about 5,000 L, about
400 L to about 4,500 L, about 400 L to about 4,000 L, about 400 L
to about 3,500 L, about 400 L to about 3,000 L, about 400 L to
about 2,500 L, about 400 L to about 2,000 L, about 400 L to about
1,500 L, about 400 L to about 1,000 L, about 400 L to about 900 L,
about 400 L to about 800 L, about 400 L to about 700 L, about 400 L
to about 600 L, about 400 L to about 500 L, about 500 L to about
20,000 L, about 500 L to about 18,000 L, about 500 L to about
16,000 L, about 500 L to about 14,000 L, about 500 L to about
12,000 L, about 500 L to about 10,000 L, about 500 L to about 9,500
L, about 500 L to about 9,000 L, about 500 L to about 8,500 L,
about 500 L to about 8,000 L, about 500 L to about 7,500 L, about
500 L to about 7,000 L, about 500 L to about 6,500 L, about 500 L
to about 6,000 L, about 500 L to about 5,500 L, about 500 L to
about 5,000 L, about 500 L to about 4,500 L, about 500 L to about
4,000 L, about 500 L to about 3,500 L, about 500 L to about 3,000
L, about 500 L to about 2,500 L, about 500 L to about 2,000 L,
about 500 L to about 1,500 L, about 500 L to about 1,000 L, about
500 L to about 900 L, about 500 L to about 800 L, about 500 L to
about 700 L, about 500 L to about 600 L, about 600 L to about
20,000 L, about 600 L to about 18,000 L, about 600 L to about
16,000 L, about 600 L to about 14,000 L, about 600 L to about
12,000 L, about 600 L to about 10,000 L, about 600 L to about 9,500
L, about 600 L to about 9,000 L, about 600 L to about 8,500 L,
about 600 L to about 8,000 L, about 600 L to about 7,500 L, about
600 L to about 7,000 L, about 600 L to about 6,500 L, about 600 L
to about 6,000 L, about 600 L to about 5,500 L, about 600 L to
about 5,000 L, about 600 L to about 4,500 L, about 600 L to about
4,000 L, about 600 L to about 3,500 L, about 600 L to about 3,000
L, about 600 L to about 2,500 L, about 600 L to about 2,000 L,
about 600 L to about 1,500 L, about 600 L to about 1,000 L, about
600 L to about 900 L, about 600 L to about 800 L, about 600 L to
about 700 L, about 700 L to about 20,000 L, about 700 L to about
18,000 L, about 700 L to about 16,000 L, about 700 L to about
14,000 L, about 700 L to about 12,000 L, about 700 L to about
10,000 L, about 700 L to about 9,500 L, about 700 L to about 9,000
L, about 700 L to about 8,500 L, about 700 L to about 8,000 L,
about 700 L to about 7,500 L, about 700 L to about 7,000 L, about
700 L to about 6,500 L, about 700 L to about 6,000 L, about 700 L
to about 5,500 L, about 700 L to about 5,000 L, about 700 L to
about 4,500 L, about 700 L to about 4,000 L, about 700 L to about
3,500 L, about 700 L to about 3,000 L, about 700 L to about 2,500
L, about 700 L to about 2,000 L, about 700 L to about 1,500 L,
about 700 L to about 1,000 L, about 700 L to about 900 L, about 700
L to about 800 L, about 800 L to about 20,000 L, about 800 L to
about 18,000 L, about 800 L to about 16,000 L, about 800 L to about
14,000 L, about 800 L to about 12,000 L, about 800 L to about
10,000 L, about 800 L to about 9,500 L, about 800 L to about 9,000
L, about 800 L to about 8,500 L, about 800 L to about 8,000 L,
about 800 L to about 7,500 L, about 800 L to about 7,000 L, about
800 L to about 6,500 L, about 800 L to about 6,000 L, about 800 L
to about 5,500 L, about 800 L to about 5,000 L, about 800 L to
about 4,500 L, about 800 L to about 4,000 L, about 800 L to about
3,500 L, about 800 L to about 3,000 L, about 800 L to about 2,500
L, about 800 L to about 2,000 L, about 800 L to about 1,500 L,
about 800 L to about 1,000 L, about 800 L to about 900 L, about 900
L to about 20,000 L, about 900 L to about 18,000 L, about 900 L to
about 16,000 L, about 900 L to about 14,000 L, about 900 L to about
12,000 L, about 900 L to about 10,000 L, about 900 L to about 9,500
L, about 900 L to about 9,000 L, about 900 L to about 8,500 L,
about 900 L to about 8,000 L, about 900 L to about 7,500 L, about
900 L to about 7,000 L, about 900 L to about 6,500 L, about 900 L
to about 6,000 L, about 900 L to about 5,500 L, about 900 L to
about 5,000 L, about 900 L to about 4,500 L, about 900 L to about
4,000 L, about 900 L to about 3,500 L, about 900 L to about 3,000
L, about 900 L to about 2,500 L, about 900 L to about 2,000 L,
about 900 L to about 1,500 L, about 900 L to about 1,000 L, about
1,000 L to about 20,000 L, about 1,000 L to about 18,000 L, about
1,000 L to about 16,000 L, about 1,000 L to about 14,000 L, about
1,000 L to about 12,000 L, about 1,000 L to about 10,000 L, about
1,000 L to about 9,500 L, about 1,000 L to about 9,000 L, about
1,000 L to about 8,500 L, about 1,000 L to about 8,000 L, about
1,000 L to about 7,500 L, about 1,000 L to about 7,000 L, about
1,000 L to about 6,500 L, about 1,000 L to about 6,000 L, about
1,000 L to about 5,500 L, about 1,000 L to about 5,000 L, about
1,000 L to about 4,500 L, about 1,000 L to about 4,000 L, about
1,000 L to about 3,500 L, about 1,000 L to about 3,000 L, about
1,000 L to about 2,500 L, about 1,000 L to about 2,000 L, about
1,000 L to about 1,500 L, about 1,500 L to about 20,000 L, about
1,500 L to about 18,000 L, about 1,500 L to about 16,000 L, about
1,500 L to about 14,000 L, about 1,500 L to about 12,000 L, about
1,500 L to about 10,000 L, about 1,500 L to about 9,500 L, about
1,500 L to about 9,000 L, about 1,500 L to about 8,500 L, about
1,500 L to about 8,000 L, about 1,500 L to about 7,500 L, about
1,500 L to about 7,000 L, about 1,500 L to about 6,500 L, about
1,500 L to about 6,000 L, about 1,500 L to about 5,500 L, about
1,500 L to about 5,000 L, about 1,500 L to about 4,500 L, about
1,500 L to about 4,000 L, about 1,500 L to about 3,500 L, about
1,500 L to about 3,000 L, about 1,500 L to about 2,500 L, about
1,500 L to about 2,000 L, about 2,000 L to about
20,000 L, about 2,000 L to about 18,000 L, about 2,000 L to about
16,000 L, about 2,000 L to about 14,000 L, about 2,000 L to about
12,000 L, about 2,000 L to about 10,000 L, about 2,000 L to about
9,500 L, about 2,000 L to about 9,000 L, about 2,000 L to about
8,500 L, about 2,000 L to about 8,000 L, about 2,000 L to about
7,500 L, about 2,000 L to about 7,000 L, about 2,000 L to about
6,500 L, about 2,000 L to about 6,000 L, about 2,000 L to about
5,500 L, about 2,000 L to about 5,000 L, about 2,000 L to about
4,500 L, about 2,000 L to about 4,000 L, about 2,000 L to about
3,500 L, about 2,000 L to about 3,000 L, about 2,000 L to about
2,500 L, about 2,500 L to about 20,000 L, about 2,500 L to about
18,000 L, about 2,500 L to about 16,000 L, about 2,500 L to about
14,000 L, about 2,500 L to about 12,000 L, about 2,500 L to about
10,000 L, about 2,500 L to about 9,500 L, about 2,500 L to about
9,000 L, about 2,500 L to about 8,500 L, about 2,500 L to about
8,000 L, about 2,500 L to about 7,500 L, about 2,500 L to about
7,000 L, about 2,500 L to about 6,500 L, about 2,500 L to about
6,000 L, about 2,500 L to about 5,500 L, about 2,500 L to about
5,000 L, about 2,500 L to about 4,500 L, about 2,500 L to about
4,000 L, about 2,500 L to about 3,500 L, about 2,500 L to about
3,000 L, about 3,000 L to about 20,000 L, about 3,000 L to about
18,000 L, about 3,000 L to about 16,000 L, about 3,000 L to about
14,000 L, about 3,000 L to about 12,000 L, about 3,000 L to about
10,000 L, about 3,000 L to about 9,500 L, about 3,000 L to about
9,000 L, about 3,000 L to about 8,500 L, about 3,000 L to about
8,000 L, about 3,000 L to about 7,500 L, about 3,000 L to about
7,000 L, about 3,000 L to about 6,500 L, about 3,000 L to about
6,000 L, about 3,000 L to about 5,500 L, about 3,000 L to about
5,000 L, about 3,000 L to about 4,500 L, about 3,000 L to about
4,000 L, about 3,000 L to about 3,500 L, about 3,500 L to about
20,000 L, about 3,500 L to about 18,000 L, about 3,500 L to about
16,000 L, about 3,500 L to about 14,000 L, about 3,500 L to about
12,000 L, about 3,500 L to about 10,000 L, about 3,500 L to about
9,500 L, about 3,500 L to about 9,000 L, about 3,500 L to about
8,500 L, about 3,500 L to about 8,000 L, about 3,500 L to about
7,500 L, about 3,500 L to about 7,000 L, about 3,500 L to about
6,500 L, about 3,500 L to about 6,000 L, about 3,500 L to about
5,500 L, about 3,500 L to about 5,000 L, about 3,500 L to about
4,500 L, about 3,500 L to about 4,000 L, about 4,000 L to about
20,000 L, about 4,000 L to about 18,000 L, about 4,000 L to about
16,000 L, about 4,000 L to about 14,000 L, about 4,000 L to about
12,000 L, about 4,000 L to about 10,000 L, about 4,000 L to about
9,500 L, about 4,000 L to about 9,000 L, about 4,000 L to about
8,500 L, about 4,000 L to about 8,000 L, about 4,000 L to about
7,500 L, about 4,000 L to about 7,000 L, about 4,000 L to about
6,500 L, about 4,000 L to about 6,000 L, about 4,000 L to about
5,500 L, about 4,000 L to about 5,000 L, about 4,000 L to about
4,500 L, about 4,500 L to about 20,000 L, about 4,500 L to about
18,000 L, about 4,500 L to about 16,000 L, about 4,500 L to about
14,000 L, about 4,500 L to about 12,000 L, about 4,500 L to about
10,000 L, about 4,500 L to about 9,500 L, about 4,500 L to about
9,000 L, about 4,500 L to about 8,500 L, about 4,500 L to about
8,000 L, about 4,500 L to about 7,500 L, about 4,500 L to about
7,000 L, about 4,500 L to about 6,500 L, about 4,500 L to about
6,000 L, about 4,500 L to about 5,500 L, about 4,500 L to about
5,000 L, about 5,000 L to about 20,000 L, about 5,000 L to about
18,000 L, about 5,000 L to about 16,000 L, about 5,000 L to about
14,000 L, about 5,000 L to about 12,000 L, about 5,000 L to about
10,000 L, about 5,000 L to about 9,500 L, about 5,000 L to about
9,000 L, about 5,000 L to about 8,500 L, about 5,000 L to about
8,000 L, about 5,000 L to about 7,500 L, about 5,000 L to about
7,000 L, about 5,000 L to about 6,500 L, about 5,000 L to about
6,000 L, about 5,000 L to about 5,500 L, about 5,500 L to about
20,000 L, about 5,500 L to about 18,000 L, about 5,500 L to about
16,000 L, about 5,500 L to about 14,000 L, about 5,500 L to about
12,000 L, about 5,500 L to about 10,000 L, about 5,500 L to about
9,500 L, about 5,500 L to about 9,000 L, about 5,500 L to about
8,500 L, about 5,500 L to about 8,000 L, about 5,500 L to about
7,500 L, about 5,500 L to about 7,000 L, about 5,500 L to about
6,500 L, about 5,500 L to about 6,000 L, about 6,000 L to about
20,000 L, about 6,000 L to about 18,000 L, about 6,000 L to about
16,000 L, about 6,000 L to about 14,000 L, about 6,000 L to about
12,000 L, about 6,000 L to about 10,000 L, about 6,000 L to about
9,500 L, about 6,000 L to about 9,000 L, about 6,000 L to about
8,500 L, about 6,000 L to about 8,000 L, about 6,000 L to about
7,500 L, about 6,000 L to about 7,000 L, about 6,000 L to about
6,500 L, about 6,500 L to about 20,000 L, about 6,500 L to about
18,000 L, about 6,500 L to about 16,000 L, about 6,500 L to about
14,000 L, about 6,500 L to about 12,000 L, about 6,500 L to about
10,000 L, about 6,500 L to about 9,500 L, about 6,500 L to about
9,000 L, about 6,500 L to about 8,500 L, about 6,500 L to about
8,000 L, about 6,500 L to about 7,500 L, about 6,500 L to about
7,000 L, about 7,000 L to about 20,000 L, about 7,000 L to about
18,000 L, about 7,000 L to about 16,000 L, about 7,000 L to about
14,000 L, about 7,000 L to about 12,000 L, about 7,000 L to about
10,000 L, about 7,000 L to about 9,500 L, about 7,000 L to about
9,000 L, about 7,000 L to about 8,500 L, about 7,000 L to about
8,000 L, about 7,000 L to about 7,500 L, about 7,500 L to about
20,000 L, about 7,500 L to about 18,000 L, about 7,500 L to about
16,000 L, about 7,500 L to about 14,000 L, about 7,500 L to about
12,000 L, about 7,500 L to about 10,000 L, about 7,500 L to about
9,500 L, about 7,500 L to about 9,000 L, about 7,500 L to about
8,500 L, about 7,500 L to about 8,000 L, about 8,000 L to about
20,000 L, about 8,000 L to about 18,000 L, about 8,000 L to about
16,000 L, about 8,000 L to about 14,000 L, about 8,000 L to about
12,000 L, about 8,000 L to about 10,000 L, about 8,000 L to about
9,500 L, about 8,000 L to about 9,000 L, about 8,000 L to about
8,500 L, about 8,500 L to about 20,000 L, about 8,500 L to about
18,000 L, about 8,500 L to about 16,000 L, about 8,500 L to about
14,000 L, about 8,500 L to about 12,000 L, about 8,500 L to about
10,000 L, about 8,500 L to about 9,500 L, about 8,500 L to about
9,000 L, about 9,000 L to about 20,000 L, about 9,000 L to about
18,000 L, about 9,000 L to about 16,000 L, about 9,000 L to about
14,000 L, about 9,000 L to about 12,000 L, about 9,000 L to about
10,000 L, about 9,000 L to about 9,500 L, about 9,500 L to about
20,000 L, about 9,500 L to about 18,000 L, about 9,500 L to about
16,000 L, about 9,500 L to about 14,000 L, about 9,500 L to about
12,000 L, about 9,500 L to about 10,000 L, about 10,000 L to about
20,000 L, about 10,000 L to about 18,000 L, about 10,000 L to about
16,000 L, about 10,000 L to about 14,000 L, about 10,000 L to about
12,000 L, about 12,000 L to about 20,000 L, about 12,000 L to about
18,000 L, about 12,000 L to about 16,000 L, about 12,000 L to about
14,000 L, about 14,000 L to about 20,000 L, about 14,000 L to about
18,000 L, about 14,000 L to about 16,000 L, about 16,000 L to about
20,000 L, about 16,000 L to about 18,000 L, or about 18,000 L to
about 20,000 L.
[0156] In some embodiments, the operation parameters can be any
parameter that are related to cell viability or cell death (e.g.,
shear stress, concentration of a sensitizer, concentration of a
protectant, temperature, pH, nutrient composition, biomass loading
and feeding strategy). The cell viability or cell death rate can be
measured by using cells that can express lactate dehydrogenase
(LDH). When the cell that can express LDH dies, LDH in the cell is
released into the liquid culture medium. Thus, the cell viability
or the cell death rate can be measured orthogonally with LDH, and
is proportional to specific LDH production (sLDHp) (FIG. 3).
[0157] FIG. 4 shows how Pluronic.RTM. (e.g., protectants),
sensitizer, and shear (e.g., agitation) can affect specific LDH
production. As shown in FIG. 4, protectants can decrease
cell-bubble attachment and increase cell durability, thus
maintaining high cell viability and protecting cells from death. In
contrast, sensitizers can increase cell-bubble attachments and
decrease cell durability, thus decreasing cell viability. As these
parameters are interconnected with each other, it is often very
difficult to predict the exact effects of sensitizers, agitation,
and/or protectants by traditional models.
[0158] FIG. 5 shows that the specific LDH production in the small
scale model is a function of sensitizer concentration, protectant
concentration, and agitation. Because of the interaction among
there parameters, the specific LDH production usually does not have
a linear relationship with any of these parameters. The effects of
these parameters on the specific LDH production can be determined
by factorial design, e.g., full factorial design, or fractional
factorial design.
[0159] Factorial design involves performing experiments under a set
of conditions, and the conditions are defined by a set of
parameters (e.g., operation parameters). In the set of conditions,
each parameter can have two or more levels. The purpose of
factorial design is to study the main effects and the interaction
effects among different parameters (e.g., sensitizer concentration,
protectant concentration, and agitation). The term "full factorial
design" refers to an experiment design that includes all possible
combinations of different levels across all parameters. For
example, with three parameters (i.e., factors) each taking two
levels, a full factorial design will have 8 combinations in total,
and is usually called a 2.sup.3 factorial design (FIG. 6). In
contrast, the term "fractional factorial designs" refers to
experimental designs consisting of a carefully chosen subset
(fraction) of the experimental runs of a full factorial design. The
subset is chosen so as to exploit the sparsity-of-effects principle
to expose information about the most important features of the
problem studied, while using a fraction of the effort of a full
factorial design in terms of experimental runs and resources. By
performing factorial design, the inoperable region (e.g., the
combination of values of parameters that make the operation fail)
can be identified. The factorial design is known in the art, and is
described, e.g., in Box, G. E.; Hunter, W. G.; Hunter, J. S.
(2005). Statistics for Experimenters: Design, Innovation, and
Discovery (2nd ed.). Wiley. ISBN 0-471-71813-0; Vicente, Gemma, et
al. "Application of the factorial design of experiments and
response surface methodology to optimize biodiesel production."
Industrial crops and products 8.1 (1998): 29-35; Vandervoort, Jo,
and Annick Ludwig. "Biocompatible stabilizers in the preparation of
PLGA nanoparticles: a factorial design study." International
journal of pharmaceutics 238.1-2 (2002): 77-92; each of which is
incorporated by reference herein in its entirety.
[0160] In a typical factorial design, the center points (e.g.,
regions of interest) are often poorly predicted. Thus, in some
embodiments, the factorial design can be used in combination with
center composite design (e.g., inscribed center composite design,
circumscribed center composite design, or face centered composite
design) (FIG. 7). A center composite design is an experimental
design that contains an imbedded factorial or fractional factorial
design with center points that is augmented with a group of axial
points that allow estimation of curvature. The composite design is
known in the art, and a detailed description regarding center
composite design can be found, e.g., in Box, G. E.; Hunter, W. G.;
Hunter, J. S. (2005). Statistics for Experimenters: Design,
Innovation, and Discovery (2nd ed.). Wiley. ISBN 0-471-71813-0;
Ahmadi, M., et al. "Application of the central composite design and
response surface methodology to the advanced treatment of olive oil
processing wastewater using Fenton's peroxidation." Journal of
Hazardous Materials 123.1-3 (2005): 187-195; each of which is
incorporated herein by reference in its entirety.
[0161] In some embodiments, the testing conditions are selected
based on full factorial screening and center composite design
(e.g., inscribed center composite design). By combining the full
factorial screening and center composite design, the selected
testing conditions can maximize regions of interest and minimize
regions of potential failure (FIG. 8 and FIG. 9).
[0162] The experiment results of these test conditions in test
culture vessels provide an estimated contour that illustrates the
relationship among the selected parameters. Once the contour of the
small scale model has been determined, the small scale model can be
used to predict the missing values. For example, with the knowledge
of antifoam concentration or shear stress, it can predict how much
Pluronic.RTM. is required for operations. This relationship is
determined by correlating cell growth rate and death rates across
different scales (e.g., small scale, benchtop, production,
etc.).
[0163] In some embodiments, the estimated contour of the small
scale model can be used to match the data points collected at a
different scale (e.g., the production scale). Because the
concentration of sensitizer and protectants are known, it is
possible to determine the shear stress in the small scale model
that corresponds to the shear stress in the production bioreactor
from the contour (FIG. 10). Based on shear stress that corresponds
to the shear stress in the production bioreactor and the
concentration of sensitizer, the effect of protectant concentration
change (A[Protectant]) can be determined from the estimated contour
(FIG. 11). For example, as shown in FIG. 11, the far left point
(circle), the left center point (circle), the right center point
(circle), and the far right point (circle) all have the same shear
stress and sensitizer concentration. The shaded region is
inoperable space. By increasing the protectant concentration (from
the right center point (circle) to the far right point (circle),
with an arrow depicted between these two points), the acceptable
range of sensitizer concentration can be increased, leading to
better resilience to sensitizer.
[0164] Thus, in some embodiments, the model as described herein can
be used to determine interactive effects among different
parameters. The traditional models usually study the effect of each
parameter individually. The models as described herein can study
the effects of three parameters at once rather than the effect of
each parameter individually. Thus, this model described herein can
predict where the expected cell culture performance will be if any
single parameter is changed.
[0165] Furthermore, those traditional scale-up parameters usually
do not provide cell culture performance predictions. The model
described herein can predict an operating range with better
guidance which leads to a lower chance of failure when moving to a
production scale, and it provides the ability to scale up once
other traditional parameters have been met (P/V, tip speed, mixing
time, KLa).
[0166] In some other embodiments, the models as described herein
can be used to establish an operating design space for reactors at
any scale given any individual parameter or combination of
parameters (e.g., Pluronic.RTM., antifoam, and shear). The model
can be used to determine maximum amount of antifoam agents that the
cell culture can tolerate before failure for a given shear
condition.
[0167] In some embodiments, the model can be used to determine
maximum amount of antifoam agents that the Pluronic.RTM.
concentration can sustain before losing its efficacy for a given
shear condition.
[0168] In some embodiments, the model can be used to test different
types of antifoam agents (antifoam C, autoclaved antifoam,
irradiated antifoams, etc.) against different Pluronic.RTM..
[0169] In some embodiments, the model can be cell-density-range
independent, and need not apply only to intensified perfusion
cultures, as it uses LDH to normalize across scales.
[0170] In some embodiments, the model can also test antifoam
concentrations either with absolute addition (1 time bolus at
beginning) or daily addition.
[0171] In some embodiments, the model can be used to test for the
kinetics behind antifoam deactivation and its impact to cell
culture.
Cells
[0172] The methods provided herein can be used to culture a variety
of different cells (e.g., mammalian cells, bacterial cells, and
yeast cells).
