U.S. patent application number 16/798014 was filed with the patent office on 2020-10-08 for ph adjustment to improve thaw recovery of cell banks.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Kara CALHOUN, Marcia COYNE, Phillip DUFFY, Angela MEIER, Steven J. MEIER.
Application Number | 20200315162 16/798014 |
Document ID | / |
Family ID | 1000004899703 |
Filed Date | 2020-10-08 |
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United States Patent
Application |
20200315162 |
Kind Code |
A1 |
MEIER; Angela ; et
al. |
October 8, 2020 |
pH ADJUSTMENT TO IMPROVE THAW RECOVERY OF CELL BANKS
Abstract
Provided herein are methods of freezing mammalian cells for
storage or improving thaw recovery of cell banks comprising
freezing mammalian cells in a freezing medium having a pH of 6.7 to
8.5.
Inventors: |
MEIER; Angela; (San
Francisco, CA) ; MEIER; Steven J.; (Burlingame,
CA) ; DUFFY; Phillip; (Brisbane, CA) ; COYNE;
Marcia; (San Mateo, CA) ; CALHOUN; Kara;
(South San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
1000004899703 |
Appl. No.: |
16/798014 |
Filed: |
February 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15399666 |
Jan 5, 2017 |
10602739 |
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16798014 |
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PCT/US2015/039757 |
Jul 9, 2015 |
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15399666 |
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62022392 |
Jul 9, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2510/02 20130101;
C12N 5/0018 20130101; A01N 1/0221 20130101; C12N 5/0682 20130101;
A01N 1/0284 20130101 |
International
Class: |
A01N 1/02 20060101
A01N001/02; C12N 5/00 20060101 C12N005/00; C12N 5/071 20060101
C12N005/071 |
Claims
1-48. (canceled)
49: A method of freezing mammalian cells for storage comprising (a)
adjusting pH of a freezing medium to a pH of 7.5 to 8.5, wherein
the freezing medium comprises a buffered solution and a
cryoprotective agent; (b) combining the mammalian cells with the
freezing medium to form a cell pool; and (c) freezing the mammalian
cells in the cell pool.
50. (canceled)
51: The method of claim 49, wherein the pH is adjusted to a pH of
7.5.
52: The method of claim 49, wherein the adjusted pH is a target pH
or a measured pH.
53: The method of claim 52, wherein the target pH is 7.7 to
8.3.
54: The method of claim 52, wherein the measured pH is 7.7 to
8.3.
55: The method of claim 49, further comprising a step of measuring
the adjusted pH of the freezing medium.
56: The method of claim 55, wherein if the measured pH of the
freezing medium is below a target pH, repeating the adjusting step
and measuring step until the adjusted pH of the freezing medium is
7.5 to 8.5.
57: The method of claim 49, wherein the pH is adjusted by adding a
base.
58: The method of claim 57, wherein the base is selected from the
group consisting of sodium carbonate, sodium bicarbonate, HEPES
sodium salt, sodium hydroxide, and potassium hydroxide.
59: The method of claim 49, wherein the mammalian cells are in a
medium having a pH of about 6.2 to about 6.6 before the mammalian
cells are combined with the freezing medium.
60: The method of claim 49, wherein the cryoprotective agent is
DMSO, glycerol, propanediol, ethylene glycol, or a sugar.
61: The method of claim 60, wherein the cryoprotective agent is
DMSO or glycerol, and wherein the DMSO or glycerol in the cell pool
is at a concentration of about 5% to about 12.5% by volume.
62: The method of claim 61, wherein the DMSO or glycerol in the
cell pool is at a concentration of about 5% to about 10% by
volume.
63: The method of claim 49, wherein the cell pool contains the
mammalian cells at a cell density of 8% to 28% packed cell volume
(PCV).
64: The method of claim 49, further comprising a step of cooling
cell culture fluid during cell harvest and concentration process
before the mammalian cells are combined with a freezing medium.
65: The method of claim 64, wherein the cell culture fluid is
cooled to a temperature at or below about 20.degree. C.
66: The method of claim 64, wherein the cell culture fluid is
cooled to a temperature at or below about 10.degree. C.
67-81. (canceled)
82: The method of claim 49, wherein the pH of the freezing medium
is adjusted to a pH of 7.7 to 8.3 in step a.
83: The method of claim 49, wherein the pH of the freezing medium
is adjusted to a pH of 7.8 to 8.3 in step a.
84: The method of claim 49, wherein the mammalian cell is a CHO
cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
provisional application Ser. No. 62/022,392, filed Jul. 9, 2014,
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of freezing cells
(such as mammalian cells) for banking and freezing media for use in
freezing cells.
BACKGROUND OF THE INVENTION
[0003] Cell banks are produced by first accumulating cells in a
batch/perfusion cell culture process and then harvesting cells for
banking. The process involves three stages: cell accumulation,
harvest and cell concentration, and cell banking. A harvest process
step serves to concentrate the final cell culture fluid or to
extract the cells from the cell culture fluid using centrifugation.
A subsequent pooling and filling process serves to prepare cell
bank ampoules for long-term storage.
[0004] These traditional methods may result in inconsistent or poor
viability post-thaw for select cell lines. Imperative to the cell
banking process is the need for high cell viability after thawing
of the frozen cells. Thus, improved methods of freezing cells for
cell banking are desirable. Cell culture medium for freezing cells
that allows for greater cell viability when thawed after banking
would be beneficial.
[0005] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes.
BRIEF SUMMARY OF THE INVENTION
[0006] In one aspect, provided herein is a method of improving thaw
recovery of cell banks comprising freezing eukaryotic cells (e.g.,
mammalian cells, insect cells, etc.) for banking in a freezing
medium, wherein the freezing medium comprises a buffered solution
and a cryoprotective agent, and wherein the freezing medium has a
pH of about 6.7 to about 8.5 prior to freezing or has been adjusted
to a pH of about 6.7 to about 8.5 prior to freezing.
[0007] In another aspect, provided herein is a method of freezing
eukaryotic cells (e.g., mammalian cells, insect cells, etc.) for
storage comprising freezing the cells in a freezing medium, wherein
the freezing medium comprises a buffered solution and a
cryoprotective agent, and wherein the freezing medium has a pH of
about 6.7 to about 8.5 prior to freezing or has been adjusted to a
pH of about 6.7 to about 8.5 prior to freezing.
[0008] In some embodiments of the methods described above or
herein, the freezing medium has a pH of about 6.7 to about 8.3,
about 6.8 to about 8.3, about 6.9 to about 8.3, about 7.0 to about
8.3, about 7.1 to about 8.3, about 7.2 to about 8.3, about 7.3 to
about 8.3, about 7.4 to about 8.3, about 7.5 to about 8.3, about
7.2 to about 8.0, about 7.2 to about 7.8, or about 7.5 prior to
freezing.
[0009] In some embodiments of the methods described above or
herein, the pH of the freezing medium has been adjusted to a pH of
about 6.7 to about 8.5, about 6.7 to about 8.3, about 6.8 to about
8.3, about 6.9 to about 8.3, about 7.0 to about 8.3, about 7.1 to
about 8.3, about 7.2 to about 8.3, about 7.3 to about 8.3, about
7.4 to about 8.3, about 7.5 to about 8.3, about 7.2 to about 8.0,
about 7.2 to about 7.8, or about 7.5. In some embodiments of the
methods described above or herein, the cells (e.g., mammalian cells
or insect cells) are combined with a freezing medium before and/or
after pH adjustment. In some embodiments, the adjusted pH is a
target pH or a measured pH. In some embodiments, the target pH is
about 6.7 to about 8.5, or any of the pH or in any pH ranges
described herein. In some embodiments, the measured pH is about 6.7
to about 8.5, or any of the pH or in any pH ranges described
herein. In some embodiments of the methods described above or
herein, the method further comprises a step of measuring an initial
pH of the freezing medium containing the cells (e.g., mammalian
cells or insect cells) prior to adjusting pH of the freezing
medium. In some embodiments of the methods described above or
herein, the method further comprises a step of measuring the
adjusted pH of the freezing medium. In some embodiments of the
methods described above or herein, if the measured pH of the
freezing medium is below a target pH, the method comprises
repeating the adjusting step and measuring step until the adjusted
pH of the freezing medium is about 6.7 to about 8.5, or any of the
pH or in any pH ranges described herein.
[0010] In some embodiments of the methods described herein or
above, the pH is adjusted by adding a base. In some embodiments,
the base is selected from the group consisting of sodium carbonate,
sodium bicarbonate, HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) sodium salt,
sodium hydroxide, and potassium hydroxide. In some embodiments, the
pH of the freezing medium is adjusted by adding a base to the
freezing medium according to the following formula
V.sub.base=C.sub.base*V.sub.p (pH.sub.t-pH.sub.i), wherein
C.sub.base is a base-specific coefficient, V.sub.base is a volume
of the base to add to the freezing medium, V.sub.p is the volume of
the freezing medium, pH.sub.t is the target pH, and pH.sub.i is the
initial pH. In some embodiments, the pH of the freezing medium is
adjusted by adding sodium carbonate to the freezing medium
according to the following formula V.sub.Na2CO3=0.0085V.sub.p
(pH.sub.t-pH.sub.i), wherein V.sub.Na2CO3 is a volume of 1M sodium
carbonate to add to the freezing medium, V.sub.p is the volume of
the freezing medium, pH.sub.t is the target pH, and pH.sub.i is the
initial pH. In some embodiments, the target pH is a pH between
about 6.7 to about 8.5, about 7.2 to about 8.3, or any of the pH or
in any pH ranges described herein. In some embodiments, the target
pH is 7.5.
[0011] In some embodiments of the methods described above or
herein, the cells (e.g., mammalian cells or insect cells) are in a
medium having a pH of about 6.2 to about 6.6 before the cells are
combined with a freezing medium.