[0173] Non-limiting examples of bacterial cells include E. coli,
Clostridium difficile, Clostridium sporogenes, Clostridium
beijerinkii, and Clostridium acetobutylicum. Exemplary commercially
available bacterial cells that can be used in any of the methods
described herein include BL21(DE3) (Novagen), BL21(DE3)-pLysS
(Novagen), BL21 Star-pLysS (Invitrogen), BL21-SI (Invitrogen),
BL21-AI (Invitrogen), Tuner (Novagen), Tuner pLysS (Novagen),
Origami (Novagen), Origami D (Novagen), Origami B pLysS (Novagen),
Rosetta (Novagen), Rosetta pLysS (Novagen), Rosetta-gami-pLysS
(Novagen), BL21 CodonPlus (Stratagene), AD494 (Novagen), BL21trxB
(Novagen), HMS174 (Novagen), NovaBlue (DE3) (Novagen), BLR
(Novagen), C41(DE3) (Lucigen), C43(DE3) (Lucigen), Lemo21(DE3) (New
England BioLabs), SHuffle T7 (New England BioLabs), ArticExpress
(Agilent Technologies), and Artic Express (DE3) (Agilent
Technologies).
[0174] Non-limiting examples of yeast cells include: S. cerevisiae,
S. exiguus, and Pichia pastoris. Additional examples of yeast cells
that can be used in any of the methods described herein include
those from the genera Saccharomyces, Pichia, Kluyveromyces,
Hansenula, and Yarrowia. Additional examples of yeast cells that
can be used in any of the methods described herein are described in
Nielsen, Methods in Enzymology 536:133-147, 2014.
[0175] Non-limiting examples of mammalian cells that can be
cultured using any of the methods described herein include: Chinese
hamster ovary (CHO) cells (e.g., CHO DG44 cells, CHO-Kls cells, and
CHO-DXB11 cells), Sp2.0, myeloma cells (e.g., NS/0), B-cells,
hybridoma cells, T-cells, human embryonic kidney (HEK) cells (e.g,
HEK 293E and HEK 293F), African green monkey kidney epithelial
cells (Vero) cells, and Madin-Darby Canine (Cocker Spaniel) kidney
epithelial cells (MDCK) cells. Additional mammalian cells that can
be cultured using the methods described herein are known in the
art. In non-limiting examples of any of the methods described
herein, the concentration of mammalian cells present in a test
culture vessel and/or a production bioreactor at the start of
culturing is about 0.1.times.10.sup.6 cells/mL to about
10.times.10.sup.6 cells/mL (e.g., about 0.1.times.10.sup.6 cells/mL
to about 9.times.10.sup.6 cells/mL, about 0.1.times.10.sup.6
cells/mL to about 8.times.10.sup.6 cells/mL, about
0.1.times.10.sup.6 cells/mL to about 7.times.10.sup.6 cells/mL,
about 0.1.times.10.sup.6 cells/mL to about 6.times.10.sup.6
cells/mL, about 0.1.times.10.sup.6 cells/mL to about
5.times.10.sup.6 cells/mL, about 0.1.times.10.sup.6 cells/mL to
about 4.times.10.sup.6 cells/mL, about 0.1.times.10.sup.6 cells/mL
to about 3.times.10.sup.6 cells/mL, about 0.1.times.10.sup.6
cells/mL to about 2.times.10.sup.6 cells/mL, about
0.1.times.10.sup.6 cells/mL to about 1.5.times.10.sup.6 cells/mL,
about 0.1.times.10.sup.6 cells/mL to about 1.0.times.10.sup.6
cells/mL, about 0.1.times.10.sup.6 cells/mL to about
0.8.times.10.sup.6 cells/mL, about 0.1.times.10.sup.6 cells/mL to
about 0.5.times.10.sup.6 cells/mL, about 0.1.times.10.sup.6
cells/mL to about 0.3.times.10.sup.6 cells/mL, about
0.2.times.10.sup.6 cells/mL to about 10.times.10.sup.6 cells/mL,
about 0.2.times.10.sup.6 cells/mL to about 9.times.10.sup.6
cells/mL, about 0.2.times.10.sup.6 cells/mL to about
8.times.10.sup.6 cells/mL, about 0.2.times.10.sup.6 cells/mL to
about 7.times.10.sup.6 cells/mL, about 0.2.times.10.sup.6 cells/mL
to about 6.times.10.sup.6 cells/mL, about 0.2.times.10.sup.6
cells/mL to about 5.times.10.sup.6 cells/mL, about
0.2.times.10.sup.6 cells/mL to about 4.times.10.sup.6 cells/mL,
about 0.2.times.10.sup.6 cells/mL to about 3.times.10.sup.6
cells/mL, about 0.2.times.10.sup.6 cells/mL to about
2.times.10.sup.6 cells/mL, about 0.2.times.10.sup.6 cells/mL to
about 1.5.times.10.sup.6 cells/mL, about 0.2.times.10.sup.6
cells/mL to about 1.0.times.10.sup.6 cells/mL, about
0.2.times.10.sup.6 cells/mL to about 0.8.times.10.sup.6 cells/mL,
about 0.2.times.10.sup.6 cells/mL to about 0.5.times.10.sup.6
cells/mL, about 0.3.times.10.sup.6 cells/mL to about
10.times.10.sup.6 cells/mL, about 0.3.times.10.sup.6 cells/mL to
about 9.times.10.sup.6 cells/mL, about 0.3.times.10.sup.6 cells/mL
to about 8.times.10.sup.6 cells/mL, about 0.3.times.10.sup.6
cells/mL to about 7.times.10.sup.6 cells/mL, about
0.3.times.10.sup.6 cells/mL to about 6.times.10.sup.6 cells/mL,
about 0.3.times.10.sup.6 cells/mL to about 5.times.10.sup.6
cells/mL, about 0.3.times.10.sup.6 cells/mL to about
4.times.10.sup.6 cells/mL, about 0.3.times.10.sup.6 cells/mL to
about 3.times.10.sup.6 cells/mL, about 0.3.times.10.sup.6 cells/mL
to about 2.times.10.sup.6 cells/mL, about 0.3.times.10.sup.6
cells/mL to about 1.5.times.10.sup.6 cells/mL, about
0.3.times.10.sup.6 cells/mL to about 1.0.times.10.sup.6 cells/mL,
about 0.3.times.10.sup.6 cells/mL to about 0.8.times.10.sup.6
cells/mL, about 0.3.times.10.sup.6 cells/mL to about
0.5.times.10.sup.6 cells/mL, about 0.5.times.10.sup.6 cells/mL to
about 10.times.10.sup.6 cells/mL, about 0.5.times.10.sup.6 cells/mL
to about 9.times.10.sup.6 cells/mL, about 0.5.times.10.sup.6
cells/mL to about 8.times.10.sup.6 cells/mL, about
0.5.times.10.sup.6 cells/mL to about 7.times.10.sup.6 cells/mL,
about 0.5.times.10.sup.6 cells/mL to about 6.times.10.sup.6
cells/mL, about 0.5.times.10.sup.6 cells/mL to about
5.times.10.sup.6 cells/mL, about 0.5.times.10.sup.6 cells/mL to
about 4.times.10.sup.6 cells/mL, about 0.5.times.10.sup.6 cells/mL
to about 3.times.10.sup.6 cells/mL, about 0.5.times.10.sup.6
cells/mL to about 2.times.10.sup.6 cells/mL, about
0.5.times.10.sup.6 cells/mL to about 1.5.times.10.sup.6 cells/mL,
about 0.5.times.10.sup.6 cells/mL to about 1.0.times.10.sup.6
cells/mL, about 0.5.times.10.sup.6 cells/mL to about
0.8.times.10.sup.6 cells/mL, about 0.8.times.10.sup.6 cells/mL to
about 10.times.10.sup.6 cells/mL, about 0.8.times.10.sup.6 cells/mL
to about 9.times.10.sup.6 cells/mL, about 0.8.times.10.sup.6
cells/mL to about 8.times.10.sup.6 cells/mL, about
0.8.times.10.sup.6 cells/mL to about 7.times.10.sup.6 cells/mL,
about 0.8.times.10.sup.6 cells/mL to about 6.times.10.sup.6
cells/mL, about 0.8.times.10.sup.6 cells/mL to about
5.times.10.sup.6 cells/mL, about 0.8.times.10.sup.6 cells/mL to
about 4.times.10.sup.6 cells/mL, about 0.8.times.10.sup.6 cells/mL
to about 3.times.10.sup.6 cells/mL, about 0.8.times.10.sup.6
cells/mL to about 2.times.10.sup.6 cells/mL, about
0.8.times.10.sup.6 cells/mL to about 1.5.times.10.sup.6 cells/mL,
about 0.8.times.10.sup.6 cells/mL to about 1.0.times.10.sup.6
cells/mL, about 1.0.times.10.sup.6 cells/mL to about
10.times.10.sup.6 cells/mL, about 1.0.times.10.sup.6 cells/mL to
about 9.times.10.sup.6 cells/mL, about 1.0.times.10.sup.6 cells/mL
to about 8.times.10.sup.6 cells/mL, about 1.0.times.10.sup.6
cells/mL to about 7.times.10.sup.6 cells/mL, about
1.0.times.10.sup.6 cells/mL to about 6.times.10.sup.6 cells/mL,
about 1.0.times.10.sup.6 cells/mL to about 5.times.10.sup.6
cells/mL, about 1.0.times.10.sup.6 cells/mL to about
4.times.10.sup.6 cells/mL, about 1.0.times.10.sup.6 cells/mL to
about 3.times.10.sup.6 cells/mL, about 1.0.times.10.sup.6 cells/mL
to about 2.times.10.sup.6 cells/mL, about 1.0.times.10.sup.6
cells/mL to about 1.5.times.10.sup.6 cells/mL, about
1.5.times.10.sup.6 cells/mL to about 10.times.10.sup.6 cells/mL,
about 1.5.times.10.sup.6 cells/mL to about 9.times.10.sup.6
cells/mL, about 1.5.times.10.sup.6 cells/mL to about
8.times.10.sup.6 cells/mL, about 1.5.times.10.sup.6 cells/mL to
about 7.times.10.sup.6 cells/mL, about 1.5.times.10.sup.6 cells/mL
to about 6.times.10.sup.6 cells/mL, about 1.5.times.10.sup.6
cells/mL to about 5.times.10.sup.6 cells/mL, about
1.5.times.10.sup.6 cells/mL to about 4.times.10.sup.6 cells/mL,
about 1.5.times.10.sup.6 cells/mL to about 3.times.10.sup.6
cells/mL, about 1.5.times.10.sup.6 cells/mL to about
2.times.10.sup.6 cells/mL, about 2.times.10.sup.6 cells/mL to about
10.times.10.sup.6 cells/mL, about 2.times.10.sup.6 cells/mL to
about 9.times.10.sup.6 cells/mL, about 2.times.10.sup.6 cells/mL to
about 8.times.10.sup.6 cells/mL, about 2.times.10.sup.6 cells/mL to
about 7.times.10.sup.6 cells/mL, about 2.times.10.sup.6 cells/mL to
about 6.times.10.sup.6 cells/mL, about 2.times.10.sup.6 cells/mL to
about 5.times.10.sup.6 cells/mL, about 2.times.10.sup.6 cells/mL to
about 4.times.10.sup.6 cells/mL, about 2.times.10.sup.6 cells/mL to
about 3.times.10.sup.6 cells/mL, about 3.times.10.sup.6 cells/mL to
about 10.times.10.sup.6 cells/mL, about 3.times.10.sup.6 cells/mL
to about 9.times.10.sup.6 cells/mL, about 3.times.10.sup.6 cells/mL
to about 8.times.10.sup.6 cells/mL, about 3.times.10.sup.6 cells/mL
to about 7.times.10.sup.6 cells/mL, about 3.times.10.sup.6 cells/mL
to about 6.times.10.sup.6 cells/mL, about 3.times.10.sup.6 cells/mL
to about 5.times.10.sup.6 cells/mL, about 3.times.10.sup.6 cells/mL
to about 4.times.10.sup.6 cells/mL, about 4.times.10.sup.6 cells/mL
to about 10.times.10.sup.6 cells/mL, about 4.times.10.sup.6
cells/mL to about 9.times.10.sup.6 cells/mL, about 4.times.10.sup.6
cells/mL to about 8.times.10.sup.6 cells/mL, about 4.times.10.sup.6
cells/mL to about 7.times.10.sup.6 cells/mL, about 4.times.10.sup.6
cells/mL to about 6.times.10.sup.6 cells/mL, about 4.times.10.sup.6
cells/mL to about 5.times.10.sup.6 cells/mL, about 5.times.10.sup.6
cells/mL to about 10.times.10.sup.6 cells/mL, about
5.times.10.sup.6 cells/mL to about 9.times.10.sup.6 cells/mL, about
5.times.10.sup.6 cells/mL to about 8.times.10.sup.6 cells/mL, about
5.times.10.sup.6 cells/mL to about 7.times.10.sup.6 cells/mL, about
5.times.10.sup.6 cells/mL to about 6.times.10.sup.6 cells/mL, about
6.times.10.sup.6 cells/mL to about 10.times.10.sup.6 cells/mL,
about 6.times.10.sup.6 cells/mL to about 9.times.10.sup.6 cells/mL,
about 6.times.10.sup.6 cells/mL to about 8.times.10.sup.6 cells/mL,
about 6.times.10.sup.6 cells/mL to about 7.times.10.sup.6 cells/mL,
about 7.times.10.sup.6 cells/mL to about 10.times.10.sup.6
cells/mL, about 7.times.10.sup.6 cells/mL to about 9.times.10.sup.6
cells/mL, about 7.times.10.sup.6 cells/mL to about 8.times.10.sup.6
cells/mL, about 8.times.10.sup.6 cells/mL to about
10.times.10.sup.6 cells/mL, about 8.times.10.sup.6 cells/mL to
about 9.times.10.sup.6 cells/mL, or about 9.times.10.sup.6 cells/mL
to about 10.times.10.sup.6 cells/mL).
[0176] The cell (e.g., mammalian cell) can contain a recombinant
nucleic acid (e.g., a nucleic acid stably integrated in the cell's
genome) that encodes a recombinant therapeutic protein (e.g., a
recombinant therapeutic protein that is secreted by the cell).
Non-limiting examples of recombinant therapeutic proteins are
described herein and are known in the art.
Recombinant Therapeutic Proteins
[0177] A recombinant therapeutic protein can be a secreted protein
that is released by the cell (e.g., mammalian cell) into the liquid
culture medium of the cell culture. For example, a nucleic acid
sequence encoding a soluble recombinant protein can contain a
sequence that encodes a secretion signal peptide at the N- or
C-terminus of the recombinant protein, which is cleaved by an
enzyme present in the cell (e.g., mammalian cell), and subsequently
released into the liquid culture medium of the cell culture. In
other instances, the recombinant protein is a soluble protein that
is not secreted, and the recombinant protein is recovered from
within the cell (e.g., mammalian cell).
[0178] Non-limiting examples of recombinant proteins include
immunoglobulins (including light and heavy chain immunoglobulins,
antibodies, or antibody fragments (e.g., any of the antibody
fragments described herein), enzymes (e.g., a galactosidase (e.g.,
an alpha-galactosidase), Myozyme, or Cerezyme), proteins (e.g.,
human erythropoietin, tumor necrosis factor (TNF), or an interferon
alpha or beta), or immunogenic or antigenic proteins or protein
fragments (e.g., proteins for use in a vaccine). In some
embodiments, the recombinant protein is an engineered
antigen-binding polypeptide that contains at least one
multifunctional recombinant protein scaffold (see, e.g., the
recombinant antigen-binding proteins described in Gebauer et al.,
Current Opin. Chem. Biol. 13:245-255, 2009; and U.S. Patent
Application Publication No. 2012/0164066 (herein incorporated by
reference in its entirety)). Non-limiting examples of recombinant
proteins that are antibodies include: panitumumab, omalizumab,
abagovomab, abciximab, actoxumab, adalimumab, adecatumumab,
afelimomab, afutuzumab, alacizumab, alacizumab, alemtuzumab,
alirocumab, altumomab, amatuximab, anatumomab, apolizumab,
atinumab, tocilizumab, basilizimab, bectumomab, belimumab,
bevacizumab, biciromab, canakinumab, cetuximab, daclizumab,
densumab, eculizumab, edrecolomab, efalizumab, efungumab,
ertumaxomab, etaracizumab, golimumab, infliximab, natalizumab,
palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab,
tocilizumab, and trastuzumab. Additional examples of therapeutic
antibodies are known in the art. Additional non-limiting examples
of recombinant proteins include: alglucosidase alfa, laronidase,
abatacept, galsulfase, lutropin alfa, antihemophilic factor,
agalsidase beta, interferon beta-la, darbepoetin alfa,
tenecteplase, etanercept, coagulation factor IX, follicle
stimulating hormone, interferon beta-la, imiglucerase, dornase
alfa, epoetin alfa, and alteplase.
[0179] A secreted, soluble recombinant protein can be recovered
from the liquid culture medium. A variety of different methods for
removing liquid culture medium from cells (e.g., mammalian cells)
are known in the art, including, for example, centrifugation,
filtration, pipetting, and/or aspiration. The secreted recombinant
protein can then be recovered and further purified from the liquid
culture medium using a variety of biochemical techniques including
various types of chromatography (e.g., affinity chromatography,
molecular sieve chromatography, cation exchange chromatography, or
anion exchange chromatography) and/or filtration (e.g., molecular
weight cut-off filtration).
[0180] To recover an intracellular recombinant protein, the cell
(e.g., mammalian cell) can be lysed. A wide variety of methods for
lysing cells (e.g., mammalian cells) are known in the art,
including, for example, sonication and/or detergent, enzymatic,
and/or chemical lysis. A recombinant protein can be purified from a
cell lysate (e.g., a mammalian cell lysate) using a variety of
biochemical methods known in the art, typically starting with a
step of centrifugation to remove the cellular debris, and then one
or more additional steps (e.g., one or more types of chromatography
(e.g., affinity chromatography, molecular sieve chromatography,
cation exchange chromatography, or anion exchange chromatography)
and/or filtration (e.g., molecular weight cut-off filtration)).
[0181] In some embodiments, the recovered recombinant protein is at
least or about 50% pure by weight, e.g., at least or about 55% pure
by weight, at least 60% pure by weight, at least 65% pure by
weight, at least 70% pure by weight, at least 75% pure by weight,
at least 80% pure by weight, at least 85% pure by weight, at least
90% pure by weight, at least 95% pure by weight, at least 96% pure
by weight, at least 97% pure by weight, at least 98% pure by
weight, or at least or about 99% pure by weight, or greater than
99% pure by weight.
[0182] In some embodiments, the recovered recombinant protein is a
recombinant human therapeutic protein that has one or more
different biophysical properties as compared to the same native
protein in a human (e.g., differences in the type or amount of
glycosylation, differences in phosphorylation, differences in
acylation, differences in metallation or metal stoichiometry,
and/or differences in cofactor binding).
[0183] Also provided herein is a recombinant therapeutic protein
produced by any of the methods described herein.
Liquid Culture Media
[0184] Liquid culture media are known in the art. The liquid
culture medium can be supplemented with a mammalian serum (e.g.,
fetal calf serum and bovine serum), and/or a growth hormone or
growth factor (e.g., insulin, transferrin, and epidermal growth
factor). Alternatively or in addition, liquid culture medium can be
a chemically-defined liquid culture medium, an animal-derived
component free liquid culture medium, a serum-free liquid culture
medium, or a serum-containing liquid culture medium. Non-limiting
examples of chemically-defined liquid culture media, animal-derived
component free liquid culture media, serum-free liquid culture
media, and serum-containing liquid culture media are commercially
available.
[0185] A liquid culture medium typically contains an energy source
(e.g., a carbohydrate, such as glucose), essential amino acids
(e.g., the basic set of twenty amino acids plus cysteine), vitamins
and/or other organic compounds required at low concentrations, free
fatty acids, and/or trace elements. The liquid culture medium can,
if desired, be supplemented with, e.g., a mammalian hormone or
growth factor (e.g., insulin, transferrin, or epidermal growth
factor), salts and buffers (e.g., calcium, magnesium, and phosphate
salts), nucleosides and bases (e.g., adenosine, thymidine, and
hypoxanthine), protein and tissue hydrolysates, and/or any
combination of these or other additives.
[0186] Non-limiting examples of liquid culture media that are
particularly useful in the presently described methods include,
e.g., CD CHO, Opti CHO, and Forti CHO (all available from Life
Technologies; Grand Island, N.Y.), Hycell CHO medium (Thermo Fisher
Scientific, Inc.; Waltham, Mass.), Ex-cell CD CHO Fusion medium
(Sigma-Aldrich Co.; St. Louis, Mo.), and PowerCHO medium (Lonza
Group, Ltd.; Basel, Switzerland). Medium components that also may
be useful in the present methods include, but are not limited to,
chemically-defined (CD) hydrolysates, e.g., CD peptone, CD
polypeptides (two or more amino acids), and CD growth factors.
Additional examples of liquid tissue culture medium and medium
components are known in the art.
Perfusion Culturing
[0187] The culturing of a cell can be performed using perfusion
culturing. Perfusion culturing a cell (e.g., a mammalian cell) in a
vessel includes the removal from the vessel of a first volume of a
first liquid culture medium (e.g., containing any concentration of
mammalian cells, e.g., a first volume of a first liquid culture
medium that is substantially free of cells), and adding to the
first liquid culture medium a second volume of a second liquid
culture medium. Removal and adding can be performed simultaneously
or sequentially, or a combination of the two. Further, removal and
adding can be performed continuously (e.g., at a rate that removes
and replaces a volume of between 0.1% to 800% (e.g., between 1% and
700%, between 1% and 600%, between 1% and 500%, between 1% and
400%, between 1% and 350%, between 1% and 300%, between 1% and
250%, between 1% and 100%, between 100% and 200%, between 5% and
150%, between 10% and 50%, between 15% and 40%, between 8% and 80%,
and between 4% and 30%) of the volume of the vessel or the first
liquid culture medium volume over any given time period (e.g., over
a 24-hour period, over an incremental time period of about 1 hour
to about 24 hours, or over an incremental time period of greater
than 24 hours)) or periodically (e.g., once every third day, once
every other day, once a day, twice a day, three times a day, four
times a day, or five times a day), or any combination thereof.
Where performed periodically, the volume that is removed or
replaced (e.g., within about a 24-hour period, within an
incremental time period of about 1 hour to about 24 hours, or
within an incremental time period of greater than 24 hours) can be,
e.g., between 0.1% to 800% (e.g., between 1% and 700%, between 1%
and 600%, between 1% and 500%, between 1% and 400%, between 1% and
300%, between 1% and 200%, between 1% and 100%, between 100% and
200%, between 5% and 150%, between 10% and 50%, between 15% and
40%, between 8% and 80%, and between 4% and 30%) of the volume of
the vessel or the first liquid culture medium volume. The first
volume of the first liquid culture medium removed and the second
volume of the second liquid culture medium added can in some
instances be held approximately the same over each 24-hour period
(or, alternatively, an incremental time period of about 1 hour to
about 24 hours or an incremental time period of greater than 24
hours) over the entire or part of the culturing period. As is known
in the art, the rate at which the first volume of the first liquid
culture medium is removed (volume/unit of time) and the rate at
which the second volume of the second liquid culture medium is
added (volume/unit of time) can be varied. The rate at which the
first volume of the first liquid culture medium is removed
(volume/unit of time) and the rate at which the second volume of
the second liquid culture medium is added (volume/unit of time) can
be about the same or can be different.