[0012] In some embodiments of the methods described above or
herein, the cryoprotective agent is DMSO (dimethyl sulfoxide),
glycerol, propanediol, ethylene glycol, a macromolecule, a sugar,
or a combination thereof. In some embodiments, the DMSO or glycerol
in the freezing medium prior to freezing is at a concentration of
about 5% to about 12.5% by volume. In some embodiments, the DMSO or
glycerol in the freezing medium prior to freezing the cells (e.g.,
mammalian cells or insect cells) is at a concentration of about 5%
to about 10% by volume.
[0013] In some embodiments of the methods described above or
herein, the freezing medium containing the cells (e.g., mammalian
cells or insect cells) has a cell density of about 8% to about 28%
packed cell volume (PCV) prior to freezing.
[0014] In some embodiments of the method described above or herein,
the method further comprises a step of cooling cell culture fluid
during cell harvest and concentration process before the cells
(e.g., mammalian cells or insect cells) are combined with a
freezing medium. In some embodiments, the cell culture fluid is
cooled to a temperature at or below about 20.degree. C. In some
embodiments, the cell culture fluid is cooled to a temperature at
or below about 10.degree. C.
[0015] In another aspect, provided here is a method of freezing
eukaryotic cells (e.g., mammalian cells, insect cells, etc.) for
storage or improving thaw recovery of cell banks comprising (a)
adjusting pH of a freezing medium containing cells to a pH of about
6.7 to about 8.5, wherein the freezing medium comprises a buffered
solution and a cryoprotective agent; and (b) freezing the
cells.
[0016] In some embodiments, the pH is adjusted to a pH of about 6.7
to about 8.3, about 6.8 to about 8.3, about 6.9 to about 8.3, about
7.0 to about 8.3, about 7.1 to about 8.3, about 7.2 to about 8.3,
about 7.3 to about 8.3, about 7.4 to about 8.3, about 7.5 to about
8.3, about 7.2 to about 8.0, about 7.2 to about 7.8, or about 7.5.
In some embodiments, the adjusted pH is a target pH or a measured
pH. In some embodiments, the target pH is about 6.7 to about 8.5,
or any of the pH or in any pH ranges described herein. In some
embodiments, the target pH is about 7.5. In some embodiments, the
measured pH is about 6.7 to about 8.5, or any of the pH or in any
pH ranges described herein.
[0017] In some embodiments of the methods described above or
herein, the method further comprises a step of measuring an initial
pH of the freezing medium containing the cells (e.g., mammalian
cells or insect cells) prior to adjusting pH of the freezing
medium. In some embodiments, the method further comprises a step of
measuring the adjusted pH of the freezing medium. In some
embodiments, if the measured pH of the freezing medium is below a
target pH, the method comprises repeating the adjusting step and
measuring step until the adjusted pH of the freezing medium is
about 6.7 to about 8.5, about 7.2 to about 8.3, or any of the pH or
in any pH ranges described herein.
[0018] In some embodiments of the methods described above or
herein, the pH is adjusted by adding a base. In some embodiments,
the base is selected from the group consisting of sodium carbonate,
sodium bicarbonate, HEPES sodium salt, sodium hydroxide, and
potassium hydroxide. In some embodiments, the pH of the freezing
medium is adjusted by adding a base to the freezing medium
according to the following formula V.sub.base=C.sub.base*V.sub.p
(pH.sub.t-pH.sub.i), wherein C.sub.base is a base-specific
coefficient, V.sub.base is a volume of the base to add to the
freezing medium, V.sub.p is the volume of the freezing medium,
pH.sub.t is the target pH, and pH.sub.i is the initial pH. In some
embodiments, the pH of the freezing medium is adjusted by adding
sodium carbonate to the freezing medium according to the following
formula V.sub.Na2CO3=0.0085V.sub.p (pH.sub.t-pH.sub.i), wherein
V.sub.Na2CO3 is a volume of 1M sodium carbonate to add to the
freezing medium, V.sub.p is the volume of the freezing medium,
pH.sub.t is the target pH, and pH.sub.i is the initial pH. In some
embodiments, the target pH is about 6.7 to about 8.5, or any of the
pH or in any pH ranges described herein. In some embodiments, the
target pH is about 7.5.
[0019] In some embodiments, the cells (e.g., mammalian cells or
insect cells) are in a medium having a pH of about 6.2 to about 6.6
before the cells are combined with a freezing medium.
[0020] In some embodiments, the cryoprotective agent in the
freezing medium is DMSO, glycerol, propanediol, ethylene glycol, a
macromolecule, a sugar, or a combination thereof. In some
embodiments, the DMSO or glycerol in the freezing medium containing
the cells (e.g., mammalian cells or insect cells) prior to freezing
is at a concentration of about 5% to about 12.5% by volume. In some
embodiments, the DMSO or glycerol in the freezing medium containing
the cells (e.g., mammalian cells or insect cells) prior to freezing
the cells is at a concentration of about 5% to about 10% by
volume.