[0188] Alternatively, the volume removed and added can change
(e.g., gradually increase) over each 24-hour period (or
alternatively, an incremental time period of between 1 hour and
about 24 hours or an incremental time period of greater than 24
hours) during the culturing period. For example the volume of the
first liquid culture medium removed and the volume of the second
liquid culture medium added within each 24-hour period (or
alternatively, an incremental time period of between about 1 hour
and above 24 hours or an incremental time period of greater than 24
hours) over the culturing period can be increased (e.g., gradually
or through staggered increments) over the culturing period from a
volume that is between 0.5% to about 20% of the vessel volume or
the first liquid culture medium volume to about 25% to about 150%
of the vessel volume or the first liquid culture medium volume.
[0189] Skilled practitioners will appreciate that the first liquid
culture medium and the second liquid culture medium can be the same
type of media. In other instances, the first liquid culture medium
and the second liquid culture medium can be different.
[0190] The first volume of the first liquid culture medium can be
removed, e.g., by a mechanical system that can remove the first
volume of the first liquid culture medium from the vessel (e.g.,
the first volume of the first liquid culture medium that is
substantially free of cells from the bioreactor). Alternatively or
in addition, the first volume of the first liquid culture medium
can be removed by seeping or gravity flow of the first volume of
the first liquid culture medium through a sterile membrane with a
molecular weight cut-off that excludes the cell (e.g., mammalian
cell).
[0191] The second volume of the second liquid culture medium can be
added to the first liquid culture medium in an automated fashion,
e.g., by perfusion pump.
[0192] In some instances, removing the first volume of the first
liquid culture medium (e.g., a first volume of the first liquid
culture medium that is substantially free of mammalian cells) and
adding to the first liquid culture medium a second volume of the
second liquid culture medium does not occur within at least 1 hour
(e.g., within 2 hours, within 3 hours, within 4 hours, within 5
hours, within 6 hours, within 7 hours, within 8 hours, within 9
hours, within 10 hours, within 12 hours, within 14 hours, within 16
hours, within 18 hours, within 24 hours, within 36 hours, within 48
hours, within 72 hours, within 96 hours, or after 96 hours) of the
seeding of the vessel with a cell (e.g., a mammalian cell).
Fed-Batch Culturing
[0193] The culturing of a cell (e.g., a mammalian cell) can be
performed using fed-batch culturing. Fed-batch culturing a cell
(e.g., a mammalian cell) includes, over the majority of the
culturing period, the addition (e.g., periodic or continuous
addition) to a first liquid culture medium of a second volume of a
second liquid culture medium. The adding of the second liquid
culture medium can be performed continuously (e.g., at a rate that
adds a volume of between 0.1% to 300% (e.g., between 1% and 250%,
between 1% and 100%, between 100% and 200%, between 5% and 150%,
between 10% and 50%, between 15% and 40%, between 8% and 80%, and
between 4% and 30%) of the volume of the vessel or the first liquid
culture medium volume over any given time period (e.g., over a
24-hour period, over an incremental time period of about 1 hour to
about 24 hours, or over an incremental time period of greater than
24 hours)) or periodically (e.g., once every third day, once every
other day, once a day, twice a day, three times a day, four times a
day, or five times a day), or any combination thereof. Where
performed periodically, the volume that is added (e.g., within
about a 24-hour period, within an incremental time period of about
1 hour to about 24 hours, or within an incremental time period of
greater than 24 hours) can be, e.g., between 0.1% to 300% (e.g.,
between 1% and 200%, between 1% and 100%, between 100% and 200%,
between 5% and 150%, between 10% and 50%, between 15% and 40%,
between 8% and 80%, and between 4% and 30%) of the volume of the
vessel or the first liquid culture medium volume. The second volume
of the second liquid culture medium added can in some instances be
held approximately the same over each 24-hour period (or,
alternatively, an incremental time period of about 1 hour to about
24 hours or an incremental time period of greater than 24 hours)
over the entire or part of the culturing period. As is known in the
art, the rate at which the second volume of the second liquid
culture medium is added (volume/unit of time) can be varied over
the entire or part of the culturing period. For example, the volume
of the second liquid culture medium added can change (e.g.,
gradually increase) over each 24-hour period (or alternatively, an
incremental time period of between 1 hour and about 24 hours or an
incremental time period of greater than 24 hours) during the
culturing period. For example the volume of the second liquid
culture medium added within each 24-hour period (or alternatively,
an incremental time period of between about 1 hour and above 24
hours or an incremental time period of greater than 24 hours) over
the culturing period can be increased (e.g., gradually or through
staggered increments) over the culturing period from a volume that
is between 0.5% to about 20% of the vessel volume or the first
liquid culture medium volume to about 25% to about 150% of the
vessel volume or the first liquid culture medium volume. The rate
at which the second volume of the second liquid culture medium is
added (volume/unit of time) can be about the same over the entire
or part of the culturing period.
[0194] Skilled practitioners will appreciate that the first liquid
culture medium and the second liquid culture medium can be the same
type of media. In other instances, the first liquid culture medium
and the second liquid culture medium can be different. The volume
of the second liquid culture medium can be added to the first
liquid culture medium in an automated fashion, e.g., by perfusion
pump.
[0195] In some instances, adding to the first liquid culture medium
a second volume of the second liquid culture medium does not occur
within at least 1 hour (e.g., within 2 hours, within 3 hours,
within 4 hours, within 5 hours, within 6 hours, within 7 hours,
within 8 hours, within 9 hours, within 10 hours, within 12 hours,
within 14 hours, within 16 hours, within 18 hours, within 24 hours,
within 36 hours, within 48 hours, within 72 hours, within 96 hours,
or after 96 hours) of the seeding of the vessel with a cell (e.g.,
a mammalian cell). The cell culture medium in fed-batch cultures is
typically harvested at the end of culture period and used in any of
the processes described herein, however, the cell culture medium in
fed-batch cultures can also be harvested at one or more time points
during the culturing period and used in any of the processes
described herein.
[0196] Skilled practitioners will appreciate that any of the
various culture parameters (e.g., vessels, volumes, rates or
frequencies of replacing culture volumes, agitation frequencies,
temperatures, media, and CO.sub.2 concentrations) can be used in
any combination in to perform these methods.
Temperature
[0197] Culturing can be performed at a temperature of 32.degree. C.
to about 39.degree. C., e.g., about 32.degree. C. to about
37.degree. C. Skilled practitioners will appreciate that the
temperature can be changed at specific time point(s) in the
culturing method (e.g., during one or more of the first time
period, the second time period, and the third time period), e.g.,
on an hourly or daily basis. For example, the temperature can be
changed or shifted (e.g., increased or decreased) at about one day,
two days, three days, four days, five days, six days, seven days,
eight days, nine days, ten days, eleven days, twelve days, fourteen
days, fifteen days, sixteen days, seventeen days, eighteen days,
nineteen days, or about twenty days or more after the initial
seeding of the shake flask with the mammalian cell) or at any time
point within the first, second, and/or third time periods described
herein. For example, the temperature can be shifted upwards (e.g.,
a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,
7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0.degree. C.). For example, the
temperature can be shifted downwards (e.g., a change of up to or
about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0,
2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,
9.0, 9.5, or 10.degree. C.).
CO.sub.2
[0198] Culturing can include incubating a cell culture in an
atmosphere containing at most or about 1% to 15% CO.sub.2 (e.g., at
most or about 14% CO.sub.2, 12% CO.sub.2, 10% CO.sub.2, 8%
CO.sub.2, 6% CO.sub.2, 5% CO.sub.2, 4% CO.sub.2, 3% CO.sub.2, 2%
CO.sub.2, or at most or about 1% CO.sub.2).
[0199] Culturing can include incubating a cell culture in a
humidified atmosphere (e.g., at least or about 20%, 30%, 40%, 50%,
60%, 70%, 85%, 80%, 85%, 90%, or at least or about 95% humidity, or
about 100% humidity).
Cell Viability Measurement
[0200] A variety of methods and assays that can be used to
determine cell viability of a cell culture (e.g., a mammalian cell
culture) are known in the art. For example, lactate dehydrogenase
(LDH) can be detected in a liquid culture medium of a cell culture.
Commercial kits for detection of LDH in a liquid culture medium of
a cell culture are known in the art.
[0201] A variety of different dyes which are excluded by viable
cells, but selectively taken up by non-viable cells are used to
determine cell viability. Non-limiting examples of these dyes
include propidium iodide, trypan blue, and 7-aminoactinomycin D.
The viability of the cells is determined by visualizing or
otherwise detecting the staining of the cells after incubation with
the dye.
[0202] Additional assays for determining cell viability of a
culture of cells include the detection of mitochondrial activity or
caspase activity in the cells (e.g., Resazurin and Formazan).
Additional methods for determining cell viability in a culture of
cells are known in the art.
Methods of Predicting the Effect of a Concentration of a Sensitizer
on Cell Viability in a Production Bioreactor
[0203] Provided herein are methods of predicting the effect of a
concentration of a sensitizer (e.g., any of the sensitizers
described herein or known in the art) on cell viability in a
production bioreactor that include: (a) determining cell viability
under a plurality of test conditions in a test culture vessel
(e.g., any of the test culture vessels described herein), where the
plurality of test conditions are defined by a set of parameters;
and (b) based on the cell viability in the test culture vessel
under the plurality of conditions, predicting the effect of a
concentration of a sensitizer on cell viability in a production
bioreactor (e.g., any of the production bioreactors described
herein).
[0204] In some embodiments, the set of parameters includes,
consists, or consists essentially of shear stress, concentration of
the sensitizer, and concentration of a protectant (e.g., any of the
protectants described herein). In some embodiments of these
methods, the plurality of testing conditions are selected based on
full factorial screening and/or inscribed center composite design.
In some embodiments of any of these methods, the sensitizer is an
antifoam (e.g., a polydimethylsiloxane-based antifoam, simethicone,
or any other antifoam described herein or known in the art).
[0205] In some embodiments, the test culture vessel (e.g., any of
the exemplary test culture vessels described herein or known in the
art) has a volume of about 5 mL to about 5 L (or any of the
subranges of this range described herein). In some embodiments, the
test culture vessel is a shake flask, e.g., a baffled shake
flask.
[0206] In some embodiments, the production bioreactor (e.g., any of
the exemplary production bioreactors described herein or known in
the art) has a volume of about 5 L to about 20,000 L (or any of the
subranges of this range described herein). In some examples, the
production bioreactor is a perfusion bioreactor. In other examples,
the production bioreactor is a fed batch bioreactor.
[0207] The specific cell death rate as dsescribed herein is defined
by the formula below:
[0208] Specific cell death rate=(specific LDH production)/(LDH
content per cell).
[0209] Some embodiments of these methods further include culturing
a cell (e.g., any of the cells described herein) in the production
bioreactor using a concentration of the sensitizer predicted to
have a specific cell death rate of less 40% per day, less than 39%
per day, less than 38% per day, less than 37% per day, less than
36% per day, less than 35% per day, less than 34% per day, less
than 33% per day, less than 32% per day, less than 31% per day,
less than 30% per day, less than 29% per day, less than 28% per
day, less than 27% per day, less than 26% per day, less than 25%
per day, less than 24% per day, less than 23% per day, less than
22% per day, less than 21% per day, less than 20% per day, less
than 19% per day, less than 18% per day, less than 17% per day,
less than 16% per day, less than 15% per day, less than 14% per
day, less than 13% per day, less than 12% per day, less than 11%
per day, less than 10% per day, less than 9% per day, less than 8%
per day, less than 7% per day, less than 6% per day, less than 5%
per day, less than 4% per day, less than 3% per day, less than 2%
per day, or less than 1% per day, during the culturing of the cell
(e.g., any of the cells described herein) in the production
bioreactor. Some embodiments of any of the methods described herein
further include culturing a cell (e.g., any of the cells described
herein) in the production bioreactor using a concentration of the
sensitizer predicted to have a specific cell death rate of about
0.1% to about 40% per day, about 0.1% to about 38% per day, about
0.1% to about 36% per day, about 0.1% to about 34% per day, about
0.1% to about 32% per day, about 0.1% to about 30% per day, about
0.1% to about 28% per day, about 0.1% to about 26% per day, about
0.1% to about 24% per day, about 0.1% to about 22% per day, about
0.1% to about 20% per day, about 0.1% to about 18% per day, about
0.1% to about 16% per day, about 0.1% to about 14% per day, about
0.1% to about 12% per day, about 0.1% to about 10% per day, about
0.1% to about 8% per day, about 0.1% to about 6% per day, about
0.1% to about 5% per day, about 0.1% to about 4.5% per day, about
0.1% to about 4.0% per day, about 0.1% to about 3.5% per day, about
0.1% to about 3.0% per day, about 0.1% to about 2.5% per day, about
0.1% to about 2.0% per day, about 0.1% to about 1.5% per day, about
0.1% to about 1.0% per day, about 0.1% to about 0.5% per day, about
0.5% to about 40% per day, about 0.5% to about 38% per day, about
0.5% to about 36% per day, about 0.5% to about 34% per day, about
0.5% to about 32% per day, about 0.5% to about 30% per day, about
0.5% to about 28% per day, about 0.5% to about 26% per day, about
0.5% to about 24% per day, about 0.5% to about 22% per day, about
0.5% to about 20% per day, about 0.5% to about 18% per day, about
0.5% to about 16% per day, about 0.5% to about 14% per day, about
0.5% to about 12% per day, about 0.5% to about 10% per day, about
0.5% to about 8% per day, about 0.5% to about 6% per day, about
0.5% to about 5% per day, about 0.5% to about 4.5% per day, about
0.5% to about 4.0% per day, about 0.5% to about 3.5% per day, about
0.5% to about 3.0% per day, about 0.5% to about 2.5% per day, about
0.5% to about 2.0% per day, about 0.5% to about 1.5% per day, about
0.5% to about 1.0% per day, about 1.0% to about 40% per day, about
1.0% to about 38% per day, about 1.0% to about 36% per day, about
1.0% to about 34% per day, about 1.0% to about 32% per day, about
1.0% to about 30% per day, about 1.0% to about 28% per day, about
1.0% to about 26% per day, about 1.0% to about 24% per day, about
1.0% to about 22% per day, about 1.0% to about 20% per day, about
1.0% to about 18% per day, about 1.0% to about 16% per day, about
1.0% to about 14% per day, about 1.0% to about 12% per day, about
1.0% to about 10% per day, about 1.0% to about 8% per day, about
1.0% to about 6% per day, about 1.0% to about 5% per day, about
1.0% to about 4.5% per day, about 1.0% to about 4.0% per day, about
1.0% to about 3.5% per day, about 1.0% to about 3.0% per day, about
1.0% to about 2.5% per day, about 1.0% to about 2.0% per day, about
1.0% to about 1.5% per day, about 1.5% to about 40% per day, about
1.5% to about 38% per day, about 1.5% to about 36% per day, about
1.5% to about 34% per day, about 1.5% to about 32% per day, about
1.5% to about 30% per day, about 1.5% to about 28% per day, about
1.5% to about 26% per day, about 1.5% to about 24% per day, about
1.5% to about 22% per day, about 1.5% to about 20% per day, about
1.5% to about 18% per day, about 1.5% to about 16% per day, about
1.5% to about 14% per day, about 1.5% to about 12% per day, about
1.5% to about 10% per day, about 1.5% to about 8% per day, about
1.5% to about 6% per day, about 1.5% to about 5% per day, about
1.5% to about 4.5% per day, about 1.5% to about 4.0% per day, about
1.5% to about 3.5% per day, about 1.5% to about 3.0% per day, about
1.5% to about 2.5% per day, about 1.5% to about 2.0% per day, about
2.0% to about 40% per day, about 2.0% to about 38% per day, about
2.0% to about 36% per day, about 2.0% to about 34% per day, about
2.0% to about 32% per day, about 2.0% to about 30% per day, about
2.0% to about 28% per day, about 2.0% to about 26% per day, about
2.0% to about 24% per day, about 2.0% to about 22% per day, about
2.0% to about 20% per day, about 2.0% to about 18% per day, about
2.0% to about 16% per day, about 2.0% to about 14% per day, about
2.0% to about 12% per day, about 2.0% to about 10% per day, about
2.0% to about 8% per day, about 2.0% to about 6% per day, about
2.0% to about 5% per day, about 2.0% to about 4.5% per day, about
2.0% to about 4.0% per day, about 2.0% to about 3.5% per day, about
2.0% to about 3.0% per day, about 2.0% to about 2.5% per day, about
2.5% to about 40% per day, about 2.5% to about 38% per day, about
2.5% to about 36% per day, about 2.5% to about 34% per day, about
2.5% to about 32% per day, about 2.5% to about 30% per day, about
2.5% to about 28% per day, about 2.5% to about 26% per day, about
2.5% to about 24% per day, about 2.5% to about 22% per day, about
2.5% to about 20% per day, about 2.5% to about 18% per day, about
2.5% to about 16% per day, about 2.5% to about 14% per day, about
2.5% to about 12% per day, about 2.5% to about 10% per day, about
2.5% to about 8% per day, about 2.5% to about 6% per day, about
2.5% to about 5% per day, about 2.5% to about 4.5% per day, about
2.5% to about 4.0% per day, about 2.5% to about 3.5% per day, about
2.5% to about 3.0% per day, about 3.0% to about 40% per day, about
3.0% to about 38% per day, about 3.0% to about 36% per day, about
3.0% to about 34% per day, about 3.0% to about 32% per day, about
3.0% to about 30% per day, about 3.0% to about 28% per day, about
3.0% to about 26% per day, about 3.0% to about 24% per day, about
3.0% to about 22% per day, about 3.0% to about 20% per day, about
3.0% to about 18% per day, about 3.0% to about 16% per day, about
3.0% to about 14% per day, about 3.0% to about 12% per day, about
3.0% to about 10% per day, about 3.0% to about 8% per day, about
3.0% to about 6% per day, about 3.0% to about 5% per day, about
3.0% to about 4.5% per day, about 3.0% to about 4.0% per day, about
3.0% to about 3.5% per day, about 3.5% to about 40% per day, about
3.5% to about 38% per day, about 3.5% to about 36% per day, about
3.5% to about 34% per day, about 3.5% to about 32% per day, about
3.5% to about 30% per day, about 3.5% to about 28% per day, about
3.5% to about 26% per day, about 3.5% to about 24% per day, about
3.5% to about 22% per day, about 3.5% to about 20% per day, about
3.5% to about 18% per day, about 3.5% to about 16% per day, about
3.5% to about 14% per day, about 3.5% to about 12% per day, about
3.5% to about 10% per day, about 3.5% to about 8% per day, about
3.5% to about 6% per day, about 3.5% to about 5% per day, about
3.5% to about 4.5% per day, about 3.5% to about 4.0% per day, about
4.0% to about 40% per day, about 4.0% to about 38% per day, about
4.0% to about 36% per day, about 4.0% to about 34% per day, about
4.0% to about 32% per day, about 4.0% to about 30% per day, about
4.0% to about 28% per day, about 4.0% to about 26% per day, about
4.0% to about 24% per day, about 4.0% to about 22% per day, about
4.0% to about 20% per day, about 4.0% to about 18% per day, about
4.0% to about 16% per day, about 4.0% to about 14% per day, about
4.0% to about 12% per day, about 4.0% to about 10% per day, about
4.0% to about 8% per day, about 4.0% to about 6% per day, about
4.0% to about 5% per day, about 4.0% to about 4.5% per day, about
4.5% to about 40% per day, about 4.5% to about 38% per day, about
4.5% to about 36% per day, about 4.5% to about 34% per day, about
4.5% to about 32% per day, about 4.5% to about 30% per day, about
4.5% to about 28% per day, about 4.5% to about 26% per day, about
4.5% to about 24% per day, about 4.5% to about 22% per day, about
4.5% to about 20% per day, about 4.5% to about 18% per day, about
4.5% to about 16% per day, about 4.5% to about 14% per day, about
4.5% to about 12% per day, about 4.5% to about 10% per day, about
4.5% to about 8% per day, about 4.5% to about 6% per day, about
4.5% to about 5% per day, about 5% to about 40% per day, about 5%
to about 38% per day, about 5% to about 36% per day, about 5% to
about 34% per day, about 5% to about 32% per day, about 5% to about
30% per day, about 5% to about 28% per day, about 5% to about 26%
per day, about 5% to about 24% per day, about 5% to about 22% per
day, about 5% to about 20% per day, about 5% to about 18% per day,
about 5% to about 16% per day, about 5% to about 14% per day, about
5% to about 12% per day, about 5% to about 10% per day, about 5% to
about 8% per day, about 5% to about 6% per day, about 6% to about
40% per day, about 6% to about 38% per day, about 6% to about 36%
per day, about 6% to about 34% per day, about 6% to about 32% per
day, about 6% to about 30% per day, about 6% to about 28% per day,
about 6% to about 26% per day, about 6% to about 24% per day, about
6% to about 22% per day, about 6% to about 20% per day, about 6% to
about 18% per day, about 6% to about 16% per day, about 6% to about
14% per day, about 6% to about 12% per day, about 6% to about 10%
per day, about 6% to about 8% per day, about 8% to about 40% per
day, about 8% to about 38% per day, about 8% to about 36% per day,
about 8% to about 34% per day, about 8% to about 32% per day, about
8% to about 30% per day, about 8% to about 28% per day, about 8% to
about 26% per day, about 8% to about 24% per day, about 8% to about
22% per day, about 8% to about 20% per day, about 8% to about 18%
per day, about 8% to about 16% per day, about 8% to about 14% per
day, about 8% to about 12% per day, about 8% to about 10% per day,
about 10% to about 40% per day, about 10% to about 38% per day,
about 10% to about 36% per day, about 10% to about 34% per day,
about 10% to about 32% per day, about 10% to about 30% per day,
about 10% to about 28% per day, about 10% to about 26% per day,
about 10% to about 24% per day, about 10% to about 22% per day,
about 10% to about 20% per day, about 10% to about 18% per day,
about 10% to about 16% per day, about 10% to about 14% per day,
about 10% to about 12% per day, about 12% to about 40% per day,
about 12% to about 38% per day, about 12% to about 36% per day,
about 12% to about 34% per day, about 12% to about 32% per day,
about 12% to about 30% per day, about 12% to about 28% per day,
about 12% to about 26% per day, about 12% to about 24% per day,
about 12% to about 22% per day, about 12% to about 20% per day,
about 12% to about 18% per day, about 12% to about 16% per day,
about 12% to about 14% per day, about 14% to about 40% per day,
about 14% to about 38% per day, about 14% to about 36% per day,
about 14% to about 34% per day, about 14% to about 32% per day,
about 14% to about 30% per day, about 14% to about 28% per day,
about 14% to about 26% per day, about 14% to about 24% per day,
about 14% to about 22% per day, about 14% to about 20% per day,
about 14% to about 18% per day, about 14% to about 16% per day,
about 16% to about 40% per day, about 16% to about 38% per day,
about 16% to about 36% per day, about 16% to about 34% per day,
about 16% to about 32% per day, about 16% to about 30% per day,
about 16% to about 28% per day, about 16% to about 26% per day,
about 16% to about 24% per day, about 16% to about 22% per day,
about 16% to about 20% per day, about 16% to about 18% per day,
about 18% to about 40% per day, about 18% to about 38% per day,
about 18% to about 36% per day, about 18% to about 34% per day,
about 18% to about 32% per day, about 18% to about 30% per day,
about 18% to about 28% per day, about 18% to about 26% per day,
about 18% to about 24% per day, about 18% to about 22% per day,
about 18% to about 20% per day, about 20% to about 40% per day,
about 20% to about 38% per day, about 20% to about 36% per day,
about 20% to about 34% per day, about 20% to about 32% per day,
about 20% to about 30% per day, about 20% to about 28% per day,
about 20% to about 26% per day, about 20% to about 24% per day,
about 20% to about 22% per day, about 22% to about 40% per day,
about 22% to about 38% per day, about 22% to about 36% per day,
about 22% to about 34% per day, about 22% to about 32% per day,
about 22% to about 30% per day, about 22% to about 28% per day,
about 22% to about 26% per day, about 22% to about 24% per day,
about 24% to about 40% per day, about 24% to about 38% per day,
about 24% to about 36% per day, about 24% to about 34% per day,
about 24% to about 32% per day, about 24% to about 30% per day,
about 24% to about 28% per day, about 24% to about 26% per day,
about 26% to about 40% per day, about 26% to about 38% per day,
about 26% to about 36% per day, about 26% to about 34% per day,
about 26% to about 32% per day, about 26% to about 30% per day,
about 26% to about 28% per day, about 28% to about 40% per day,
about 28% to about 38% per day, about 28% to about 36% per day,
about 28% to about 34% per day, about 28% to about 32% per day,
about 28% to about 30% per day, about 30% to about 40% per day,
about 30% to about 38% per day, about 30% to about 36% per day,
about 30% to about 34% per day, about 30% to about 32% per day,
about 32% to about 40% per day, about 32% to about 38% per day,
about 32% to about 36% per day, about 32% to about 34% per day,
about 34% to about 40% per day, about 34% to about 38% per day,
about 34% to about 36% per day, about 36% to about 40% per day,
about 36% to about 38% per day, or about 38% to about 40% per day,
during the culturing of the cell (e.g., any of the cells described
herein) in the production bioreactor.