[0021] In some embodiments, the freezing medium containing the
cells (e.g., mammalian cells or insect cells) has a cell density of
about 8% to about 28% packed cell volume (PCV) prior to
freezing.
[0022] In some embodiments, the method further comprises a step of
cooling cell culture fluid during cell harvest and concentration
process before the cells (e.g., mammalian cells or insect cells)
are combined with a freezing medium. In some embodiments, the cell
culture fluid is cooled to a temperature at or below about
20.degree. C., or at or below about 10.degree. C.
[0023] In another aspect, provided herein is a method of freezing
eukaryotic cells (e.g., mammalian cells, insect cells, etc.) for
storage or improving thaw recovery of cell banks comprising (a)
adjusting pH of a freezing medium to a pH of about 6.7 to about
8.5, wherein the freezing medium comprises a buffered solution and
a cryoprotective agent; (b) combining the cells with the freezing
medium to form a cell pool; and (c) freezing the cells in the cell
pool.
[0024] In some embodiments, the pH is adjusted to a pH of about 6.7
to about 8.3, about 6.8 to about 8.3, about 6.9 to about 8.3, about
7.0 to about 8.3, about 7.1 to about 8.3, about 7.2 to about 8.3,
about 7.3 to about 8.3, about 7.4 to about 8.3, about 7.5 to about
8.3, about 7.2 to about 8.0, about 7.2 to about 7.8, or about 7.5.
In some embodiments, the adjusted pH is a target pH or a measured
pH. In some embodiments, the target pH is about 6.7 to about 8.5,
or any of the pH or in any pH ranges described herein. In some
embodiments, the measured pH is about 6.7 to about 8.5, or any of
the pH or in any pH ranges described herein.
[0025] In some embodiments, the method further comprises a step of
measuring the adjusted pH of the freezing medium. In some
embodiments, if the measured pH of the freezing medium is below a
target pH, the method further comprises repeating the adjusting
step and measuring step until the adjusted pH of the freezing
medium is about 6.7 to about 8.5, or any of the pH or in any pH
ranges described herein.
[0026] In some embodiments of the methods described above or
herein, the pH is adjusted by adding a base. In some embodiments,
the base is selected from the group consisting of sodium carbonate,
sodium bicarbonate, HEPES sodium salt, sodium hydroxide, and
potassium hydroxide.
[0027] In some embodiments of the methods described above or
herein, the cells (e.g., mammalian cells or insect cells) are in a
medium having a pH of about 6.2 to about 6.6 before the cells are
combined with a freezing medium.
[0028] In some embodiment of the methods described above or herein,
the cryoprotective agent in the freezing medium is DMSO, glycerol,
propanediol, ethylene glycol, a macromolecule, a sugar, or a
combination thereof. In some embodiment, the DMSO or glycerol in
the cell pool is at a concentration of about 5% to about 12.5% by
volume, or about 5% to about 10% by volume.
[0029] In some embodiments of the methods described above or
herein, the cell pool contains the cells (e.g., mammalian cells or
insect cells) at a cell density of about 8% to about 28% packed
cell volume (PCV).
[0030] In some embodiments of the methods described above or
herein, the method further comprises a step of cooling cell culture
fluid during cell harvest and concentration process before the
cells (e.g., mammalian cells or insect cells) are combined with a
freezing medium. In some embodiments, the cell culture fluid is
cooled to a temperature at or below about 20.degree. C. In some
embodiments, the cell culture fluid is cooled to a temperature at
or below about 10.degree. C.
[0031] In some embodiments of the methods described above or
herein, the cells are mammalian cells, such as Chinese hamster
ovary (CHO) cells, NS0 murine myeloma cells, PER.C6.RTM. human
cells, or hybridomas. In some embodiments, the cells are insect
cells, such as High Five.TM., S2 (Schneider 2), Sf9, and Sf21. In
some embodiments of the methods described above or herein, the
cells (e.g., mammalian cells or insect cells) comprise a nucleic
acid encoding a polypeptide. In some embodiments, the polypeptide
is a therapeutic protein. In some embodiments, the therapeutic
protein is selected from the group consisting of an antibody, an
antibody fragment, an enzyme, and a receptor fusion protein.
[0032] In another aspect, provided herein is an eukaryotic cell
pool (e.g., a mammalian cell pool, or an insect cell pool) for
freezing cells comprising a buffered solution, a cryoprotective
agent, and eukaryotic cells comprising a nucleic acid encoding a
polypeptide, wherein the medium has a pH of about 6.7 to about 8.5
or about 7.2 to about 8.3 (or any of the pH or in any pH ranges
described herein) prior to freezing the cells. In some embodiments,
the cells are mammalian cells, such as Chinese hamster ovary (CHO)
cells, NS0 murine myeloma cells, PER.C6.RTM. human cells, or
hybridomas. In some embodiments, the cells are insect cells, such
as High Five.TM., S2 (Schneider 2), Sf9, and Sf21. In some
embodiments, the cells (e.g., mammalian cells or insect cells)
comprise a nucleic acid encoding a polypeptide. In some
embodiments, the polypeptide is a therapeutic protein. In some
embodiments, the therapeutic protein is selected from the group
consisting of an antibody, an antibody fragment, an enzyme, and a
receptor fusion protein.