Some embodiments of these methods further include culturing a cell
(e.g., any of the cells described herein) in the production
bioreactor (e.g., any of the production bioreactors described
herein) using a concentration of the sensitizer predicted to result
in a cell specific LDH production of less than 150 nU/cell/day,
less than 145 nU/cell/day, less than 140 nU/cell/day, less than 135
nU/cell/day, less than 130 nU/cell/day, less than 125 nU/cell/day,
less than 120 nU/cell/day, less than 115 nU/cell/day, less than 150
nU/cell/day, less than 110 nU/cell/day, less than 105 nU/cell/day,
less than 100 nU/cell/day, less than 95 nU/cell/day, less than 90
nU/cell/day, less than 85 nU/cell/day, less than 80 nU/cell/day,
less than 75 nU/cell/day, less than 70 nU/cell/day, less than 65
nU/cell/day, less than 60 nU/cell/day, less than 55 nU/cell/day,
less than 50 nU/cell/day, less than 45 nU/cell/day, less than 45
nU/cell/day, less than 40 nU/cell/day, less than 35 nU/cell/day,
less than 30 nU/cell/day, less than 25 nU/cell/day, less than 20
nU/cell/day, less than 15 nU/cell/day, less than 10 nU/cell/day,
less than 5 nU/cell/day, or less than less than 1 nU/cell/day,
during the culturing of the cell in the production bioreactor. Some
embodiments of these methods further include culturing a cell
(e.g., any of the cells described herein) in the production
bioreactor (e.g., any of the production bioreactors described
herein) using a concentration of the sensitizer predicted to result
in a cell specific LDH production of about 0.1 nU/cell/day to about
150 nU/cell/day, about 0.1 nU/cell/day to about 140 nU/cell/day,
about 0.1 nU/cell/day to about 130 nU/cell/day, about 0.1
nU/cell/day to about 120 nU/cell/day, about 0.1 nU/cell/day to
about 110 nU/cell/day, about 0.1 nU/cell/day to about 100
nU/cell/day, about 0.1 nU/cell/day to about 95 nU/cell/day, about
0.1 nU/cell/day to about 90 nU/cell/day, about 0.1 nU/cell/day to
about 85 nU/cell/day, about 0.1 nU/cell/day to about 80
nU/cell/day, about 0.1 nU/cell/day to about 75 nU/cell/day, about
0.1 nU/cell/day to about 70 nU/cell/day, about 0.1 nU/cell/day to
about 65 nU/cell/day, about 0.1 nU/cell/day to about 60
nU/cell/day, about 0.1 nU/cell/day to about 55 nU/cell/day, about
0.1 nU/cell/day to about 50 nU/cell/day, about 0.1 nU/cell/day to
about 45 nU/cell/day, about 0.1 nU/cell/day to about 40
nU/cell/day, about 0.1 nU/cell/day to about 35 nU/cell/day, about
0.1 nU/cell/day to about 30 nU/cell/day, about 0.1 nU/cell/day to
about 25 nU/cell/day, about 0.1 nU/cell/day to about 20
nU/cell/day, about 0.1 nU/cell/day to about 15 nU/cell/day, about
0.1 nU/cell/day to about 10 nU/cell/day, about 0.1 nU/cell/day to
about 5 nU/cell/day, about 0.1 nU/cell/day to about 2.5
nU/cell/day, about 0.1 nU/cell/day to about 1.0 nU/cell/day, about
1.0 nU/cell/day to about 150 nU/cell/day, about 1.0 nU/cell/day to
about 140 nU/cell/day, about 1.0 nU/cell/day to about 130
nU/cell/day, about 1.0 nU/cell/day to about 120 nU/cell/day, about
1.0 nU/cell/day to about 110 nU/cell/day, about 1.0 nU/cell/day to
about 100 nU/cell/day, about 1.0 nU/cell/day to about 95
nU/cell/day, about 1.0 nU/cell/day to about 90 nU/cell/day, about
1.0 nU/cell/day to about 85 nU/cell/day, about 1.0 nU/cell/day to
about 80 nU/cell/day, about 1.0 nU/cell/day to about 75
nU/cell/day, about 1.0 nU/cell/day to about 70 nU/cell/day, about
1.0 nU/cell/day to about 65 nU/cell/day, about 1.0 nU/cell/day to
about 60 nU/cell/day, about 1.0 nU/cell/day to about 55
nU/cell/day, about 1.0 nU/cell/day to about 50 nU/cell/day, about
1.0 nU/cell/day to about 45 nU/cell/day, about 1.0 nU/cell/day to
about 40 nU/cell/day, about 1.0 nU/cell/day to about 35
nU/cell/day, about 1.0 nU/cell/day to about 30 nU/cell/day, about
1.0 nU/cell/day to about 25 nU/cell/day, about 1.0 nU/cell/day to
about 20 nU/cell/day, about 1.0 nU/cell/day to about 15
nU/cell/day, about 1.0 nU/cell/day to about 10 nU/cell/day, about
1.0 nU/cell/day to about 5 nU/cell/day, about 1.0 nU/cell/day to
about 2.5 nU/cell/day, about 2.5 nU/cell/day to about 150
nU/cell/day, about 2.5 nU/cell/day to about 140 nU/cell/day, about
2.5 nU/cell/day to about 130 nU/cell/day, about 2.5 nU/cell/day to
about 120 nU/cell/day, about 2.5 nU/cell/day to about 110
nU/cell/day, about 2.5 nU/cell/day to about 100 nU/cell/day, about
2.5 nU/cell/day to about 95 nU/cell/day, about 2.5 nU/cell/day to
about 90 nU/cell/day, about 2.5 nU/cell/day to about 85
nU/cell/day, about 2.5 nU/cell/day to about 80 nU/cell/day, about
2.5 nU/cell/day to about 75 nU/cell/day, about 2.5 nU/cell/day to
about 70 nU/cell/day, about 2.5 nU/cell/day to about 65
nU/cell/day, about 2.5 nU/cell/day to about 60 nU/cell/day, about
2.5 nU/cell/day to about 55 nU/cell/day, about 2.5 nU/cell/day to
about 50 nU/cell/day, about 2.5 nU/cell/day to about 45
nU/cell/day, about 2.5 nU/cell/day to about 40 nU/cell/day, about
2.5 nU/cell/day to about 35 nU/cell/day, about 2.5 nU/cell/day to
about 30 nU/cell/day, about 2.5 nU/cell/day to about 25
nU/cell/day, about 2.5 nU/cell/day to about 20 nU/cell/day, about
2.5 nU/cell/day to about 15 nU/cell/day, about 2.5 nU/cell/day to
about 10 nU/cell/day, about 2.5 nU/cell/day to about 5 nU/cell/day,
about 5 nU/cell/day to about 150 nU/cell/day, about 5 nU/cell/day
to about 140 nU/cell/day, about 5 nU/cell/day to about 130
nU/cell/day, about 5 nU/cell/day to about 120 nU/cell/day, about 5
nU/cell/day to about 110 nU/cell/day, about 5 nU/cell/day to about
100 nU/cell/day, about 5 nU/cell/day to about 95 nU/cell/day, about
5 nU/cell/day to about 90 nU/cell/day, about 5 nU/cell/day to about
85 nU/cell/day, about 5 nU/cell/day to about 80 nU/cell/day, about
5 nU/cell/day to about 75 nU/cell/day, about 5 nU/cell/day to about
70 nU/cell/day, about 5 nU/cell/day to about 65 nU/cell/day, about
5 nU/cell/day to about 60 nU/cell/day, about 5 nU/cell/day to about
55 nU/cell/day, about 5 nU/cell/day to about 50 nU/cell/day, about
5 nU/cell/day to about 45 nU/cell/day, about 5 nU/cell/day to about
40 nU/cell/day, about 5 nU/cell/day to about 35 nU/cell/day, about
5 nU/cell/day to about 30 nU/cell/day, about 5 nU/cell/day to about
25 nU/cell/day, about 5 nU/cell/day to about 20 nU/cell/day, about
5 nU/cell/day to about 15 nU/cell/day, about 5 nU/cell/day to about
10 nU/cell/day, about 10 nU/cell/day to about 150 nU/cell/day,
about 10 nU/cell/day to about 140 nU/cell/day, about 10 nU/cell/day
to about 130 nU/cell/day, about 10 nU/cell/day to about 120
nU/cell/day, about 10 nU/cell/day to about 110 nU/cell/day, about
10 nU/cell/day to about 100 nU/cell/day, about 10 nU/cell/day to
about 95 nU/cell/day, about 10 nU/cell/day to about 90 nU/cell/day,
about 10 nU/cell/day to about 85 nU/cell/day, about 10 nU/cell/day
to about 80 nU/cell/day, about 10 nU/cell/day to about 75
nU/cell/day, about 10 nU/cell/day to about 70 nU/cell/day, about 10
nU/cell/day to about 65 nU/cell/day, about 10 nU/cell/day to about
60 nU/cell/day, about 10 nU/cell/day to about 55 nU/cell/day, about
10 nU/cell/day to about 50 nU/cell/day, about 10 nU/cell/day to
about 45 nU/cell/day, about 10 nU/cell/day to about 40 nU/cell/day,
about 10 nU/cell/day to about 35 nU/cell/day, about 10 nU/cell/day
to about 30 nU/cell/day, about 10 nU/cell/day to about 25
nU/cell/day, about 10 nU/cell/day to about 20 nU/cell/day, about 10
nU/cell/day to about 15 nU/cell/day, about 15 nU/cell/day to about
150 nU/cell/day, about 15 nU/cell/day to about 140 nU/cell/day,
about 15 nU/cell/day to about 130 nU/cell/day, about 15 nU/cell/day
to about 120 nU/cell/day, about 15 nU/cell/day to about 110
nU/cell/day, about 15 nU/cell/day to about 100 nU/cell/day, about
15 nU/cell/day to about 95 nU/cell/day, about 15 nU/cell/day to
about 90 nU/cell/day, about 15 nU/cell/day to about 85 nU/cell/day,
about 15 nU/cell/day to about 80 nU/cell/day, about 15 nU/cell/day
to about 75 nU/cell/day, about 15 nU/cell/day to about 70
nU/cell/day, about 15 nU/cell/day to about 65 nU/cell/day, about 15
nU/cell/day to about 60 nU/cell/day, about 15 nU/cell/day to about
55 nU/cell/day, about 15 nU/cell/day to about 50 nU/cell/day, about
15 nU/cell/day to about 45 nU/cell/day, about 15 nU/cell/day to
about 40 nU/cell/day, about 15 nU/cell/day to about 35 nU/cell/day,
about 15 nU/cell/day to about 30 nU/cell/day, about 15 nU/cell/day
to about 25 nU/cell/day, about 15 nU/cell/day to about 20
nU/cell/day, about 20 nU/cell/day to about 150 nU/cell/day, about
20 nU/cell/day to about 140 nU/cell/day, about 20 nU/cell/day to
about 130 nU/cell/day, about 20 nU/cell/day to about 120
nU/cell/day, about 20 nU/cell/day to about 110 nU/cell/day, about
20 nU/cell/day to about 100 nU/cell/day, about 20 nU/cell/day to
about 95 nU/cell/day, about 20 nU/cell/day to about 90 nU/cell/day,
about 20 nU/cell/day to about 85 nU/cell/day, about 20 nU/cell/day
to about 80 nU/cell/day, about 20 nU/cell/day to about 75
nU/cell/day, about 20 nU/cell/day to about 70 nU/cell/day, about 20
nU/cell/day to about 65 nU/cell/day, about 20 nU/cell/day to about
60 nU/cell/day, about 20 nU/cell/day to about 55 nU/cell/day, about
20 nU/cell/day to about 50 nU/cell/day, about 20 nU/cell/day to
about 45 nU/cell/day, about 20 nU/cell/day to about 40 nU/cell/day,
about 20 nU/cell/day to about 35 nU/cell/day, about 20 nU/cell/day
to about 30 nU/cell/day, about 20 nU/cell/day to about 25
nU/cell/day, about 25 nU/cell/day to about 150 nU/cell/day, about
25 nU/cell/day to about 140 nU/cell/day, about 25 nU/cell/day to
about 130 nU/cell/day, about 25 nU/cell/day to about 120
nU/cell/day, about 25 nU/cell/day to about 110 nU/cell/day, about
25 nU/cell/day to about 100 nU/cell/day, about 25 nU/cell/day to
about 95 nU/cell/day, about 25 nU/cell/day to about 90 nU/cell/day,
about 25 nU/cell/day to about 85 nU/cell/day, about 25 nU/cell/day
to about 80 nU/cell/day, about 25 nU/cell/day to about 75
nU/cell/day, about 25 nU/cell/day to about 70 nU/cell/day, about 25
nU/cell/day to about 65 nU/cell/day, about 25 nU/cell/day to about
60 nU/cell/day, about 25 nU/cell/day to about 55 nU/cell/day, about
25 nU/cell/day to about 50 nU/cell/day, about 25 nU/cell/day to
about 45 nU/cell/day, about 25 nU/cell/day to about 40 nU/cell/day,
about 25 nU/cell/day to about 35 nU/cell/day, about 25 nU/cell/day
to about 30 nU/cell/day, about 30 nU/cell/day to about 150
nU/cell/day, about 30 nU/cell/day to about 140 nU/cell/day, about
30 nU/cell/day to about 130 nU/cell/day, about 30 nU/cell/day to
about 120 nU/cell/day, about 30 nU/cell/day to about 110
nU/cell/day, about 30 nU/cell/day to about 100 nU/cell/day, about
30 nU/cell/day to about 95 nU/cell/day, about 30 nU/cell/day to
about 90 nU/cell/day, about 30 nU/cell/day to about 85 nU/cell/day,
about 30 nU/cell/day to about 80 nU/cell/day, about 30 nU/cell/day
to about 75 nU/cell/day, about 30 nU/cell/day to about 70
nU/cell/day, about 30 nU/cell/day to about 65 nU/cell/day, about 30
nU/cell/day to about 60 nU/cell/day, about 30 nU/cell/day to about
55 nU/cell/day, about 30 nU/cell/day to about 50 nU/cell/day, about
30 nU/cell/day to about 45 nU/cell/day, about 30 nU/cell/day to
about 40 nU/cell/day, about 30 nU/cell/day to about 35 nU/cell/day,
about 35 nU/cell/day to about 150 nU/cell/day, about 35 nU/cell/day
to about 140 nU/cell/day, about 35 nU/cell/day to about 130
nU/cell/day, about 35 nU/cell/day to about 120 nU/cell/day, about
35 nU/cell/day to about 110 nU/cell/day, about 35 nU/cell/day to
about 100 nU/cell/day, about 35 nU/cell/day to about 95
nU/cell/day, about 35 nU/cell/day to about 90 nU/cell/day, about 35
nU/cell/day to about 85 nU/cell/day, about 35 nU/cell/day to about
80 nU/cell/day, about 35 nU/cell/day to about 75 nU/cell/day, about
35 nU/cell/day to about 70 nU/cell/day, about 35 nU/cell/day to
about 65 nU/cell/day, about 35 nU/cell/day to about 60 nU/cell/day,
about 35 nU/cell/day to about 55 nU/cell/day, about 35 nU/cell/day
to about 50 nU/cell/day, about 35 nU/cell/day to about 45
nU/cell/day, about 35 nU/cell/day to about 40 nU/cell/day, about 40
nU/cell/day to about 150 nU/cell/day, about 40 nU/cell/day to about
140 nU/cell/day, about 40 nU/cell/day to about 130 nU/cell/day,
about 40 nU/cell/day to about 120 nU/cell/day, about 40 nU/cell/day
to about 110 nU/cell/day, about 40 nU/cell/day to about 100
nU/cell/day, about 40 nU/cell/day to about 95 nU/cell/day, about 40
nU/cell/day to about 90 nU/cell/day, about 40 nU/cell/day to about
85 nU/cell/day, about 40 nU/cell/day to about 80 nU/cell/day, about
40 nU/cell/day to about 75 nU/cell/day, about 40 nU/cell/day to
about 70 nU/cell/day, about 40 nU/cell/day to about 65 nU/cell/day,
about 40 nU/cell/day to about 60 nU/cell/day, about 40 nU/cell/day
to about 55 nU/cell/day, about 40 nU/cell/day to about 50
nU/cell/day, about 40 nU/cell/day to about 45 nU/cell/day, about 45
nU/cell/day to about 150 nU/cell/day, about 45 nU/cell/day to about
140 nU/cell/day, about 45 nU/cell/day to about 130 nU/cell/day,
about 45 nU/cell/day to about 120 nU/cell/day, about 45 nU/cell/day
to about 110 nU/cell/day, about 45 nU/cell/day to about 100
nU/cell/day, about 45 nU/cell/day to about 95 nU/cell/day, about 45
nU/cell/day to about 90 nU/cell/day, about 45 nU/cell/day to about
85 nU/cell/day, about 45 nU/cell/day to about 80 nU/cell/day, about
45 nU/cell/day to about 75 nU/cell/day, about 45 nU/cell/day to
about 70 nU/cell/day, about 45 nU/cell/day to about 65 nU/cell/day,
about 45 nU/cell/day to about 60 nU/cell/day, about 45 nU/cell/day
to about 55 nU/cell/day, about 45 nU/cell/day to about 50
nU/cell/day, about 50 nU/cell/day to about 150 nU/cell/day, about
50 nU/cell/day to about 140 nU/cell/day, about 50 nU/cell/day to
about 130 nU/cell/day, about 50 nU/cell/day to about 120
nU/cell/day, about 50 nU/cell/day to about 110 nU/cell/day, about
50 nU/cell/day to about 100 nU/cell/day, about 50 nU/cell/day to
about 95 nU/cell/day, about 50 nU/cell/day to about 90 nU/cell/day,
about 50 nU/cell/day to about 85 nU/cell/day, about 50 nU/cell/day
to about 80 nU/cell/day, about 50 nU/cell/day to about 75
nU/cell/day, about 50 nU/cell/day to about 70 nU/cell/day, about 50
nU/cell/day to about 65 nU/cell/day, about 50 nU/cell/day to about
60 nU/cell/day, about 50 nU/cell/day to about 55 nU/cell/day, about
55 nU/cell/day to about 150 nU/cell/day, about 55 nU/cell/day to
about 140 nU/cell/day, about 55 nU/cell/day to about 130
nU/cell/day, about 55 nU/cell/day to about 120 nU/cell/day, about
55 nU/cell/day to about 110 nU/cell/day, about 55 nU/cell/day to
about 100 nU/cell/day, about 55 nU/cell/day to about 95
nU/cell/day, about 55 nU/cell/day to about 90 nU/cell/day, about 55
nU/cell/day to about 85 nU/cell/day, about 55 nU/cell/day to about
80 nU/cell/day, about 55 nU/cell/day to about 75 nU/cell/day, about
55 nU/cell/day to about 70 nU/cell/day, about 55 nU/cell/day to
about 65 nU/cell/day, about 55 nU/cell/day to about 60 nU/cell/day,
about 60 nU/cell/day to about 150 nU/cell/day, about 60 nU/cell/day
to about 140 nU/cell/day, about 60 nU/cell/day to about 130
nU/cell/day, about 60 nU/cell/day to about 120 nU/cell/day, about
60 nU/cell/day to about 110 nU/cell/day, about 60 nU/cell/day to
about 100 nU/cell/day, about 60 nU/cell/day to about 95
nU/cell/day, about 60 nU/cell/day to about 90 nU/cell/day, about 60
nU/cell/day to about 85 nU/cell/day, about 60 nU/cell/day to about
80 nU/cell/day, about 60 nU/cell/day to about 75 nU/cell/day, about
60 nU/cell/day to about 70 nU/cell/day, about 60 nU/cell/day to
about 65 nU/cell/day, about 65 nU/cell/day to about 150
nU/cell/day, about 65 nU/cell/day to about 140 nU/cell/day, about
65 nU/cell/day to about 130 nU/cell/day, about 65 nU/cell/day to
about 120 nU/cell/day, about 65 nU/cell/day to about 110
nU/cell/day, about 65 nU/cell/day to about 100 nU/cell/day, about
65 nU/cell/day to about 95 nU/cell/day, about 65 nU/cell/day to
about 90 nU/cell/day, about 65 nU/cell/day to about 85 nU/cell/day,
about 65 nU/cell/day to about 80 nU/cell/day, about 65 nU/cell/day
to about 75 nU/cell/day, about 65 nU/cell/day to about 70
nU/cell/day, about 70 nU/cell/day to about 150 nU/cell/day, about
70 nU/cell/day to about 140 nU/cell/day, about 70 nU/cell/day to
about 130 nU/cell/day, about 70 nU/cell/day to about 120
nU/cell/day, about 70 nU/cell/day to about 110 nU/cell/day, about
70 nU/cell/day to about 100 nU/cell/day, about 70 nU/cell/day to
about 95 nU/cell/day, about 70 nU/cell/day to about 90 nU/cell/day,
about 70 nU/cell/day to about 85 nU/cell/day, about 70 nU/cell/day
to about 80 nU/cell/day, about 70 nU/cell/day to about 75
nU/cell/day, about 75 nU/cell/day to about 150 nU/cell/day, about
75 nU/cell/day to about 140 nU/cell/day, about 75 nU/cell/day to
about 130 nU/cell/day, about 75 nU/cell/day to about 120
nU/cell/day, about 75 nU/cell/day to about 110 nU/cell/day, about
75 nU/cell/day to about 100 nU/cell/day, about 75 nU/cell/day to
about 95 nU/cell/day, about 75 nU/cell/day to about 90 nU/cell/day,
about 75 nU/cell/day to about 85 nU/cell/day, about 75 nU/cell/day
to about 80 nU/cell/day, about 80 nU/cell/day to about 150
nU/cell/day, about 80 nU/cell/day to about 140 nU/cell/day, about
80 nU/cell/day to about 130 nU/cell/day, about 80 nU/cell/day to
about 120 nU/cell/day, about 80 nU/cell/day to about 110
nU/cell/day, about 80 nU/cell/day to about 100 nU/cell/day, about
80 nU/cell/day to about 95 nU/cell/day, about 80 nU/cell/day to
about 90 nU/cell/day, about 80 nU/cell/day to about 85 nU/cell/day,
about 85 nU/cell/day to about 150 nU/cell/day, about 85 nU/cell/day
to about 140
nU/cell/day, about 85 nU/cell/day to about 130 nU/cell/day, about
85 nU/cell/day to about 120 nU/cell/day, about 85 nU/cell/day to
about 110 nU/cell/day, about 85 nU/cell/day to about 100
nU/cell/day, about 85 nU/cell/day to about 95 nU/cell/day, about 85
nU/cell/day to about 90 nU/cell/day, about 90 nU/cell/day to about
150 nU/cell/day, about 90 nU/cell/day to about 140 nU/cell/day,
about 90 nU/cell/day to about 130 nU/cell/day, about 90 nU/cell/day
to about 120 nU/cell/day, about 90 nU/cell/day to about 110
nU/cell/day, about 90 nU/cell/day to about 100 nU/cell/day, about
90 nU/cell/day to about 95 nU/cell/day, about 95 nU/cell/day to
about 150 nU/cell/day, about 95 nU/cell/day to about 140
nU/cell/day, about 95 nU/cell/day to about 130 nU/cell/day, about
95 nU/cell/day to about 120 nU/cell/day, about 95 nU/cell/day to
about 110 nU/cell/day, about 95 nU/cell/day to about 100
nU/cell/day, about 100 nU/cell/day to about 150 nU/cell/day, about
100 nU/cell/day to about 140 nU/cell/day, about 100 nU/cell/day to
about 130 nU/cell/day, about 100 nU/cell/day to about 120
nU/cell/day, about 100 nU/cell/day to about 110 nU/cell/day, about
110 nU/cell/day to about 150 nU/cell/day, about 110 nU/cell/day to
about 140 nU/cell/day, about 110 nU/cell/day to about 130
nU/cell/day, about 110 nU/cell/day to about 120 nU/cell/day, about
120 nU/cell/day to about 150 nU/cell/day, about 120 nU/cell/day to
about 140 nU/cell/day, about 120 nU/cell/day to about 130
nU/cell/day, about 130 nU/cell/day to about 150 nU/cell/day, about
130 nU/cell/day to about 140 nU/cell/day, or about 140 nU/cell/day
to about 150 nU/cell/day, during the culturing of the cell in the
production bioreactor.
[0211] The growth rate is the compound growth calculation, which
can be determined using the equation below.
Final cell density=Initial density.times.e.sup.kt,
[0212] where k is the growth constant and t is the time in
days.
[0213] The apparent growth rate is the sum of the calculated death
rate added to the growth rate.
Some embodiments of any of the methods described herein further
include culturing a cell (e.g., any of the cells described herein)
in the production bioreactor (e.g., any of the production
bioreactors described herein) using a concentration of the
sensitizer predicted to result in an apparent growth rate of
greater than 1% per day, greater than 1.5% per day, greater than 2%
per day, greater than 2.5% per day, greater than 3% per day,
greater than 3.5% per day, greater than 4.0% per day, greater than
4.5% per day, greater than 5.0% per day, greater than 5.5% per day,
greater than 6.0% per day, greater than 6.5% per day, greater than
7.0% per day, greater than 7.5% per day, greater than 8.0% per day,
greater than 8.5% per day, greater than 9.0% per day, greater than
9.5% per day, greater than 10.0% per day, greater than 10.5% per
day, greater than 11.0% per day, greater than 11.5% per day,
greater than 12.0% per day, greater than 12.5% per day, greater
than 13.0% per day, greater than 13.5% per day, greater than 14.0%
per day, greater than 14.5% per day, greater than 15.0% per day,
greater than 16% per day, greater than 17% per day, greater than
18% per day, greater than 19% per day, greater than 20% per day,
greater than 21% per day, greater than 22% per day, greater than
23% per day, greater than 24% per day, greater than 25% per day,
greater than 30% per day, greater than 35% per day, greater than
40% per day, greater than 45% per day, greater than 50% per day,
greater than 55% per day, greater than 60% per day, greater than
65% per day, greater than 70% per day, greater than 75% per day,
greater than 80% per day, greater than 85% per day, greater than
90% per day, greater than 95% per day, greater than 100% per day,
greater than 105% per day, greater than 110% per day, greater than
115% per day, greater than 120% per day, greater than 125% per day,
greater than 130% per day, greater than 135% per day, greater than
140% per day, greater than 145% per day, or greater than 150% per
day, during the culturing of the cell in the production bioreactor.
Some embodiments of any of the methods described herein further
include culturing a cell (e.g., any of the cells described herein)
in the production bioreactor (e.g., any of the production
bioreactors described herein) using a concentration of the
sensitizer predicted to result in an apparent growth rate of about
0.1% per day to about 150% per day, about 0.1% per day to about
140% per day, about 0.1% per day to about 135% per day, about 0.1%
per day to about 130% per day, about 0.1% per day to about 125% per
day, about 0.1% per day to about 120% per day, about 0.1% per day
to about 115% per day, about 0.1% per day to about 110% per day,
about 0.1% per day to about 105% per day, about 0.1% per day to
about 100% per day, about 0.1% per day to about 95% per day, about
0.1% per day to about 90% per day, about 0.1% per day to about 85%
per day, about 0.1% per day to about 80% per day, about 0.1% per
day to about 75% per day, about 0.1% to about 70% per day, about
0.1% per day to about 65% per day, about 0.1% per day to about 60%
per day, about 0.1% per day to about 55% per day, about 0.1% per
day to about 50% per day, about 0.1% per day to about 45% per day,
about 0.1% per day to about 40% per day, about 0.1% per day to
about 35% per day, about 0.1% per day to about 30% per day, about
0.1% to about 25% per day, about 0.1% per day to about 20% per day,
about 0.1% per day to about 18% per day, about 0.1% per day to
about 16% per day, about 0.1% to about 14% per day, about 0.1% per
day to about 12% per day, about 0.1% per day to about 10% per day,
about 0.1% per day to about 9.0% per day, about 0.1% per day to
about 8.0% per day, about 0.1% per day to about 7.0% per day, about
0.1% per day to about 6.0% per day, about 0.1% per day to about
5.0% per day, about 0.1% per day to about 4.5% per day, about 0.1%
per day to about 4.0% per day, about 0.1% per day to about 3.5% per
day, about 0.1% per day to about 3.0% per day, about 0.1% per day
to about 2.5% per day, about 0.1% per day to about 2.0% per day,
about 0.1% per day to about 1.5% per day, about 0.1% per day to
about 1.0% per day, about 0.1% per day to about 0.5% per day, about
0.5% per day to about 140% per day, about 0.5% per day to about
135% per day, about 0.5% per day to about 130% per day, about 0.5%
per day to about 125% per day, about 0.5% per day to about 120% per
day, about 0.5% per day to about 115% per day, about 0.5% per day
to about 110% per day, about 0.5% per day to about 105% per day,
about 0.5% per day to about 100% per day, about 0.5% per day to
about 95% per day, about 0.5% per day to about 90% per day, about
0.5% per day to about 85% per day, about 0.5% per day to about 80%
per day, about 0.5% per day to about 75% per day, about 0.5% to
about 70% per day, about 0.5% per day to about 65% per day, about
0.5% per day to about 60% per day, about 0.5% per day to about 55%
per day, about 0.5% per day to about 50% per day, about 0.5% per
day to about 45% per day, about 0.5% per day to about 40% per day,
about 0.5% per day to about 35% per day, about 0.5% per day to
about 30% per day, about 0.5% to about 25% per day, about 0.5% per
day to about 20% per day, about 0.5% per day to about 18% per day,
about 0.5% per day to about 16% per day, about 0.5% to about 14%
per day, about 0.5% per day to about 12% per day, about 0.5% per
day to about 10% per day, about 0.5% per day to about 9.0% per day,
about 0.5% per day to about 8.0% per day, about 0.5% per day to
about 7.0% per day, about 0.5% per day to about 6.0% per day, about
0.5% per day to about 5.0% per day, about 0.5% per day to about
4.5% per day, about 0.5% per day to about 4.0% per day, about 0.5%
per day to about 3.5% per day, about 0.5% per day to about 3.0% per
day, about 0.5% per day to about 2.5% per day, about 0.5% per day
to about 2.0% per day, about 0.5% per day to about 1.5% per day,
about 0.5% per day to about 1.0% per day, about 1.0% per day to
about 140% per day, about 1.0% per day to about 135% per day, about
1.0% per day to about 130% per day, about 1.0% per day to about
125% per day, about 1.0% per day to about 120% per day, about 1.0%
per day to about 115% per day, about 1.0% per day to about 110% per
day, about 1.0% per day to about 105% per day, about 1.0% per day
to about 100% per day, about 1.0% per day to about 95% per day,
about 1.0% per day to about 90% per day, about 1.0% per day to
about 85% per day, about 1.0% per day to about 80% per day, about
1.0% per day to about 75% per day, about 1.0% to about 70% per day,
about 1.0% per day to about 65% per day, about 1.0% per day to
about 60% per day, about 1.0% per day to about 55% per day, about
1.0% per day to about 50% per day, about 1.0% per day to about 45%
per day, about 1.0% per day to about 40% per day, about 1.0% per
day to about 35% per day, about 1.0% per day to about 30% per day,
about 1.0% to about 25% per day, about 1.0% per day to about 20%
per day, about 1.0% per day to about 18% per day, about 1.0% per
day to about 16% per day, about 1.0% to about 14% per day, about
1.0% per day to about 12% per day, about 1.0% per day to about 10%
per day, about 1.0% per day to about 9.0% per day, about 1.0% per
day to about 8.0% per day, about 1.0% per day to about 7.0% per
day, about 1.0% per day to about 6.0% per day, about 1.0% per day
to about 5.0% per day, about 1.0% per day to about 4.5% per day,
about 1.0% per day to about 4.0% per day, about 1.0% per day to
about 3.5% per day, about 1.0% per day to about 3.0% per day, about
1.0% per day to about 2.5% per day, about 1.0% per day to about
2.0% per day, about 1.0% per day to about 1.5% per day, about 1.5%
per day to about 140% per day, about 1.5% per day to about 135% per
day, about 1.5% per day to about 130% per day, about 1.5% per day
to about 125% per day, about 1.5% per day to about 120% per day,
about 1.5% per day to about 115% per day, about 1.5% per day to
about 110% per day, about 1.5% per day to about 105% per day, about
1.5% per day to about 100% per day, about 1.5% per day to about 95%
per day, about 1.5% per day to about 90% per day, about 1.5% per
day to about 85% per day, about 1.5% per day to about 80% per day,
about 1.5% per day to about 75% per day, about 1.5% to about 70%
per day, about 1.5% per day to about 65% per day, about 1.5% per
day to about 60% per day, about 1.5% per day to about 55% per day,
about 1.5% per day to about 50% per day, about 1.5% per day to
about 45% per day, about 1.5% per day to about 40% per day, about
1.5% per day to about 35% per day, about 1.5% per day to about 30%
per day, about 1.5% to about 25% per day, about 1.5% per day to
about 20% per day, about 1.5% per day to about 18% per day, about
1.5% per day to about 16% per day, about 1.5% to about 14% per day,
about 1.5% per day to about 12% per day, about 1.5% per day to
about 10% per day, about 1.5% per day to about 9.0% per day, about
1.5% per day to about 8.0% per day, about 1.5% per day to about
7.0% per day, about 1.5% per day to about 6.0% per day, about 1.5%
per day to about 5.0% per day, about 1.5% per day to about 4.5% per
day, about 1.5% per day to about 4.0% per day, about 1.5% per day
to about 3.5% per day, about 1.5% per day to about 3.0% per day,
about 1.5% per day to about 2.5% per day, about 1.5% per day to
about 2.0% per day, about 2.0% per day to about 140% per day, about
2.0% per day to about 135% per day, about 2.0% per day to about
130% per day, about 2.0% per day to about 125% per day, about 2.0%
per day to about 120% per day, about 2.0% per day to about 115% per
day, about 2.0% per day to about 110% per day, about 2.0% per day
to about 105% per day, about 2.0% per day to about 100% per day,
about 2.0% per day to about 95% per day, about 2.0% per day to
about 90% per day, about 2.0% per day to about 85% per day, about
2.0% per day to about 80% per day, about 2.0% per day to about 75%
per day, about 2.0% to about 70% per day, about 2.0% per day to
about 65% per day, about 2.0% per day to about 60% per day, about
2.0% per day to about 55% per day, about 2.0% per day to about 50%
per day, about 2.0% per day to about 45% per day, about 2.0% per
day to about 40% per day, about 2.0% per day to about 35% per day,
about 2.0% per day to about 30% per day, about 2.0% to about 25%
per day, about 2.0% per day to about 20% per day, about 2.0% per
day to about 18% per day, about 2.0% per day to about 16% per day,
about 2.0% to about 14% per day, about 2.0% per day to about 12%
per day, about 2.0% per day to about 10% per day, about 2.0% per
day to about 9.0% per day, about 2.0% per day to about 8.0% per
day, about 2.0% per day to about 7.0% per day, about 2.0% per day
to about 6.0% per day, about 2.0% per day to about 5.0% per day,
about 2.0% per day to about 4.5% per day, about 2.0% per day to
about 4.0% per day, about 2.0% per day to about 3.5% per day, about
2.0% per day to about 3.0% per day, about 2.0% per day to about
2.5% per day, about 2.5% per day to about 140% per day, about 2.5%
per day to about 135% per day, about 2.5% per day to about 130% per
day, about 2.5% per day to about 125% per day, about 2.5% per day
to about 120% per day, about 2.5% per day to about 115% per day,
about 2.5% per day to about 110% per day, about 2.5% per day to
about 105% per day, about 2.5% per day to about 100% per day, about
2.5% per day to about 95% per day, about 2.5% per day to about 90%
per day, about 2.5% per day to about 85% per day, about 2.5% per
day to about 80% per day, about 2.5% per day to about 75% per day,
about 2.5% to about 70% per day, about 2.5% per day to about 65%
per day, about 2.5% per day to about 60% per day, about 2.5% per
day to about 55% per day, about 2.5% per day to about 50% per day,
about 2.5% per day to about 45% per day, about 2.5% per day to
about 40% per day, about 2.5% per day to about 35% per day, about
2.5% per day to about 30% per day, about 2.5% to about 25% per day,
about 2.5% per day to about 20% per day, about 2.5% per day to
about 18% per day, about 2.5% per day to about 16% per day, about
2.5% to about 14% per day, about 2.5% per day to about 12% per day,
about 2.5% per day to about 10% per day, about 2.5% per day to
about 9.0% per day, about 2.5% per day to about 8.0% per day, about
2.5% per day to about 7.0% per day, about 2.5% per day to about
6.0% per day, about 2.5% per day to about 5.0% per day, about 2.5%
per day to about 4.5% per day, about 2.5% per day to about 4.0% per
day, about 2.5% per day to about 3.5% per day, about 2.5% per day
to about 3.0% per day, about 3.0% per day to about 140% per day,
about 3.0% per day to about 135% per day, about 3.0% per day to
about 130% per day, about 3.0% per day to about 125% per day, about
3.0% per day to about 120% per day, about 3.0% per day to about
115% per day, about 3.0% per day to about 110% per day, about 3.0%
per day to about 105% per day, about 3.0% per day to about 100% per
day, about 3.0% per day to about 95% per day, about 3.0% per day to
about 90% per day, about 3.0% per day to about 85% per day, about
3.0% per day to about 80% per day, about 3.0% per day to about 75%
per day, about 3.0% to about 70% per day, about 3.0% per day to
about 65% per day, about 3.0% per day to about 60% per day, about
3.0% per day to about 55% per day, about 3.0% per day to about 50%
per day, about 3.0% per day to about 45% per day, about 3.0% per
day to about 40% per day, about 3.0% per day to about 35% per day,
about 3.0% per day to about 30% per day, about 3.0% to about 25%
per day, about 3.0% per day to about 20% per day, about 3.0% per
day to about 18% per day, about 3.0% per day to about 16% per day,
about 3.0% to about 14% per day, about 3.0% per day to about 12%
per day, about 3.0% per day to about 10% per day, about 3.0% per
day to about 9.0% per day, about 3.0% per day to about 8.0% per
day, about 3.0% per day to about 7.0% per day, about 3.0% per day
to about 6.0% per day, about 3.0% per day to about 5.0% per day,
about 3.0% per day to about 4.5% per day, about 3.0% per day to
about 4.0% per day, about 3.0% per day to about 3.5% per day, about
3.5% per day to about 140% per day, about 3.5% per day to about
135% per day, about 3.5% per day to about 130% per day, about 3.5%
per day to about 125% per day, about 3.5% per day to about 120% per
day, about 3.5% per day to about 115% per day, about 3.5% per day
to about 110% per day, about 3.5% per day to about 105% per day,
about 3.5% per day to about 100% per day, about 3.5% per day to
about 95% per day, about 3.5% per day to about 90% per day, about
3.5% per day to about 85% per day, about 3.5% per day to about 80%
per day, about 3.5% per day to about 75% per day, about 3.5% to
about 70% per day, about 3.5% per day to about 65% per day, about
3.5% per day to about 60% per day, about 3.5% per day to about 55%
per day, about 3.5% per day to about 50% per day, about 3.5% per
day to about 45% per day, about 3.5% per day to about 40% per day,
about 3.5% per day to about 35% per day, about 3.5% per day to
about 30% per day, about 3.5% to about 25% per day, about 3.5% per
day to about 20% per day, about 3.5% per day to about 18% per day,
about 3.5% per day to about 16% per day, about 3.5% to about 14%
per day, about 3.5% per day to about 12% per day, about 3.5% per
day to about 10% per day, about 3.5% per day to about 9.0% per day,
about 3.5% per day to about 8.0% per day, about 3.5% per day to
about 7.0% per day, about 3.5% per day to about 6.0% per day, about
3.5% per day to about 5.0% per day, about 3.5% per day to about
4.5% per day, about 3.5% per day to about 4.0% per day, about 4.0%
per day to about 140% per day, about 4.0% per day to about 135% per
day, about 4.0% per day to about 130% per day, about 4.0% per day
to about 125% per day, about 4.0% per day to about 120% per day,
about 4.0% per day to about 115% per day, about 4.0% per day to
about 110% per day, about 4.0% per day to about 105% per day, about
4.0% per day to about 100% per day, about 4.0% per day to about 95%
per day, about 4.0% per day to about 90% per day, about 4.0% per
day to about 85% per day, about 4.0% per day to about 80% per day,
about 4.0% per day to about 75% per day, about 4.0% to about 70%
per day, about 4.0% per day to about 65% per day, about 4.0% per
day to about 60% per day, about 4.0% per day to about 55% per day,
about 4.0% per day to about 50% per day, about 4.0% per day to
about 45% per day, about 4.0% per day to about 40% per day, about
4.0% per day to about 35% per day, about 4.0% per day to about 30%
per day, about 4.0% to about 25% per day, about 4.0% per day to
about 20% per day, about 4.0% per day to about 18% per day, about
4.0% per day to about 16% per day, about 4.0% to about 14% per day,
about 4.0% per day to about 12% per day, about 4.0% per day to
about 10% per day, about 4.0% per day to about 9.0% per day, about
4.0% per day to about 8.0% per day, about 4.0% per day to about
7.0% per day, about 4.0% per day to about 6.0% per day, about 4.0%
per day to about 5.0% per day, about 4.0% per day to about 4.5% per
day, about 4.5% per day to about 140% per day, about 4.5% per day
to about 135% per day, about 4.5% per day to about 130% per day,
about 4.5% per day to about 125% per day, about 4.5% per day to
about 120% per day, about 4.5% per day to about 115% per day, about
4.5% per day to about 110% per day, about 4.5% per day to about
105% per day, about 4.5% per day to about 100% per day, about 4.5%
per day to about 95% per day, about 4.5% per day to about 90% per
day, about 4.5% per day to about 85% per day, about 4.5% per day to
about 80% per day, about 4.5% per day to about 75% per day, about
4.5% to about 70% per day, about 4.5% per day to about 65% per day,
about 4.5% per day to about 60% per day, about 4.5% per day to
about 55% per day, about 4.5% per day to about 50% per day, about
4.5% per day to about 45% per day, about 4.5%
per day to about 40% per day, about 4.5% per day to about 35% per
day, about 4.5% per day to about 30% per day, about 4.5% to about
25% per day, about 4.5% per day to about 20% per day, about 4.5%
per day to about 18% per day, about 4.5% per day to about 16% per
day, about 4.5% to about 14% per day, about 4.5% per day to about
12% per day, about 4.5% per day to about 10% per day, about 4.5%
per day to about 9.0% per day, about 4.5% per day to about 8.0% per
day, about 4.5% per day to about 7.0% per day, about 4.5% per day
to about 6.0% per day, about 4.5% per day to about 5.0% per day,
about 5.0% per day to about 140% per day, about 5.0% per day to
about 135% per day, about 5.0% per day to about 130% per day, about
5.0% per day to about 125% per day, about 5.0% per day to about
120% per day, about 5.0% per day to about 115% per day, about 5.0%
per day to about 110% per day, about 5.0% per day to about 105% per
day, about 5.0% per day to about 100% per day, about 5.0% per day
to about 95% per day, about 5.0% per day to about 90% per day,
about 5.0% per day to about 85% per day, about 5.0% per day to
about 80% per day, about 5.0% per day to about 75% per day, about
5.0% to about 70% per day, about 5.0% per day to about 65% per day,
about 5.0% per day to about 60% per day, about 5.0% per day to
about 55% per day, about 5.0% per day to about 50% per day, about
5.0% per day to about 45% per day, about 5.0% per day to about 40%
per day, about 5.0% per day to about 35% per day, about 5.0% per
day to about 30% per day, about 5.0% to about 25% per day, about
5.0% per day to about 20% per day, about 5.0% per day to about 18%
per day, about 5.0% per day to about 16% per day, about 5.0% to
about 14% per day, about 5.0% per day to about 12% per day, about
5.0% per day to about 10% per day, about 5.0% per day to about 9.0%
per day, about 5.0% per day to about 8.0% per day, about 5.0% per
day to about 7.0% per day, about 5.0% per day to about 6.0% per
day, about 6.0% per day to about 140% per day, about 6.0% per day
to about 135% per day, about 6.0% per day to about 130% per day,
about 6.0% per day to about 125% per day, about 6.0% per day to
about 120% per day, about 6.0% per day to about 115% per day, about
6.0% per day to about 110% per day, about 6.0% per day to about
105% per day, about 6.0% per day to about 100% per day, about 6.0%
per day to about 95% per day, about 6.0% per day to about 90% per
day, about 6.0% per day to about 85% per day, about 6.0% per day to
about 80% per day, about 6.0% per day to about 75% per day, about
6.0% to about 70% per day, about 6.0% per day to about 65% per day,
about 6.0% per day to about 60% per day, about 6.0% per day to
about 55% per day, about 6.0% per day to about 50% per day, about
6.0% per day to about 45% per day, about 6.0% per day to about 40%
per day, about 6.0% per day to about 35% per day, about 6.0% per
day to about 30% per day, about 6.0% to about 25% per day, about
6.0% per day to about 20% per day, about 6.0% per day to about 18%
per day, about 6.0% per day to about 16% per day, about 6.0% to
about 14% per day, about 6.0% per day to about 12% per day, about
6.0% per day to about 10% per day, about 6.0% per day to about 9.0%
per day, about 6.0% per day to about 8.0% per day, about 6.0% per
day to about 7.0% per day, about 7.0% per day to about 140% per
day, about 7.0% per day to about 135% per day, about 7.0% per day
to about 130% per day, about 7.0% per day to about 125% per day,
about 7.0% per day to about 120% per day, about 7.0% per day to
about 115% per day, about 7.0% per day to about 110% per day, about
7.0% per day to about 105% per day, about 7.0% per day to about
100% per day, about 7.0% per day to about 95% per day, about 7.0%
per day to about 90% per day, about 7.0% per day to about 85% per
day, about 7.0% per day to about 80% per day, about 7.0% per day to
about 75% per day, about 7.0% to about 70% per day, about 7.0% per
day to about 65% per day, about 7.0% per day to about 60% per day,
about 7.0% per day to about 55% per day, about 7.0% per day to
about 50% per day, about 7.0% per day to about 45% per day, about
7.0% per day to about 40% per day, about 7.0% per day to about 35%
per day, about 7.0% per day to about 30% per day, about 7.0% to
about 25% per day, about 7.0% per day to about 20% per day, about
7.0% per day to about 18% per day, about 7.0% per day to about 16%
per day, about 7.0% to about 14% per day, about 7.0% per day to
about 12% per day, about 7.0% per day to about 10% per day, about
7.0% per day to about 9.0% per day, about 7.0% per day to about
8.0% per day, about 8.0% per day to about 140% per day, about 8.0%
per day to about 135% per day, about 8.0% per day to about 130% per