[0033] In another aspect, provided herein is a cell bank comprising
a plurality of containers and each container contains (a) a
freezing medium comprising a buffer and a cryoprotective agent, and
(b) eukaryotic cells (e.g., mammalian cells or insect cells)
comprising a nucleic acid encoding a polypeptide, wherein the
freezing medium has a pH of about 6.7 to about 8.5 or about 7.2 to
about 8.3 (or any of the pH or in any pH ranges described herein)
prior to freezing the cell. In some embodiments, the containers are
ampoules. In some embodiments, the cells are mammalian cells, such
as Chinese hamster ovary (CHO) cells, NS0 murine myeloma cells,
PER.C6.RTM. human cells, or hybridomas. In some embodiments, the
cells are insect cells, such as High Five.TM., S2 (Schneider 2),
Sf9, and Sf21. In some embodiments, the cells (e.g., mammalian
cells or insect cells) comprise a nucleic acid encoding a
polypeptide. In some embodiments, the polypeptide is a therapeutic
protein. In some embodiments, the therapeutic protein is selected
from the group consisting of an antibody, an antibody fragment, an
enzyme, and a receptor fusion protein.
[0034] It is to be understood that one, some, or all of the
properties of the various embodiments described herein may be
combined to form other embodiments of the present invention. These
and other aspects of the invention will become apparent to one of
skill in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows an example of a cell banking process flow.
[0036] FIGS. 2A-2B show thaw passage day 1 viability (FIG. 2A) and
day 4 growth (PCV) (FIG. 2B) results for cell banks generated from
pH adjusted pools at initial pH of 6.6 or 6.3.
[0037] FIGS. 3A-3F show thaw passage day 1 viability (FIGS. 3A, 3C,
and 3E) and overall growth rate by PCV (FIGS. 3B, 3D, and 3F) vs.
banking pH and the effect of adjusting pH to a range of pH targets
across nine CHO cell lines each producing a different antibody
(antibodies 1-9).
[0038] FIGS. 4A-4B show thaw passage viable cell density (VCD) or
viable cell count (VCC) (FIG. 4A) and viability (%) (FIG. 4B)
trends, which demonstrate the effect of adjusting pH to 7.5 from an
initial pool pH of 6.3 vs. no pH adjustment.
[0039] FIG. 5 shows the ratio of viable packed cell volume (VPCV)
to viable cell count (VCC), which is an indirect means of
estimating cell size, during the pooling process. The data show the
effect of freeze media (FM) addition and pH adjustment to pH 7.3,
7.6 or 8.0 on cell size. A larger ratio indicates a larger cell
size.
[0040] FIGS. 6A-6B show thaw passage overall growth rate by PCV
results for cell banks generated from pH adjusted pools at an
initial pH of 6.4 (target 6.2) or 6.6 (target 6.7) at to (FIG. 6A)
and t2 (2 hours) (FIG. 6B).
DETAILED DESCRIPTION
[0041] Provided herein are methods of improving thaw recovery of
cell banks comprising freezing eukaryotic cells (e.g., mammalian
cells, insect cells, etc.) for banking in a freezing medium,
wherein the freezing medium comprises a buffered solution and a
cryoprotective agent, and wherein the freezing medium has a pH of
about 6.7 to about 8.5 prior to freezing.
[0042] Also provided herein are methods of freezing eukaryotic
cells (e.g., mammalian cells, insect cells, etc.) for storage
comprising freezing the cells in a freezing medium, wherein the
freezing medium comprises a buffered solution and a cryoprotective
agent, and wherein the freezing medium has a pH of about 6.7 to
about 8.5 prior to freezing.
[0043] Also provided herein are methods of freezing eukaryotic
cells (e.g., mammalian cells, insect cells, etc.) for storage or
improving thaw recovery of cell banks comprising (a) adjusting pH
of a freezing medium containing the cells to a pH of about 6.7 to
about 8.5, wherein the freezing medium comprises a buffered
solution and a cryoprotective agent; and (b) freezing the
cells.
[0044] Also provided herein are methods of freezing eukaryotic
cells (e.g., mammalian cells, insect cells, etc.) for storage or
improving thaw recovery of cell banks comprising (a) adjusting pH
of a freezing medium to a pH of about 6.7 to about 8.5, wherein the
freezing medium comprises a buffered solution and a cryoprotective
agent; (b) combining the cells with the freezing medium to form a
cell pool; and (c) freezing the cells in the cell pool.
[0045] Also provided herein are eukaryotic cell pools for freezing
eukaryotic cells (e.g., mammalian cells, insect cells, etc.)
comprising a buffered solution, a cryoprotective agent, and
eukaryotic cells comprising a nucleic acid encoding a polypeptide,
wherein the medium has a pH of about 6.7 to about 8.5 prior to
freezing the cells.
[0046] Also provided herein are cell banks comprising a plurality
of containers and each container contains (a) a freezing medium
comprising a buffer and a cryoprotective agent, and (b) eukaryotic
cells (e.g., mammalian cells, insect cells, etc.) comprising a
nucleic acid encoding a polypeptide, wherein the freezing medium
has a pH of about 6.7 to about 8.5 prior to freezing the cell.
I. Definitions
[0047] The terms "medium" and "cell culture medium" refer to a
solution used for maintaining cells. The medium may further
comprise a nutrient source used for growing cells. As is understood
by a person of skill in the art, the nutrient source may contain
components required by the cell for growth and/or survival or may
contain components that aid in cell growth and/or survival.
Vitamins, essential or non-essential amino acids, and trace
elements are examples of medium components.
[0048] A "basal nutrient medium" refers to a medium comprising the
basic nutrients required for cell growth and survival. Examples of
a basal nutrient medium include Eagle's Minimum Essential Medium
(EMEM) and Dulbecco's Modified Eagle's Medium (DMEM).
[0049] A "chemically defined cell culture medium" or "CDM" is a
medium with a specified composition that is free of products
derived from animal or plant such as for example animal serum and
plant peptone. As would be understood by a person of skill in the
art, a CDM may be used in a process of polypeptide production
whereby a cell is in contact with, and secretes a polypeptide into,
the CDM. Thus, it is understood that a composition may contain a
CDM and a polypeptide product and that the presence of the
polypeptide product does not render the CDM chemically
undefined.
[0050] A "chemically undefined cell culture medium" refers to a
medium whose chemical composition cannot be specified and which may
contain one or more products derived from animal or plant sources,
for example animal serum or plant peptone. As would be understood
by a person of skill in the art, a chemically undefined cell
culture medium may contain a product derived from an animal or a
plant as a nutrient source.
[0051] A "freezing medium", "cell freezing medium" or "cell culture
medium for freezing" refers to a buffered solution containing a
cryoprotective agent. A freezing medium may be used for freezing
cells (e.g., mammalian cells or insect cells) contained in the
freezing medium. A "buffered solution", as used herein, refers to a
water-based, isotonic, pH buffered salt solution, which acts to
preserve the integrity of the cell membrane and serves as a carrier
for one or more cryoprotective agents. A freezing medium may also
contain additional components found in cell culture medium.
Examples of buffers may include bicarbonate buffer, PBS (phosphate
buffered saline), HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS
(3-(N-morpholino)propanesulfonic acid), TES
(N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), TRIS
(tris(hydroxymethyl)aminomethane), TEST (TES/TRIS combo), and a
combination thereof. Examples of medium may include Eagle's Minimum
Essential Medium (EMEM) and Dulbecco's Modified Eagle's Medium
(DMEM). Cryoprotective agents protect cells from freezing damage
and may be classified as "permeating", able to cross the plasma
membrane (e.g., glycerol, dimethyl sulfoxide (DMSO), propanediol,
ethylene glycol, etc.), or "non-permeating" (e.g., macromolecules,
sugars, etc.).
[0052] "Culturing" a cell refers to contacting a cell with a cell
culture medium under conditions suitable to the survival and/or
growth and/or proliferation of the cell.
[0053] "Batch culture" refers to a culture in which all components
for cell culturing (including the cells and all culture nutrients)
are supplied to the culturing vessel at the start of the culturing
process.
[0054] The phrase "fed batch cell culture," as used herein refers
to a batch culture wherein the cells and culture medium are
supplied to the culturing vessel initially, and additional culture
nutrients are fed, continuously or in discrete increments, to the
culture during the culturing process, with or without periodic cell
and/or product harvest before termination of culture.
[0055] "Perfusion culture" is a culture by which the cells are
restrained in the culture by, e.g., filtration, encapsulation,
anchoring to microcarriers, etc., and the culture medium is
continuously or intermittently introduced and removed from the
culturing vessel.
[0056] "Cell banking" or "banking" is a process by which cells are
frozen to sub-zero temperatures (cryopreserved) to halt
enzymatic/chemical reactions, thus maintaining cells in a viable
state for later use. The frozen cells may be stored at less than
about 0.degree. C. (e.g., at -20.degree. C., -70.degree. C.,
-80.degree. C., or lower) for later use. For example, cells may be
stored in ampoules placed in the vapor phase within a freezer
containing liquid nitrogen at -196.degree. C.
[0057] "Culturing vessel" refers to a container used for culturing
a cell. The culturing vessel can be of any size so long as it is
useful for the culturing of cells.
[0058] The term "titer" as used herein refers to the total amount
of recombinantly expressed polypeptide produced by a cell culture
divided by a given amount of medium volume. Titer is typically
expressed in units of milligrams of polypeptide per milliliter of
medium.