day, about 8.0% per day to about 125% per day, about 8.0% per day
to about 120% per day, about 8.0% per day to about 115% per day,
about 8.0% per day to about 110% per day, about 8.0% per day to
about 105% per day, about 8.0% per day to about 100% per day, about
8.0% per day to about 95% per day, about 8.0% per day to about 90%
per day, about 8.0% per day to about 85% per day, about 8.0% per
day to about 80% per day, about 8.0% per day to about 75% per day,
about 8.0% to about 70% per day, about 8.0% per day to about 65%
per day, about 8.0% per day to about 60% per day, about 8.0% per
day to about 55% per day, about 8.0% per day to about 50% per day,
about 8.0% per day to about 45% per day, about 8.0% per day to
about 40% per day, about 8.0% per day to about 35% per day, about
8.0% per day to about 30% per day, about 8.0% to about 25% per day,
about 8.0% per day to about 20% per day, about 8.0% per day to
about 18% per day, about 8.0% per day to about 16% per day, about
8.0% to about 14% per day, about 8.0% per day to about 12% per day,
about 8.0% per day to about 10% per day, about 8.0% per day to
about 9.0% per day, about 9.0% per day to about 140% per day, about
9.0% per day to about 135% per day, about 9.0% per day to about
130% per day, about 9.0% per day to about 125% per day, about 9.0%
per day to about 120% per day, about 9.0% per day to about 115% per
day, about 9.0% per day to about 110% per day, about 9.0% per day
to about 105% per day, about 9.0% per day to about 100% per day,
about 9.0% per day to about 95% per day, about 9.0% per day to
about 90% per day, about 9.0% per day to about 85% per day, about
9.0% per day to about 80% per day, about 9.0% per day to about 75%
per day, about 9.0% to about 70% per day, about 9.0% per day to
about 65% per day, about 9.0% per day to about 60% per day, about
9.0% per day to about 55% per day, about 9.0% per day to about 50%
per day, about 9.0% per day to about 45% per day, about 9.0% per
day to about 40% per day, about 9.0% per day to about 35% per day,
about 9.0% per day to about 30% per day, about 9.0% to about 25%
per day, about 9.0% per day to about 20% per day, about 9.0% per
day to about 18% per day, about 9.0% per day to about 16% per day,
about 9.0% to about 14% per day, about 9.0% per day to about 12%
per day, about 9.0% per day to about 10% per day, about 10% per day
to about 140% per day, about 10% per day to about 135% per day,
about 10% per day to about 130% per day, about 10% per day to about
125% per day, about 10% per day to about 120% per day, about 10%
per day to about 115% per day, about 10% per day to about 110% per
day, about 10% per day to about 105% per day, about 10% per day to
about 100% per day, about 10% per day to about 95% per day, about
10% per day to about 90% per day, about 10% per day to about 85%
per day, about 10% per day to about 80% per day, about 10% per day
to about 75% per day, about 10% to about 70% per day, about 10% per
day to about 65% per day, about 10% per day to about 60% per day,
about 10% per day to about 55% per day, about 10% per day to about
50% per day, about 10% per day to about 45% per day, about 10% per
day to about 40% per day, about 10% per day to about 35% per day,
about 10% per day to about 30% per day, about 10% to about 25% per
day, about 10% per day to about 20% per day, about 10% per day to
about 18% per day, about 10% per day to about 16% per day, about
10% to about 14% per day, about 10% per day to about 12% per day,
about 12% per day to about 140% per day, about 12% per day to about
135% per day, about 12% per day to about 130% per day, about 12%
per day to about 125% per day, about 12% per day to about 120% per
day, about 12% per day to about 115% per day, about 12% per day to
about 110% per day, about 12% per day to about 105% per day, about
12% per day to about 100% per day, about 12% per day to about 95%
per day, about 12% per day to about 90% per day, about 12% per day
to about 85% per day, about 12% per day to about 80% per day, about
12% per day to about 75% per day, about 12% to about 70% per day,
about 12% per day to about 65% per day, about 12% per day to about
60% per day, about 12% per day to about 55% per day, about 12% per
day to about 50% per day, about 12% per day to about 45% per day,
about 12% per day to about 40% per day, about 12% per day to about
35% per day, about 12% per day to about 30% per day, about 12% to
about 25% per day, about 12% per day to about 20% per day, about
12% per day to about 18% per day, about 12% per day to about 16%
per day, about 12% to about 14% per day, about 14% per day to about
140% per day, about 14% per day to about 135% per day, about 14%
per day to about 130% per day, about 14% per day to about 125% per
day, about 14% per day to about 120% per day, about 14% per day to
about 115% per day, about 14% per day to about 110% per day, about
14% per day to about 105% per day, about 14% per day to about 100%
per day, about 14% per day to about 95% per day, about 14% per day
to about 90% per day, about 14% per day to about 85% per day, about
14% per day to about 80% per day, about 14% per day to about 75%
per day, about 14% to about 70% per day, about 14% per day to about
65% per day, about 14% per day to about 60% per day, about 14% per
day to about 55% per day, about 14% per day to about 50% per day,
about 14% per day to about 45% per day, about 14% per day to about
40% per day, about 14% per day to about 35% per day, about 14% per
day to about 30% per day, about 14% to about 25% per day, about 14%
per day to about 20% per day, about 14% per day to about 18% per
day, about 14% per day to about 16% per day, about 16% per day to
about 140% per day, about 16% per day to about 135% per day, about
16% per day to about 130% per day, about 16% per day to about 125%
per day, about 16% per day to about 120% per day, about 16% per day
to about 115% per day, about 16% per day to about 110% per day,
about 16% per day to about 105% per day, about 16% per day to about
100% per day, about 16% per day to about 95% per day, about 16% per
day to about 90% per day, about 16% per day to about 85% per day,
about 16% per day to about 80% per day, about 16% per day to about
75% per day, about 16% to about 70% per day, about 16% per day to
about 65% per day, about 16% per day to about 60% per day, about
16% per day to about 55% per day, about 16% per day to about 50%
per day, about 16% per day to about 45% per day, about 16% per day
to about 40% per day, about 16% per day to about 35% per day, about
16% per day to about 30% per day, about 16% to about 25% per day,
about 16% per day to about 20% per day, about 16% per day to about
18% per day, about 18% per day to about 140% per day, about 18% per
day to about 135% per day, about 18% per day to about 130% per day,
about 18% per day to about 125% per day, about 18% per day to about
120% per day, about 18% per day to about 115% per day, about 18%
per day to about 110% per day, about 18% per day to about 105% per
day, about 18% per day to about 100% per day, about 18% per day to
about 95% per day, about 18% per day to about 90% per day, about
18% per day to about 85% per day, about 18% per day to about 80%
per day, about 18% per day to about 75% per day, about 18% to about
70% per day, about 18% per day to about 65% per day, about 18% per
day to about 60% per day, about 18% per day to about 55% per day,
about 18% per day to about 50% per day, about 18% per day to about
45% per day, about 18% per day to about 40% per day, about 18% per
day to about 35% per day, about 18% per day to about 30% per day,
about 18% to about 25% per day, about 18% per day to about 20% per
day, about 20% per day to about 140% per day, about 20% per day to
about 135% per day, about 20% per day to about 130% per day, about
20% per day to about 125% per day, about 20% per day to about 120%
per day, about 20% per day to about 115% per day, about 20% per day
to about 110% per day, about 20% per day to about 105% per day,
about 20% per day to about 100% per day, about 20% per day to about
95% per day, about 20% per day to about 90% per day, about 20% per
day to about 85% per day, about 20% per day to about 80% per day,
about 20% per day to about 75% per day, about 20% to about 70% per
day, about 20% per day to about 65% per day, about 20% per day to
about 60% per day, about 20% per day to about 55% per day, about
20% per day to about 50% per day, about 20% per day to about 45%
per day, about 20% per day to about 40% per day, about 20% per day
to about 35% per day, about 20% per day to about 30% per day, about
20% to about 25% per day, about 25% per day to about 140% per day,
about 25% per day to about 135% per day, about 25% per day to about
130% per day, about 25% per day to about 125% per day, about 25%
per day to about 120% per day, about 25% per day to about 115% per
day, about 25% per day to about 110% per day, about 25% per day to
about 105% per day, about 25% per day to about 100% per day, about
25% per day to about 95% per day, about 25% per day to about 90%
per day, about 25% per day to about 85% per day, about 25% per day
to about 80% per day, about 25% per day to about 75% per day, about
25% to about 70% per day, about 25% per day to about 65% per day,
about 25% per day to about 60% per day, about 25% per day to about
55% per day, about 25% per day to about 50% per day, about 25% per
day to about 45% per day, about 25% per day to about 40% per day,
about 25% per day to about 35% per day, about 25% per day to about
30% per day, about 30% per day to about 140% per day, about 30% per
day to about 135% per day, about 30% per day to about 130% per day,
about 30% per day to about 125% per day, about 30% per day to about
120% per day, about 30% per day to about 115% per day, about 30%
per day to about 110% per day, about 30% per day to about 105% per
day, about 30% per day to about 100% per day, about 30% per day to
about 95% per day, about 30% per day to about 90% per day, about
30% per day to about 85% per day, about 30% per day to about 80%
per day, about 30% per day to about 75% per day, about 30% to about
70% per day, about 30% per day to about 65% per day, about 30% per
day to about 60% per day, about 30% per day to about 55% per day,
about 30% per day to about 50% per day, about 30% per day to about
45% per day, about 30% per day to about 40% per day, about 30% per
day to about 35% per day, about 35% per day to about 140% per day,
about 35% per day to about 135% per day, about 35% per day to about
130% per day, about 35% per day to about 125% per day, about 35%
per day to about 120% per day, about 35% per day to about 115% per
day, about 35% per day to about 110% per day, about 35% per day to
about 105% per day, about 35% per day to about 100% per day, about
35% per day to about 95% per day, about 35% per day to about 90%
per day, about 35% per day to about 85% per day, about 35% per day
to about 80% per day, about 35% per day to about 75% per day, about
35% to about 70% per day, about 35% per day to about 65% per day,
about 35% per day to about 60% per day, about 35% per day to about
55% per day, about 35% per day to about 50% per day, about 35% per
day to about 45% per day, about 35% per day to about 40% per day,
about 40% per day to about 140% per day, about 40% per day to about
135% per day, about 40% per day to about 130% per day, about 40%
per day to about 125% per day, about 40% per day to about 120% per
day, about 40% per day to about 115% per day, about 40% per day to
about 110% per day, about 40% per day to about 105% per day, about
40% per day to about 100% per day, about 40% per day to about 95%
per day, about 40% per day to about 90% per day, about 40% per day
to about 85% per day, about 40% per day to about 80% per day, about
40% per day to about 75% per day, about 40% to about 70% per day,
about 40% per day to about 65% per day, about 40% per day to about
60% per day, about 40% per day to about 55% per day, about 40% per
day to about 50% per day, about 40% per day to about 45% per day,
about 45% per day to about 140% per day, about 45% per day to about
135% per day, about 45% per day to about 130% per day, about 45%
per day to about 125% per day, about 45% per day to about 120% per
day, about 45% per day to about 115% per day, about 45% per day to
about 110% per day, about 45% per day to about 105% per day, about
45% per day to about 100% per day, about 45% per day to about 95%
per day, about 45% per day to about 90% per day, about 45% per day
to about 85% per day, about 45% per day to about 80% per day, about
45% per day to about 75% per day, about 45% to about 70% per day,
about 45% per day to about 65% per day, about 45% per day to about
60% per day, about 45% per day to about 55% per day, about 45% per
day to about 50% per day, about 50% per day to about 140% per day,
about 50% per day to about 135% per day, about 50% per day to about
130% per day, about 50% per day to about 125% per day, about 50%
per day to about 120% per day, about 50% per day to about 115% per
day, about 50% per day to about 110% per day, about 50% per day to
about 105% per day, about 50% per day to about 100% per day, about
50% per day to about 95% per day, about 50% per day to about 90%
per day, about 50% per day to about 85% per day, about
50% per day to about 80% per day, about 50% per day to about 75%
per day, about 50% to about 70% per day, about 50% per day to about
65% per day, about 50% per day to about 60% per day, about 50% per
day to about 55% per day, about 55% per day to about 140% per day,
about 55% per day to about 135% per day, about 55% per day to about
130% per day, about 55% per day to about 125% per day, about 55%
per day to about 120% per day, about 55% per day to about 115% per
day, about 55% per day to about 110% per day, about 55% per day to
about 105% per day, about 55% per day to about 100% per day, about
55% per day to about 95% per day, about 55% per day to about 90%
per day, about 55% per day to about 85% per day, about 55% per day
to about 80% per day, about 55% per day to about 75% per day, about
55% to about 70% per day, about 55% per day to about 65% per day,
about 55% per day to about 60% per day, about 60% per day to about
140% per day, about 60% per day to about 135% per day, about 60%
per day to about 130% per day, about 60% per day to about 125% per
day, about 60% per day to about 120% per day, about 60% per day to
about 115% per day, about 650% per day to about 110% per day, about
60% per day to about 105% per day, about 60% per day to about 100%
per day, about 60% per day to about 95% per day, about 60% per day
to about 90% per day, about 60% per day to about 85% per day, about
60% per day to about 80% per day, about 60% per day to about 75%
per day, about 60% to about 70% per day, about 60% per day to about
65% per day, about 65% per day to about 140% per day, about 65% per
day to about 135% per day, about 65% per day to about 130% per day,
about 65% per day to about 125% per day, about 65% per day to about
120% per day, about 65% per day to about 115% per day, about 65%
per day to about 110% per day, about 65% per day to about 105% per
day, about 65% per day to about 100% per day, about 65% per day to
about 95% per day, about 65% per day to about 90% per day, about
65% per day to about 85% per day, about 65% per day to about 80%
per day, about 65% per day to about 75% per day, about 65% to about
70% per day, about 70% per day to about 140% per day, about 70% per
day to about 135% per day, about 70% per day to about 130% per day,
about 70% per day to about 125% per day, about 70% per day to about
120% per day, about 70% per day to about 115% per day, about 70%
per day to about 110% per day, about 70% per day to about 105% per
day, about 70% per day to about 100% per day, about 70% per day to
about 95% per day, about 70% per day to about 90% per day, about
70% per day to about 85% per day, about 70% per day to about 80%
per day, about 70% per day to about 75% per day, about 75% per day
to about 140% per day, about 75% per day to about 135% per day,
about 75% per day to about 130% per day, about 75% per day to about
125% per day, about 75% per day to about 120% per day, about 75%
per day to about 115% per day, about 75% per day to about 110% per
day, about 75% per day to about 105% per day, about 75% per day to
about 100% per day, about 75% per day to about 95% per day, about
75% per day to about 90% per day, about 75% per day to about 85%
per day, about 75% per day to about 80% per day, about 80% per day
to about 140% per day, about 80% per day to about 135% per day,
about 80% per day to about 130% per day, about 80% per day to about
125% per day, about 80% per day to about 120% per day, about 80%
per day to about 115% per day, about 80% per day to about 110% per
day, about 80% per day to about 105% per day, about 80% per day to
about 100% per day, about 80% per day to about 95% per day, about
80% per day to about 90% per day, about 80% per day to about 85%
per day, about 85% per day to about 140% per day, about 85% per day
to about 135% per day, about 85% per day to about 130% per day,
about 85% per day to about 125% per day, about 85% per day to about
120% per day, about 85% per day to about 115% per day, about 85%
per day to about 110% per day, about 85% per day to about 105% per
day, about 85% per day to about 100% per day, about 85% per day to
about 95% per day, about 85% per day to about 90% per day, about
90% per day to about 140% per day, about 90% per day to about 135%
per day, about 90% per day to about 130% per day, about 90% per day
to about 125% per day, about 90% per day to about 120% per day,
about 90% per day to about 115% per day, about 90% per day to about
110% per day, about 90% per day to about 105% per day, about 90%
per day to about 100% per day, about 90% per day to about 95% per
day, about 95% per day to about 140% per day, about 95% per day to
about 135% per day, about 95% per day to about 130% per day, about
95% per day to about 125% per day, about 95% per day to about 120%
per day, about 95% per day to about 115% per day, about 95% per day
to about 110% per day, about 95% per day to about 105% per day,
about 95% per day to about 100% per day, about 100% per day to
about 140% per day, about 100% per day to about 135% per day, about
100% per day to about 130% per day, about 100% per day to about
125% per day, about 100% per day to about 120% per day, about 100%
per day to about 115% per day, about 100% per day to about 110% per
day, about 100% per day to about 105% per day, about 105% per day
to about 140% per day, about 105% per day to about 135% per day,
about 105% per day to about 130% per day, about 105% per day to
about 125% per day, about 105% per day to about 120% per day, about
105% per day to about 115% per day, about 105% per day to about
110% per day, about 110% per day to about 140% per day, about 110%
per day to about 135% per day, about 110% per day to about 130% per
day, about 110% per day to about 125% per day, about 110% per day
to about 120% per day, about 110% per day to about 115% per day,
about 115% per day to about 140% per day, about 115% per day to
about 135% per day, about 115% per day to about 130% per day, about
115% per day to about 125% per day, about 115% per day to about
120% per day, about 120% per day to about 140% per day, about 120%
per day to about 135% per day, about 120% per day to about 130% per
day, about 120% per day to about 125% per day, about 125% per day
to about 140% per day, about 125% per day to about 135% per day,
about 125% per day to about 130% per day, about 130% per day to
about 140% per day, about 130% per day to about 135% per day, or
about 135% per day to about 140% per day, during the culturing of
the cell in the production bioreactor.
[0215] In some embodiments of these methods, the cell contains a
nucleic acid encoding a recombinant therapeutic protein. Some
embodiments of these methods further include collecting or
recovering the recombinant therapeutic protein. Some embodiments of
these methods further include purifying the recombinant therapeutic
protein. Some embodiments of these methods further include
formulating the purified recombinant therapeutic protein.
[0216] In some embodiments of these methods, the cell is a
bacterium (e.g., any of the exemplary bacteria described herein or
known in the art), a yeast (e.g., any of the exemplary yeast cells
described herein or known in the art), or a mammalian cell (e.g.,
any of the exemplary mammalian cells described herein or known in
the art).
[0217] In some embodiments of these methods, the cell viability is
determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel.
[0218] In some embodiments, the test culture vessel is a baffled
shake flask (e.g., any of the baffled shake flasks described herein
or known in the art) and the plurality of test conditions include
one or more of: (a) a rotary agitation of about 125 RPM to about
400 RPM (or any of the subranges of this range described herein);
(b) a concentration of the sensitizer (e.g., any of the sensitizers
described herein or known in the art) of about 0 ppm to about 120
ppm (or any of the subranges of this range described herein); and
(c) a concentration of a protectant (e.g., any of the protectants
described herein or known in the art) that is about 1 g/L to about
10 g/L (or any of the subranges of this range described herein). In
some embodiments, the protectant is a poloxamer (e.g.,
poloxamer-188, poloxamer-401, poloxamer-402, or poloxamer-407), a
poloxamine (e.g., poloxamine-904 or poloxamine-908), or a non-ionic
surfactant Pluronic.RTM..
Methods of Improving Cell Viability in a Production Bioreactor
[0219] Also provided herein are methods of improving cell viability
in a production bioreactor (e.g., any of the production bioreactors
described herein) that include: (a) determining cell viability
under a plurality of test conditions in a test culture vessel,
where the plurality of conditions are defined by a set of
parameters; (b) based on the cell viability in the test culture
vessel under the plurality of conditions, selecting a condition
that is optimal for cell viability in a production bioreactor; and
(c) culturing a cell in the production bioreactor under the
selected condition.
[0220] In some embodiments of these methods, the set of parameter
comprises, consists essentially of, or consists of shear stress,
concentration of a sensitizer (e.g., any of the sensitizers
described herein or known in the art), and concentration of a
protectant (e.g., any of the protectants described herein or known
in the art). In some embodiments of these methods, the plurality of
testing conditions are selected based on full factorial screening
and/or inscribed center composite design. In some embodiments of
these methods, the sensitizer is an antifoam (e.g., a
polydimethylsiloxane-based antifoam, simethicone, or any other
antifoam described herein or known in the art).