[0059] A "nucleic acid," as used interchangeably herein, refer to
polymers of nucleotides of any length, and include DNA and RNA. The
nucleotides can be deoxyribonucleotides, ribonucleotides, modified
nucleotides or bases, and/or their analogs, or any substrate that
can be incorporated into a polymer by DNA or RNA polymerase, or by
a synthetic reaction. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and their analogs. If
present, modification to the nucleotide structure may be imparted
before or after assembly of the polymer.
[0060] An "isolated nucleic acid" means and encompasses a
non-naturally occurring, recombinant or a naturally occurring
sequence outside of or separated from its usual context. An
isolated nucleic acid molecule is other than in the form or setting
in which it is found in nature. Isolated nucleic acid molecules
therefore are distinguished from the nucleic acid molecule as it
exists in natural cells. However, an isolated nucleic acid molecule
includes a nucleic acid molecule contained in cells that ordinarily
express the protein where, for example, the nucleic acid molecule
is in a chromosomal location different from that of natural
cells.
[0061] An "isolated" protein (e.g., an isolated antibody) is one
which has been identified and separated and/or recovered from a
component of its natural environment. Contaminant components of its
natural environment are materials which would interfere with
research, diagnostic or therapeutic uses for the protein, and may
include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. Isolated protein includes the protein in
situ within recombinant cells since at least one component of the
protein's natural environment will not be present. Ordinarily,
however, isolated protein will be prepared by at least one
purification step.
[0062] A "purified" polypeptide means that the polypeptide has been
increased in purity, such that it exists in a form that is more
pure than it exists in its natural environment and/or when
initially produced and/or synthesized and/or amplified under
laboratory conditions. Purity is a relative term and does not
necessarily mean absolute purity.
[0063] "Contaminants" refer to materials that are different from
the desired polypeptide product. The contaminant includes, without
limitation: host cell materials, such as host cell protein; nucleic
acid; a variant, fragment, aggregate or derivative of the desired
polypeptide; another polypeptide; endotoxin; viral contaminant;
cell culture media component, etc. Contaminants may also include
materials introduced by purification process, such as leached
Protein A.
[0064] The terms "polypeptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified naturally or by intervention; for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labeling component. Also included within the definition are, for
example, polypeptides containing one or more analogs of an amino
acid (including, for example, unnatural amino acids, etc.), as well
as other modifications known in the art. Examples of polypeptides
encompassed within the definition herein include mammalian
proteins, such as, e.g., renin; a growth hormone, including human
growth hormone and bovine growth hormone; growth hormone releasing
factor; parathyroid hormone; thyroid stimulating hormone;
lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin
B-chain; proinsulin; follicle stimulating hormone; calcitonin;
luteinizing hormone; glucagon; clotting factors such as factor
VIIIC, factor IX, tissue factor, and von Willebrands factor;
anti-clotting factors such as Protein C; atrial natriuretic factor;
lung surfactant; a plasminogen activator, such as urokinase or
human urine or tissue-type plasminogen activator (t-PA); bombesin;
thrombin; hemopoietic growth factor; tumor necrosis factor-alpha
and -beta; enkephalinase; RANTES (regulated on activation normally
T-cell expressed and secreted); human macrophage inflammatory
protein (MIP-1-alpha); a serum albumin such as human serum albumin;
Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; a microbial
protein, such as beta-lactamase; DNase; IgE; a cytotoxic
T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;
activin; vascular endothelial growth factor (VEGF); receptors for
hormones or growth factors; protein A or D; rheumatoid factors; a
neurotrophic factor such as bone-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6),
or a nerve growth factor such as NGF-b; platelet-derived growth
factor (PDGF); fibroblast growth factor such as aFGF and bFGF;
epidermal growth factor (EGF); transforming growth factor (TGF)
such as TGF-alpha and TGF-beta, including TGF-.beta.1, TGF-.beta.2,
TGF-.beta., TGF-.beta.4, or TGF-.beta.5; insulin-like growth
factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I),
insulin-like growth factor binding proteins (IGFBPs); CD proteins
such as CD3, CD4, CD8, CD19 and CD20; erythropoietin;
osteoinductive factors; immunotoxins; a bone morphogenetic protein
(BMP); an interferon such as interferon-alpha, -beta, and -gamma;
colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;
interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase;
T-cell receptors; surface membrane proteins; decay accelerating
factor; viral antigen such as, for example, a portion of the AIDS
envelope; transport proteins; homing receptors; addressins;
regulatory proteins; integrins such as CDT11a, CD11b, CD11c, CD18,
an ICAM, VLA-4 and VCAM; a tumor associated antigen such as CA125
(ovarian cancer antigen) or HER2, HER3 or HER4 receptor;
immunoadhesins; and fragments and/or variants of any of the
above-listed proteins as well as antibodies, including antibody
fragments, binding to a protein, including, for example, any of the
above-listed proteins.