[0221] In some embodiments of these methods, the test culture
vessel (e.g., any of the test culture vessels described herein or
known in the art) has a volume of about 5 mL to about 5 L (or any
of the subranges of this range described herein). In some
embodiments of these methods, the test culture vessel is a shake
flask, e.g., a baffled shake flask (e.g., any of the baffled shake
flasks described herein or known in the art).
[0222] In some embodiments of these methods, the production
bioreactor (e.g., any of the production bioreactors described
herein) has a volume of about 5 L to about 20,000 L (or any of the
subranges of this range described herein). In some examples, the
production bioreactor is a perfusion bioreactor. In other examples,
the production bioreactor is a fed batch bioreactor.
[0223] In some embodiments, step (c) results in a specific cell
death rate of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,
24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,
37%, 38%, 39%, or 40% per day, during the culturing of the cells in
the production bioreactor. In some embodiments, step (c) results in
a specific cell death rate of about 0.1% to about 40% per day (or
any of the subranges of this range described herein) during the
culturing of the cells in the production bioreactor.
[0224] In some embodiments, step (c) results in a cell specific LDH
production of less than 150 nU/cell/day, less than 145 nU/cell/day,
less than 140 nU/cell/day, less than 135 nU/cell/day, less than 130
nU/cell/day, less than 125 nU/cell/day, less than 120 nU/cell/day,
less than 115 nU/cell/day, less than 150 nU/cell/day, less than 110
nU/cell/day, less than 105 nU/cell/day, less than 100 nU/cell/day,
less than 95 nU/cell/day, less than 90 nU/cell/day, less than 85
nU/cell/day, less than 80 nU/cell/day, less than 75 nU/cell/day,
less than 70 nU/cell/day, less than 65 nU/cell/day, less than 60
nU/cell/day, less than 55 nU/cell/day, less than 50 nU/cell/day,
less than 45 nU/cell/day, less than 45 nU/cell/day, less than 40
nU/cell/day, less than 35 nU/cell/day, less than 30 nU/cell/day,
less than 25 nU/cell/day, less than 20 nU/cell/day, less than 15
nU/cell/day, less than 10 nU/cell/day, less than 5 nU/cell/day, or
less than less than 1 nU/cell/day, during the culturing of the cell
in the production bioreactor. In some embodiments of these methods,
step (c) results in a cell specific LDH production of about 0.1
nU/cell/day to about 150 nU/cell/day (or any of the subranges of
this range described herein) during the culturing of the cell in
the production bioreactor.
[0225] In some embodiments, step (c) results in an apparent growth
rate of greater than 1% per day, greater than 1.5% per day, greater
than 2% per day, greater than 2.5% per day, greater than 3% per
day, greater than 3.5% per day, greater than 4.0% per day, greater
than 4.5% per day, greater than 5.0% per day, greater than 5.5% per
day, greater than 6.0% per day, greater than 6.5% per day, greater
than 7.0% per day, greater than 7.5% per day, greater than 8.0% per
day, greater than 8.5% per day, greater than 9.0% per day, greater
than 9.5% per day, greater than 10.0% per day, greater than 10.5%
per day, greater than 11.0% per day, greater than 11.5% per day,
greater than 12.0% per day, greater than 12.5% per day, greater
than 13.0% per day, greater than 13.5% per day, greater than 14.0%
per day, greater than 14.5% per day, greater than 15.0% per day,
greater than 16% per day, greater than 17% per day, greater than
18% per day, greater than 19% per day, greater than 20% per day,
greater than 21% per day, greater than 22% per day, greater than
23% per day, greater than 24% per day, greater than 25% per day,
greater than 30% per day, greater than 35% per day, greater than
40% per day, greater than 45% per day, greater than 50% per day,
greater than 55% per day, greater than 60% per day, greater than
65% per day, greater than 70% per day, greater than 75% per day,
greater than 80% per day, greater than 85% per day, greater than
90% per day, greater than 95% per day, greater than 100% per day,
greater than 105% per day, greater than 110% per day, greater than
115% per day, greater than 120% per day, greater than 125% per day,
greater than 130% per day, greater than 135% per day, greater than
140% per day, greater than 145% per day, or greater than 150% per
day, during the culturing of the cell in the production bioreactor.
In some embodiments of these methods, step (c) results in an
apparent growth rate of about 0.1% per day to about 150% per day
(or any of the subranges of this range described herein) during the
culturing of the cell in the production bioreactor.
[0226] In some embodiments of these methods, the cell (e.g., any of
the cells described herein) contains a nucleic acid encoding a
recombinant therapeutic protein (e.g., any of the recombinant
therapeutic proteins described herein). Some embodiments of these
methods further include collecting or recovering the recombinant
therapeutic protein. Some embodiments of these methods further
include purifying the recombinant therapeutic protein. Some
embodiments of these methods further include formulating the
purified recombinant therapeutic protein. In some embodiments of
these methods, the cell is a bacterium (e.g., any of the exemplary
bacteria described herein or known in the art), a yeast cell (e.g.,
any of the exemplary yeast cells described herein or known in the
art), or a mammalian cell (e.g., any of the exemplary mammalian
cells described herein or known in the art).
[0227] In some embodiments of these methods, the cell viability is
determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel. In some embodiments
of these methods, the test culture vessel is a baffled shake flask
and the plurality of test conditions include (or consist) of one or
more (e.g., two or three) of: (a) a rotary agitation of about 125
RPM to about 400 RPM (or any of the subranges of this range
described herein); (b) a concentration of a sensitizer (e.g., any
of the sensitizers described herein or known in the art) of about 0
ppm to about 120 ppm (or any of the subranges of this range
described herein); and (c) a concentration of a protectant (e.g.,
any of the protectants described herein or known in the art) that
is about 1 g/L to about 10 g/L (or any of the subranges of this
range described herein). In some embodiments of these methods, the
protectant is a poloxamer (e.g., poloxamer-188, poloxamer-401,
poloxamer-402, or poloxamer-407), a poloxamine (e.g.,
poloxamine-904 or poloxamine-908), or a non-ionic surfactant
Pluronic.RTM..
Methods for Predicting Cell Viability in a Production
Bioreactor
[0228] Also provided herein are methods of predicting cell
viability in a production bioreactor (e.g., any of the production
bioreactors described herein) that include: (a) selecting a set of
parameters comprising at least two parameters (e.g., 2, 3, 4, 5, or
6); (b) determining cell viability under a plurality of test
conditions as defined by the set of parameters in a test culture
vessel (e.g., any of the text culture vessels described herein);
and (c) based on the cell viability in the test culture vessel
under the plurality of conditions, predicting cell viability in the
production bioreactor.
[0229] In some embodiments of these methods, the set of parameters
comprises, consists essentially of, or consists of shear stress,
concentration of a sensitizer (e.g., any of the exemplary
sensitizers described herein or known in the art), and
concentration of a protectant (e.g., any of the protectants
described herein or known in the art). In some embodiments of these
methods, the plurality of test conditions are selected based on
full factorial screening and/or inscribed center composite
design.
[0230] In some embodiments of these methods, the sensitizer is an
antifoam (e.g., a polydimethylsiloxane-based antifoam, simethicone,
or any other antifoam described herein or known in the art).
[0231] In some embodiments of these methods, the test culture
vessel (e.g., any of the test culture vessels described herein or
known in the art) has a volume of about 5 mL to about 5 L (or any
of the subranges of this range described herein). In some
embodiments of these methods, the test culture vessel is a shake
flask, e.g., a baffled shake flask (e.g., any of the baffled shake
flasks described herein or known in the art).
[0232] In some embodiments of these methods, the production
bioreactor (e.g., any of the production bioreactors described
herein) has a volume of about 5 L to about 20,000 L (or any of the
subranges of this range described herein). In some examples, the
production bioreactor is a perfusion bioreactor. In other examples,
the production bioreactor is a fed batch bioreactor.
[0233] Some embodiments of these methods further include culturing
a cell in the production bioreactor using a condition predicted to
result in a specific cell death rate of less than 1%, 2%, 3%, 4%,
5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,
19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,
32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% per day, during the
culturing of the cells in the production bioreactor. Some
embodiments of these methods further include culturing a cell in
the production bioreactor using a condition predicted to result in
a specific cell death rate of about 0.1% to about 40% per day (or
any of the subranges of this range described herein) during the
culturing of the cells in the production bioreactor.
[0234] Some embodiments of these methods further include culturing
a cell in the production bioreactor using a condition predicted to
result in a cell specific LDH production of less than 150
nU/cell/day, less than 145 nU/cell/day, less than 140 nU/cell/day,
less than 135 nU/cell/day, less than 130 nU/cell/day, less than 125
nU/cell/day, less than 120 nU/cell/day, less than 115 nU/cell/day,
less than 150 nU/cell/day, less than 110 nU/cell/day, less than 105
nU/cell/day, less than 100 nU/cell/day, less than 95 nU/cell/day,
less than 90 nU/cell/day, less than 85 nU/cell/day, less than 80
nU/cell/day, less than 75 nU/cell/day, less than 70 nU/cell/day,
less than 65 nU/cell/day, less than 60 nU/cell/day, less than 55
nU/cell/day, less than 50 nU/cell/day, less than 45 nU/cell/day,
less than 45 nU/cell/day, less than 40 nU/cell/day, less than 35
nU/cell/day, less than 30 nU/cell/day, less than 25 nU/cell/day,
less than 20 nU/cell/day, less than 15 nU/cell/day, less than 10
nU/cell/day, less than 5 nU/cell/day, or less than less than 1
nU/cell/day, during the culturing of the cell in the production
bioreactor. Some embodiments of these methods further include
culturing a cell in the production bioreactor using a condition
predicted to result in a cell specific LDH production of about 0.1
nU/cell/day to about 150 nU/cell/day (or any of the subranges of
this range described herein) during the culturing of the cell in
the production bioreactor.
[0235] Some embodiments of these methods further include culturing
a cell in the production bioreactor using a condition predicted to
result in an apparent growth rate of greater than 1% per day,
greater than 1.5% per day, greater than 2% per day, greater than
2.5% per day, greater than 3% per day, greater than 3.5% per day,
greater than 4.0% per day, greater than 4.5% per day, greater than
5.0% per day, greater than 5.5% per day, greater than 6.0% per day,
greater than 6.5% per day, greater than 7.0% per day, greater than
7.5% per day, greater than 8.0% per day, greater than 8.5% per day,
greater than 9.0% per day, greater than 9.5% per day, greater than
10.0% per day, greater than 10.5% per day, greater than 11.0% per
day, greater than 11.5% per day, greater than 12.0% per day,
greater than 12.5% per day, greater than 13.0% per day, greater
than 13.5% per day, greater than 14.0% per day, greater than 14.5%
per day, greater than 15.0% per day, greater than 16% per day,
greater than 17% per day, greater than 18% per day, greater than
19% per day, greater than 20% per day, greater than 21% per day,
greater than 22% per day, greater than 23% per day, greater than
24% per day, greater than 25% per day, greater than 30% per day,
greater than 35% per day, greater than 40% per day, greater than
45% per day, greater than 50% per day, greater than 55% per day,
greater than 60% per day, greater than 65% per day, greater than
70% per day, greater than 75% per day, greater than 80% per day,
greater than 85% per day, greater than 90% per day, greater than
95% per day, greater than 100% per day, greater than 105% per day,
greater than 110% per day, greater than 115% per day, greater than
120% per day, greater than 125% per day, greater than 130% per day,
greater than 135% per day, greater than 140% per day, greater than
145% per day, or greater than 150% per day, during the culturing of
the cell in the production bioreactor. Some embodiments of these
methods further include culturing a cell in the production
bioreactor using a condition predicted to result in an apparent
growth rate of about 0.1% per day to about 150% per day (or any of
the subranges of this range described herein) during the culturing
of the cell in the production bioreactor.
[0236] In some embodiments of these methods, the cell (e.g., any of
the cells described herein) contains a nucleic acid encoding a
recombinant therapeutic protein (e.g., any of the recombinant
therapeutic proteins described herein). Some embodiments of these
methods further include collecting or recovering the recombinant
therapeutic protein. Some embodiments of these methods further
include purifying the recombinant therapeutic protein. Some
embodiments of these methods further include formulating the
purified recombinant therapeutic protein. In some embodiments of
these methods, the cell is a bacterium (e.g., any of the exemplary
bacteria described herein or known in the art), a yeast cell (e.g.,
any of the exemplary yeast cells described herein or known in the
art), or a mammalian cell (e.g., any of the exemplary mammalian
cells described herein or known in the art).
[0237] In some embodiments of these methods, the cell viability is
determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel. In some embodiments
of these methods, the test culture vessel is a baffled shake flask
and the plurality of test conditions include (or consist) of one or
more (e.g., two or three) of: (a) a rotary agitation of about 125
RPM to about 400 RPM (or any of the subranges of this range
described herein); (b) a concentration of a sensitizer (e.g., any
of the sensitizers described herein or known in the art) of about 0
ppm to about 120 ppm (or any of the subranges of this range
described herein); and (c) a concentration of a protectant (e.g.,
any of the protectants described herein or known in the art) that
is about 1 g/L to about 10 g/L (or any of the subranges of this
range described herein). In some embodiments of these methods, the
protectant is a poloxamer (e.g., poloxamer-188, poloxamer-401,
poloxamer-402, or poloxamer-407), a poloxamine (e.g.,
poloxamine-904 or poloxamine-908), or a non-ionic surfactant
Pluronic.RTM..
Methods for Culturing a Cell in a Production Bioreactor
[0238] Also provided herein are methods of culturing a cell (e.g.,
any of the cells described herein) in a production bioreactor that
include culturing the cell in a liquid culture medium under one or
more (e.g., two, three, or four) of the following conditions:
[0239] (a) the production bioreactor has a P/V (W/m.sup.3) from
about 1.0 to about 110 (e.g., from about 3.0 to about 7.0, from
about 4.0 to about 6.0, from about 5.0 to about 6.0, or about 6.0)
(e.g., about 1.0 to about 110, about 1.0 to about 95, about 1.0 to
about 90, about 1.0 to about 85, about 1.0 to about 80, about 1.0
to about 75, about 1.0 to about 70, about 1.0 to about 65, about
1.0 to about 60, about 1.0 to about 55, about 1.0 to about 50,
about 1.0 to about 45, about 1.0 to about 40, about 1.0 to about
35, about 1.0 to about 30, about 1.0 to about 25, about 1.0 to
about 20, about 1.0 to about 18, about 1.0 to about 16, about 1.0
to about 14, about 1.0 to about 12, about 1.0 to about 10, about
1.0 to about 9.0, about 1.0 to about 8.0, about 1.0 to about 7.0,
about 1.0 to about 6.0, about 1.0 to about 5.0, about 1.0 to about
4.0, about 1.0 to about 3.0, to about 1.0 to about 2.0, about 2.0
to about 110, about 2.0 to about 95, about 2.0 to about 90, about
2.0 to about 85, about 2.0 to about 80, about 2.0 to about 75,
about 2.0 to about 70, about 2.0 to about 65, about 2.0 to about
60, about 2.0 to about 55, about 2.0 to about 50, about 2.0 to
about 45, about 2.0 to about 40, about 2.0 to about 35, about 2.0
to about 30, about 2.0 to about 25, about 2.0 to about 20, about
2.0 to about 18, about 2.0 to about 16, about 2.0 to about 14,
about 2.0 to about 12, about 2.0 to about 10, about 2.0 to about
9.0, about 2.0 to about 8.0, about 2.0 to about 7.0, about 2.0 to
about 6.0, about 2.0 to about 5.0, about 2.0 to about 4.0, about
2.0 to about 3.0, about 3.0 to about 110, about 3.0 to about 95,
about 3.0 to about 90, about 3.0 to about 85, about 3.0 to about
80, about 3.0 to about 75, about 3.0 to about 70, about 3.0 to
about 65, about 3.0 to about 60, about 3.0 to about 55, about 3.0
to about 50, about 3.0 to about 45, about 3.0 to about 40, about
3.0 to about 35, about 3.0 to about 30, about 3.0 to about 25,
about 3.0 to about 20, about 3.0 to about 18, about 3.0 to about
16, about 3.0 to about 14, about 3.0 to about 12, about 3.0 to
about 10, about 3.0 to about 9.0, about 3.0 to about 8.0, about 3.0
to about 7.0, about 3.0 to about 6.0, about 3.0 to about 5.0, about
3.0 to about 4.0, about 4.0 to about 110, about 4.0 to about 95,
about 4.0 to about 90, about 4.0 to about 85, about 4.0 to about
80, about 4.0 to about 75, about 4.0 to about 70, about 4.0 to
about 65, about 4.0 to about 60, about 4.0 to about 55, about 4.0
to about 50, about 4.0 to about 45, about 4.0 to about 40, about
4.0 to about 35, about 4.0 to about 30, about 4.0 to about 25,
about 4.0 to about 20, about 4.0 to about 18, about 4.0 to about
16, about 4.0 to about 14, about 4.0 to about 12, about 4.0 to
about 10, about 4.0 to about 9.0, about 4.0 to about 8.0, about 4.0
to about 7.0, about 4.0 to about 6.0, about 4.0 to about 5.0, about
5.0 to about 110, about 5.0 to about 95, about 5.0 to about 90,
about 5.0 to about 85, about 5.0 to about 80, about 5.0 to about
75, about 5.0 to about 70, about 5.0 to about 65, about 5.0 to
about 60, about 5.0 to about 55, about 5.0 to about 50, about 5.0
to about 45, about 5.0 to about 40, about 5.0 to about 35, about
5.0 to about 30, about 5.0 to about 25, about 5.0 to about 20,
about 5.0 to about 18, about 5.0 to about 16, about 5.0 to about
14, about 5.0 to about 12, about 5.0 to about 10, about 5.0 to
about 9.0, about 5.0 to about 8.0, about 5.0 to about 7.0, about
5.0 to about 6.0, about 6.0 to about 110, about 6.0 to about 95,
about 6.0 to about 90, about 6.0 to about 85, about 6.0 to about
80, about 6.0 to about 75, about 6.0 to about 70, about 6.0 to
about 65, about 6.0 to about 60, about 6.0 to about 55, about 6.0
to about 50, about 6.0 to about 45, about 6.0 to about 40, about
6.0 to about 35, about 6.0 to about 30, about 6.0 to about 25,
about 6.0 to about 20, about 6.0 to about 18, about 6.0 to about
16, about 6.0 to about 14, about 6.0 to about 12, about 6.0 to
about 10, about 6.0 to about 9.0, about 6.0 to about 8.0, about 6.0
to about 7.0, about 7.0 to about 110, about 7.0 to about 95, about
7.0 to about 90, about 7.0 to about 85, about 7.0 to about 80,
about 7.0 to about 75, about 7.0 to about 70, about 7.0 to about
65, about 7.0 to about 60, about 7.0 to about 55, about 7.0 to
about 50, about 7.0 to about 45, about 7.0 to about 40, about 7.0
to about 35, about 7.0 to about 30, about 7.0 to about 25, about
7.0 to about 20, about 7.0 to about 18, about 7.0 to about 16,
about 7.0 to about 14, about 7.0 to about 12, about 7.0 to about
10, about 7.0 to about 9.0, about 7.0 to about 8.0, about 8.0 to
about 110, about 8.0 to about 95, about 8.0 to about 90, about 8.0
to about 85, about 8.0 to about 80, about 8.0 to about 75, about
8.0 to about 70, about 8.0 to about 65, about 8.0 to about 60,
about 8.0 to about 55, about 8.0 to about 50, about 8.0 to about
45, about 8.0 to about 40, about 8.0 to about 35, about 8.0 to
about 30, about 8.0 to about 25, about 8.0 to about 20, about 8.0
to about 18, about 8.0 to about 16, about 8.0 to about 14, about
8.0 to about 12, about 8.0 to about 10, about 8.0 to about 9.0,
about 9.0 to about 110, about 9.0 to about 95, about 9.0 to about
90, about 9.0 to about 85, about 9.0 to about 80, about 9.0 to
about 75, about 9.0 to about 70, about 9.0 to about 65, about 9.0
to about 60, about 9.0 to about 55, about 9.0 to about 50, about
9.0 to about 45, about 9.0 to about 40, about 9.0 to about 35,
about 9.0 to about 30, about 9.0 to about 25, about 9.0 to about
20, about 9.0 to about 18, about 9.0 to about 16, about 9.0 to
about 14, about 9.0 to about 12, about 9.0 to about 10, about 10 to
about 110, about 10 to about 95, about 10 to about 90, about 10 to
about 85, about 10 to about 80, about 10 to about 75, about 10 to
about 70, about 10 to about 65, about 10 to about 60, about 10 to
about 55, about 10 to about 50, about 10 to about 45, about 10 to
about 40, about 10 to about 35, about 10 to about 30, about 10 to
about 25, about 10 to about 20, about 10 to about 18, about 10 to
about 16, about 10 to about 14, about 10 to about 12, about 12 to
about 110, about 12 to about 95, about 12 to about 90, about 12 to
about 85, about 12 to about 80, about 12 to about 75, about 12 to
about 70, about 12 to about 65, about 12 to about 60, about 12 to
about 55, about 12 to about 50, about 12 to about 45, about 12 to
about 40, about 12 to about 35, about 12 to about 30, about 12 to
about 25, about 12 to about 20, about 12 to about 18, about 12 to
about 16, about 12 to about 14, about 14 to about 110, about 14 to
about 95, about 14 to about 90, about 14 to about 85, about 14 to
about 80, about 14 to about 75, about 14 to about 70, about 14 to
about 65, about 14 to about 60, about 14 to about 55, about 14 to
about 50, about 14 to about 45, about 14 to about 40, about 14 to
about 35, about 14 to about 30, about 14 to about 25, about 14 to
about 20, about 14 to about 18, about 14 to about 16, about 16 to
about 110, about 16 to about 95, about 16 to about 90, about 16 to
about 85, about 16 to about 80, about 16 to about 75, about 16 to
about 70, about 16 to about 65, about 16 to about 60, about 16 to
about 55, about 16 to about 50, about 16 to about 45, about 16 to
about 40, about 16 to about 35, about 16 to about 30, about 16 to
about 25, about 16 to about 20, about 16 to about 18, about 18 to
about 110, about 18 to about 95, about 18 to about 90, about 18 to
about 85, about 18 to about 80, about 18 to about 75, about 18 to
about 70, about 18 to about 65, about 18 to about 60, about 18 to
about 55, about 18 to about 50, about 18 to about 45, about 18 to
about 40, about 18 to about 35, about 18 to about 30, about 18 to
about 25, about 18 to about 20, about 20 to about 110, about 20 to
about 95, about 20 to about 90, about 20 to about 85, about 20 to
about 80, about 20 to about 75, about 20 to about 70, about 20 to
about 65, about 20 to about 60, about 20 to about 55, about 20 to
about 50, about 20 to about 45, about 20 to about 40, about 20 to
about 35, about 20 to about 30, about 20 to about 25, about 25 to