[0065] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired biological activity. An antibody
can be human, humanized and/or affinity matured.
[0066] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that can be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they can be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling et al, in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),
recombinant DNA methods in bacterial, eukaryotic animal or plant
cells (see, e.g., U.S. Pat. No. 4,816,567); phage-display
technologies (see, e.g., Clackson et al., Nature, 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et
al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol.
Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci.
USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
Methods 284(1-2): 119-132 (2004) and technologies for producing
human or human-like antibodies in animals that have parts or all of
the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad.
Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258
(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg
et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813
(1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996);
Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and
Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0067] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of the active ingredient to be effective, and which
contains no additional components which are unacceptably toxic to a
subject to which the formulation would be administered. Such
formulations are sterile.
[0068] "Pharmaceutically acceptable" carriers, excipients, or
stabilizers are ones which are nontoxic to the cell or mammal being
exposed thereto at the dosages and concentrations employed
(Remington's Pharmaceutical Sciences (20.sup.th edition), ed. A.
Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia,
Pa.). Often the physiologically acceptable carrier is an aqueous pH
buffered solution. Examples of physiologically acceptable carriers
include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as Tween.TM., polyethylene glycol (PEG), and
Pluronics.TM..
[0069] As used in this specification and the appended claims, the
singular forms "a", "an" and "the" include plural referents unless
the content clearly dictates otherwise. Thus, for example,
reference to "a compound" optionally includes a combination of two
or more such compounds, and the like.
[0070] It is understood that aspect and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
[0071] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X." Numeric ranges are inclusive of the
numbers defining the range.
[0072] Where aspects or embodiments of the invention are described
in terms of a Markush group or other grouping of alternatives, the
present invention encompasses not only the entire group listed as a
whole, but each member of the group individually and all possible
subgroups of the main group, but also the main group absent one or
more of the group members. The present invention also envisages the
explicit exclusion of one or more of any of the group members in
the claimed invention.
II. Methods and Uses of the Invention
[0073] Provided herein are methods of freezing cells for banking or
storage in a cell freezing medium. Also provided herein are methods
of improving thaw recovery of cell banks. Methods comprise a step
of freezing cells in a freezing medium, wherein the freezing medium
comprises a buffered solution and a cryoprotective agent, and
wherein the freezing medium containing the cells has a pH of about
6.7 to about 8.5 or about 6.7 to about 8.3 prior to freezing. The
methods may further comprise a step of adjusting the pH of the
freezing medium to about 6.7 to about 8.5 or about 6.7 to about
8.3. The methods provided herein are useful for preparing master
cell banks (MCBs) and working cell banks (WCBs). In some
embodiments, the methods described herein improve cell viability
and/or cell growth after thawing.
[0074] Eukaryotic cells (e.g., mammalian cells, insect cells, etc.)
to be used in freezing and banking process may be prepared by a
process involving cell culture and concentration protocols known in
the art. The method may include cell accumulation, harvest, and
cell concentration before cell banking. Cell accumulation may occur
by several methods. One example may use a process controlled
bioreactor for cell accumulation; however, other methods/culture
vessels may be used as well (e.g. T-flasks, shake flasks, roller
bottles, spinner vessels, etc.). Harvest and cell concentration may
be performed by centrifugation followed by resuspension of the cell
pellet in a freezing medium. In another example, cells may be
harvested and concentrated in a single step via a hollow fiber
filter (HFF). Cell concentration may also be achieved by use of
alternative perfusion membrane/devices to remove medium from cell
culture fluid (e.g. floating perfusion membranes, cell settlers,
continuous circulating centrifuges, etc.). In some embodiments of
the method described herein, the harvesting and cell concentration
process or the harvested cell culture fluid is cooled to a
temperature at or below about 20.degree. C. (e.g., at or below
about any of 19.degree. C., 18.degree. C., 17.degree. C.,
16.degree. C., 15.degree. C., 14.degree. C., 13.degree. C.,
12.degree. C., 11.degree. C., and 10.degree. C.).
[0075] Pelleted or concentrated cells may then be combined with a
freezing medium before freezing the cells. In some embodiments,
pelleted cells can be resuspended in a freezing medium. In some
embodiments, a freezing medium containing concentrated
cryoprotective agent can be added into the harvested and
concentrated cells or the harvested and concentrated cells can be
added into a freezing medium containing concentrated cryoprotective
agent for cell banking. A freezing medium may comprise a buffer
solution and a cryoprotective agent. In some embodiments, the
buffer in the medium may comprise a zwitterionic buffer. In some
embodiments, the buffer in the medium may comprise a buffer
selected from bicarbonate buffer, PBS (phosphate buffered saline),
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS
(3-(N-morpholino)propanesulfonic acid), TES
(N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), TRIS
(tris(hydroxymethyl)aminomethane), TEST (TES/TRIS combo), and a
combination thereof. In some embodiments, buffer concentration in
the freezing medium