about 110, about 25 to about 95, about 25 to about 90, about 25 to
about 85, about 25 to about 80, about 25 to about 75, about 25 to
about 70, about 25 to about 65, about 25 to about 60, about 25 to
about 55, about 25 to about 50, about 25 to about 45, about 25 to
about 40, about 25 to about 35, about 25 to about 30, about 30 to
about 110, about 30 to about 95, about 30 to about 90, about 30 to
about 85, about 30 to about 80, about 30 to about 75, about 30 to
about 70, about 30 to about 65, about 30 to about 60, about 30 to
about 55, about 30 to about 50, about 30 to about 45, about 30 to
about 40, about 30 to about 35, about 35 to about 110, about 35 to
about 95, about 35 to about 90, about 35 to about 85, about 35 to
about 80, about 35 to about 75, about 35 to about 70, about 35 to
about 65, about 35 to about 60, about 35 to about 55, about 35 to
about 50, about 35 to about 45, about 35 to about 40, about 40 to
about 110, about 40 to about 95, about 40 to about 90, about 40 to
about 85, about 40 to about 80, about 40 to about 75, about 40 to
about 70, about 40 to about 65, about 40 to about 60, about 40 to
about 55, about 40 to about 50, about 40 to about 45, about 45 to
about 110, about 45 to about 95, about 45 to about 90, about 45 to
about 85, about 45 to about 80, about 45 to about 75, about 45 to
about 70, about 45 to about 65, about 45 to about 60, about 45 to
about 55, about 45 to about 50, about 50 to about 110, about 50 to
about 95, about 50 to about 90, about 50 to about 85, about 50 to
about 80, about 50 to about 75, about 50 to about 70, about 50 to
about 65, about 50 to about 60, about 50 to about 55, about 55 to
about 110, about 55 to about 95, about 55 to about 90, about 55 to
about 85, about 55 to about 80, about 55 to about 75, about 55 to
about 70, about 55 to about 65, about 55 to about 60, about 60 to
about 110, about 60 to about 95, about 60 to about 90, about 60 to
about 85, about 60 to about 80, about 60 to about 75, about 60 to
about 70, about 60 to about 65, about 65 to about 110, about 65 to
about 95, about 65 to about 90, about 65 to about 85, about 65 to
about 80, about 65 to about 75, about 65 to about 70, about 70 to
about 110, about 70 to about 95, about 70 to about 90, about 70 to
about 85, about 70 to about 80, about 70 to about 75, about 75 to
about 110, about 75 to about 95, about 75 to about 90, about 75 to
about 85, about 75 to about 80, about 80 to about 110, about 80 to
about 95, about 80 to about 90, about 80 to about 85, about 85 to
about 110, about 85 to about 95, about 85 to about 90, about 90 to
about 110, about 90 to about 95, about 95 to about 110, about 95 to
about 105, about 95 to about 100, about 100 to about 110, about 100
to about 105, or about 105 to about 110);
[0240] (b) the production bioreactor has a volume of air per volume
of liquid per minute (VVM) (min.sup.-1) from about 0.01 to about
0.10 (e.g., from about 0.02 to about 0.08, from about 0.04 to about
0.06, or about 0.06) (e.g., about 0.01 to about 0.10, about 0.01 to
about 0.09, about 0.01 to about 0.08, about 0.01 to about 0.07,
about 0.01 to about 0.06, about 0.01 to about 0.05, about 0.01 to
about 0.04, about 0.01 to about 0.03, about 0.01 to about 0.02,
about 0.02 to about 0.10, about 0.02 to about 0.09, about 0.02 to
about 0.08, about 0.02 to about 0.07, about 0.02 to about 0.06,
about 0.02 to about 0.05, about 0.02 to about 0.04, about 0.02 to
about 0.03, about 0.03 to about 0.10, about 0.03 to about 0.09,
about 0.03 to about 0.08, about 0.03 to about 0.07, about 0.03 to
about 0.06, about 0.03 to about 0.05, about 0.03 to about 0.04,
about 0.04 to about 0.10, about 0.04 to about 0.09, about 0.04 to
about 0.08, about 0.04 to about 0.07, about 0.04 to about 0.06,
about 0.04 to about 0.05, about 0.05 to about 0.10, about 0.05 to
about 0.09, about 0.05 to about 0.08, about 0.05 to about 0.07,
about 0.05 to about 0.06, about 0.06 to about 0.10, about 0.06 to
about 0.09, about 0.06 to about 0.08, about 0.06 to about 0.07,
about 0.07 to about 0.10, about 0.07 to about 0.09, about 0.07 to
about 0.08, about 0.08 to about 0.10, about 0.08 to about 0.09, or
about 0.09 to about 0.10);
[0241] (c) the medium comprises a sensitizer (e.g., any of the
sensitizers described herein or known in the art) that has a
concentration of about 0 ppm to about 5,000 ppm (or any of the
subranges of this range described herein); and
[0242] (d) the liquid culture medium comprises a protectant (e.g.,
any of the protectants described herein or known in the art) that
has a concentration that is from about 1 g/L to about 15 g/L (e.g.,
any of the subranges of this range described herein, e.g., about 1
g/L to about 15 g/L, about 1 g/L to about 14 g/L, about 1 g/L to
about 13 g/L, about 1 g/L to about 12 g/L, about 1 g/L to about 11
g/L, about 2 g/L to about 15 g/L, about 2 g/L to about 14 g/L,
about 2 g/L to about 13 g/L, about 2 g/L to about 12 g/L, about 2
g/L to about 11 g/L, about 3 g/L to about 15 g/L, about 3 g/L to
about 14 g/L, about 3 g/L to about 13 g/L, about 3 g/L to about 12
g/L, about 3 g/L to about 11 g/L, about 4 g/L to about 15 g/L,
about 4 g/L to about 14 g/L, about 4 g/L to about 13 g/L, about 4
g/L to about 12 g/L, about 4 g/L to about 11 g/L, about 5 g/L to
about 15 g/L, about 5 g/L to about 14 g/L, about 5 g/L to about 13
g/L, about 5 g/L to about 12 g/L, about 5 g/L to about 11 g/L,
about 6 g/L to about 15 g/L, about 6 g/L to about 14 g/L, about 6
g/L to about 13 g/L, about 6 g/L to about 12 g/L, about 6 g/L to
about 11 g/L, about 7 g/L to about 15 g/L, about 7 g/L to about 14
g/L, about 7 g/L to about 13 g/L, about 7 g/L to about 12 g/L,
about 7 g/L to about 11 g/L, about 8 g/L to about 15 g/L, about 8
g/L to about 14 g/L, about 8 g/L to about 13 g/L, about 8 g/L to
about 12 g/L, about 8 g/L to about 11 g/L, about 9 g/L to about 15
g/L, about 9 g/L to about 14 g/L, about 9 g/L to about 13 g/L,
about 9 g/L to about 12 g/L, about 9 g/L to about 11 g/L, about 10
g/L to about 15 g/L, about 10 g/L to about 14 g/L, about 10 g/L to
about 13 g/L, about 10 g/L to about 12 g/L, about 10 g/L to about
11 g/L, about 11 g/L to about 15 g/L, about 11 g/L to about 14 g/L,
about 11 g/L to about 13 g/L, about 11 g/L to about 12 g/L, about
12 g/L to about 15 g/L, about 12 g/L to about 14 g/L, about 12 g/L
to about 13 g/L, about 13 g/L to about 15 g/L, about 13 g/L to
about 14 g/L, or about 14 g/L to about 15 g/L).
[0243] Under the optimized conditions, the specific cell death rate
can be less than or predicted to be less than 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% per day. Under optimized
conditions, the specific cell death rate can be about 0.1% to about
40% (or any of the subranges of this range described herein).
[0244] In some embodiments, the cell viability can be determined by
measuring the concentration of lactic acid dehydrogenase (LDH). The
cell specific LDH production (nU/cell/day) can be less than or
predicted to be less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, or 200 (e.g., 150). In some embodiments, the LDH
production (nU/cell/day) can be about 0.1 to about 200 (e.g., any
of the subranges of this range described herein, e.g., about 0.1 to
about 200, about 0.1 to about 190, about 0.1 to about 180, about
0.1 to about 170, about 0.1 to about 160, about 0.5 to about 200,
about 0.5 to about 190, about 0.5 to about 180, about 0.5 to about
170, about 0.5 to about 160, about 1.0 to about 200, about 1.0 to
about 190, about 1.0 to about 180, about 1.0 to about 170, about
1.0 to about 160, about 2.5 to about 200, about 2.5 to about 190,
about 2.5 to about 180, about 2.5 to about 170, about 2.5 to about
160, about 5.0 to about 200, about 5.0 to about 190, about 5.0 to
about 180, about 5.0 to about 170, about 5.0 to about 160, about 10
to about 200, about 10 to about 190, about 10 to about 180, about
10 to about 170, about 10 to about 160, about 20 to about 200,
about 20 to about 190, about 20 to about 180, about 20 to about
170, about 20 to about 160, about 30 to about 200, about 30 to
about 190, about 30 to about 180, about 30 to about 170, about 30
to about 160, about 40 to about 200, about 40 to about 190, about
40 to about 180, about 40 to about 170, about 40 to about 160,
about 50 to about 200, about 50 to about 190, about 50 to about
180, about 50 to about 170, about 50 to about 160, about 60 to
about 200, about 60 to about 190, about 60 to about 180, about 60
to about 170, about 60 to about 160, about 70 to about 200, about
70 to about 190, about 70 to about 180, about 70 to about 170,
about 70 to about 160, about 80 to about 200, about 80 to about
190, about 80 to about 180, about 80 to about 170, about 80 to
about 160, about 90 to about 200, about 90 to about 190, about 90
to about 180, about 90 to about 170, about 90 to about 160, about
100 to about 200, about 100 to about 190, about 100 to about 180,
about 100 to about 170, about 100 to about 160, about 110 to about
200, about 110 to about 190, about 110 to about 180, about 110 to
about 170, about 110 to about 160, about 120 to about 200, about
120 to about 190, about 120 to about 180, about 120 to about 170,
about 120 to about 160, about 130 to about 200, about 130 to about
190, about 130 to about 180, about 130 to about 170, about 130 to
about 160, about 140 to about 200, about 140 to about 190, about
140 to about 180, about 140 to about 170, about 140 to about 160,
about 150 to about 200, about 150 to about 190, about 150 to about
180, about 150 to about 170, about 150 to about 160, about 160 to
about 200, about 160 to about 190, about 160 to about 180, about
160 to about 170, about 170 to about 200, about 170 to about 190,
about 170 to about 180, about 180 to about 200, about 180 to about
190, or about 190 to about 200).
[0245] In some embodiments, the apparent growth rate can be more
than or predicted to be greater than 1% per day, greater than 1.5%
per day, greater than 2% per day, greater than 2.5% per day,
greater than 3% per day, greater than 3.5% per day, greater than
4.0% per day, greater than 4.5% per day, greater than 5.0% per day,
greater than 5.5% per day, greater than 6.0% per day, greater than
6.5% per day, greater than 7.0% per day, greater than 7.5% per day,
greater than 8.0% per day, greater than 8.5% per day, greater than
9.0% per day, greater than 9.5% per day, greater than 10.0% per
day, greater than 10.5% per day, greater than 11.0% per day,
greater than 11.5% per day, greater than 12.0% per day, greater
than 12.5% per day, greater than 13.0% per day, greater than 13.5%
per day, greater than 14.0% per day, greater than 14.5% per day,
greater than 15.0% per day, greater than 16% per day, greater than
17% per day, greater than 18% per day, greater than 19% per day,
greater than 20% per day, greater than 21% per day, greater than
22% per day, greater than 23% per day, greater than 24% per day,
greater than 25% per day, greater than 30% per day, greater than
35% per day, greater than 40% per day, greater than 45% per day,
greater than 50% per day, greater than 55% per day, greater than
60% per day, greater than 65% per day, greater than 70% per day,
greater than 75% per day, greater than 80% per day, greater than
85% per day, greater than 90% per day, greater than 95% per day,
greater than 100% per day, greater than 105% per day, greater than
110% per day, greater than 115% per day, greater than 120% per day,
greater than 125% per day, greater than 130% per day, greater than
135% per day, greater than 140% per day, greater than 145% per day,
or greater than 150% per day. In some embodiments, the apparent
growth rate can be about 0.1% per day to about 150% per day (e.g.,
any of the subranges of this range described herein, e.g., about
0.1% to about 48%, about 0.1% to about 46%, about 0.1% to about
44%, about 0.1% to about 42%, about 0.5% to about 50%, about 0.5%
to about 48%, about 0.5% to about 46%, about 0.5% to about 44%,
about 0.5% to about 42%, about 1.0% to about 50%, about 1.0% to
about 48%, about 1.0% to about 46%, about 1.0% to about 44%, about
1.0% to about 42%, about 2.0% to about 50%, about 2.0% to about
48%, about 2.0% to about 46%, about 2.0% to about 44%, about 2.0%
to about 42%, about 4.0% to about 50%, about 4.0% to about 48%,
about 4.0% to about 46%, about 4.0% to about 44%, about 4.0% to
about 42%, about 6.0% to about 50%, about 6.0% to about 48%, about
6.0% to about 46%, about 6.0% to about 44%, about 6.0% to about
42%, about 8.0% to about 50%, about 8.0% to about 48%, about 8.0%
to about 46%, about 8.0% to about 44%, about 8.0% to about 42%,
about 10% to about 50%, about 10% to about 48%, about 10% to about
46%, about 10% to about 44%, about 10% to about 42%, about 12% to
about 50%, about 12% to about 48%, about 12% to about 46%, about
12% to about 44%, about 12% to about 42%, about 14% to about 50%,
about 14% to about 48%, about 14% to about 46%, about 14% to about
44%, about 14% to about 42%, about 16% to about 50%, about 16% to
about 48%, about 16% to about 46%, about 16% to about 44%, about
16% to about 42%, about 18% to about 50%, about 18% to about 48%,
about 18% to about 46%, about 18% to about 44%, about 18% to about
42%, about 20% to about 50%, about 20% to about 48%, about 20% to
about 46%, about 20% to about 44%, about 20% to about 42%, about
22% to about 50%, about 22% to about 48%, about 22% to about 46%,
about 22% to about 44%, about 22% to about 42%, about 24% to about
50%, about 24% to about 48%, about 24% to about 46%, about 24% to
about 44%, about 24% to about 42%, about 26% to about 50%, about
26% to about 48%, about 26% to about 46%, about 26% to about 44%,
about 26% to about 42%, about 28% to about 50%, about 28% to about
48%, about 28% to about 46%, about 28% to about 44%, about 28% to
about 42%, about 30% to about 50%, about 30% to about 48%, about
30% to about 46%, about 30% to about 44%, about 30% to about 42%,
about 32% to about 50%, about 32% to about 48%, about 32% to about
46%, about 32% to about 44%, about 32% to about 42%, about 34% to
about 50%, about 34% to about 48%, about 34% to about 46%, about
34% to about 44%, about 34% to about 42%, about 36% to about 50%,
about 36% to about 48%, about 36% to about 46%, about 36% to about
44%, about 36% to about 42%, about 38% to about 50%, about 38% to
about 48%, about 38% to about 46%, about 38% to about 44%, about
38% to about 42%, about 40% to about 50%, about 40% to about 48%,
about 40% to about 46%, about 40% to about 44%, about 40% to about
42%, about 42% to about 50%, about 42% to about 48%, about 42% to
about 46%, about 42% to about 44%, about 44% to about 50%, about
44% to about 48%, about 44% to about 46%, about 46% to about 50%,
about 46% to about 48%, or about 48% to about 50%).
[0246] In some embodiments of these methods, the sensitizer is an
antifoam agent (e.g., a polydimethylsiloxane-based antifoam,
simethicone, or any of the other antifoams described herein or
known in the art). In some embodiments, the protectant is a
poloxamer (e.g., poloxamer-188, poloxamer-401, poloxamer-402, or
poloxamer-407), a poloxamine (e.g., poloxamine-904 or
poloxamine-908), or a non-ionic surfactant Pluronic.RTM..
[0247] In some embodiments of these methods, the production
bioreactor (e.g., any of the exemplary production bioreactors
described herein or known in the art) has a volume of about 5 L to
about 20,000 L (or any of the subranges of this range described
herein). In some embodiments of these methods, the production
bioreactor is a perfusion bioreactor. In some embodiments of these
methods, the production bioreactor is a fed batch bioreactor.
[0248] In some embodiments, the method results in a specific cell
death rate of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,
24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,
37%, 38%, 39%, or 40% per day, during the culturing of the cell in
the production bioreactor. In some embodiments, the method results
in a specific cell death rate of about 0.1% to about 40% per day
(or any of the subranges of this range described herein) during the
culturing of the cell in the production bioreactor.
[0249] In some embodiments, the method results in a cell specific
LDH production of less than 150 nU/cell/day, less than 145
nU/cell/day, less than 140 nU/cell/day, less than 135 nU/cell/day,
less than 130 nU/cell/day, less than 125 nU/cell/day, less than 120
nU/cell/day, less than 115 nU/cell/day, less than 150 nU/cell/day,
less than 110 nU/cell/day, less than 105 nU/cell/day, less than 100
nU/cell/day, less than 95 nU/cell/day, less than 90 nU/cell/day,
less than 85 nU/cell/day, less than 80 nU/cell/day, less than 75
nU/cell/day, less than 70 nU/cell/day, less than 65 nU/cell/day,
less than 60 nU/cell/day, less than 55 nU/cell/day, less than 50
nU/cell/day, less than 45 nU/cell/day, less than 45 nU/cell/day,
less than 40 nU/cell/day, less than 35 nU/cell/day, less than 30
nU/cell/day, less than 25 nU/cell/day, less than 20 nU/cell/day,
less than 15 nU/cell/day, less than 10 nU/cell/day, less than 5
nU/cell/day, or less than less than 1 nU/cell/day, during the
culturing of the cell in the production bioreactor. In some
embodiments of these methods, the method results in a cell specific
LDH production of about 0.1 nU/cell/day to about 150 nU/cell/day
(or any of the subranges of this range described herein) during the
culturing of the cell in the production bioreactor.
[0250] In some embodiments, the method results in an apparent
growth rate of greater than 1% per day, greater than 1.5% per day,
greater than 2% per day, greater than 2.5% per day, greater than 3%
per day, greater than 3.5% per day, greater than 4.0% per day,
greater than 4.5% per day, greater than 5.0% per day, greater than
5.5% per day, greater than 6.0% per day, greater than 6.5% per day,
greater than 7.0% per day, greater than 7.5% per day, greater than
8.0% per day, greater than 8.5% per day, greater than 9.0% per day,
greater than 9.5% per day, greater than 10.0% per day, greater than
10.5% per day, greater than 11.0% per day, greater than 11.5% per
day, greater than 12.0% per day, greater than 12.5% per day,
greater than 13.0% per day, greater than 13.5% per day, greater
than 14.0% per day, greater than 14.5% per day, greater than 15.0%
per day, greater than 16% per day, greater than 17% per day,
greater than 18% per day, greater than 19% per day, greater than
20% per day, greater than 21% per day, greater than 22% per day,
greater than 23% per day, greater than 24% per day, greater than
25% per day, greater than 30% per day, greater than 35% per day,
greater than 40% per day, greater than 45% per day, greater than
50% per day, greater than 55% per day, greater than 60% per day,
greater than 65% per day, greater than 70% per day, greater than
75% per day, greater than 80% per day, greater than 85% per day,
greater than 90% per day, greater than 95% per day, greater than
100% per day, greater than 105% per day, greater than 110% per day,
greater than 115% per day, greater than 120% per day, greater than
125% per day, greater than 130% per day, greater than 135% per day,
greater than 140% per day, greater than 145% per day, or greater
than 150% per day, during the culturing of the cell in the
production bioreactor. In some embodiments of these methods, the
method results in an apparent growth rate of about 0.1% per day to
about 150% per day (or any of the subranges of this range described
herein) during the culturing of the cell in the production
bioreactor.
[0251] In some embodiments of these methods, the cell (e.g., any of
the cells described herein) contains a nucleic acid encoding a
recombinant therapeutic protein (e.g., any of the recombinant
therapeutic proteins described herein). Some embodiments of these
methods further include collecting or recovering the recombinant
therapeutic protein.
[0252] Some embodiments of these methods further include purifying
the recombinant therapeutic protein. Some embodiments of these
methods further include formulating the purified recombinant
therapeutic protein.
[0253] In some embodiments of these methods, the cell is a
bacterium (e.g., any of the exemplary bacteria described herein or
known in the art), a yeast cell (e.g., any of the yeast cells
described herein or known in the art), or a mammalian cell (e.g.,
any of the mammalian cells described herein or known in the
art).
[0254] In some embodiments of these methods, the cell viability is
determined by measuring a concentration of lactic acid
dehydrogenase (LDH) in the test culture vessel.
EXAMPLES
[0255] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1: Full Factorial Screening Combined with Inscribed Center
Composite Design for Test Culture Vessels
[0256] The rotary agitation (RPM), the concentration of the
sensitizer, and the concentration of a protectant were selected as
parameters. Full factorial design combined with inscribed center
composite design were used to determine a set of test conditions
(FIG. 7). The specific LDH production values were measured at each
test condition. The results were used to provide an estimated
contour of the selected parameters. Regions of poor performance and
regions of operation failure were identified (FIG. 9).
Example 2: Predicting Specific LDH Production Values at Benchtop
Scale
[0257] Proof-of-concept experiments were performed at the benchtop
scale to determine the accuracy of the prediction methods as
described herein.
[0258] The concentration of protectants in the benchtop scale were
set to three different levels. For each level, two benchtop
culturing bioreactors were tested. The peak specific LDH production
values were measured due to perfusion process, and were compared
against the predicted values.
[0259] The actual observed values from the benchtop scale
experiments matched very well with those predicted values
(P<0.0001, R.sup.2=0.84) (FIG. 13A and FIG. 13C). The results
demonstrated the methods as described herein can be successfully
used to predict the effects of various parameter at a higher
scale.
[0260] Experiments were performed maintaining an antifoam
concentration range of 30-40 ppm antifoam addition per day.
Pluronic.RTM. concentrations were adjusted from 1.8 g/L (dashed
line), 2.8 g/L (solid line, closed circles), 3.8 g/L (solid line,
open circles). Experiment was performed in replicate. This
demonstrates the models capability of predicting specific LDH
production when changing Pluronic.RTM. concentrations.
[0261] Experiments were also performed to increase the
concentration of the antifoam agent from 30 ppm to 80 ppm per day.
The predicted LDH range was 90.19.about.96.38, which matched very
well with the actual observed LDH range 94.74.about.99.36 (FIG.
14). The results showed the methods as described herein can be
successfully used to predict the effects of antifoam agents.
Example 3: Predicting Specific LDH Production Values at Production
Scale
[0262] Experiments were performed at the production scale to
determine the accuracy of the model as described herein.
[0263] Three, 50-L production bioreactors and two 100-L production
bioreactors were tested. The experiments tested poloxamer-188
(Pluronic.RTM. F68) concentrations that ranged from 1.8 g/L to 5.8
g/L.
[0264] The actual observed values for specific LDH production
matched well with these predicted values (P<0.0001,
R.sup.2=0.84) (FIG. 15A and FIG. 15B). The results demonstrated
that the methods as described herein can be successfully used to
predict the effects of selected parameters at the production scale
(production bioreactor scale).
OTHER EMBODIMENTS
[0265] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *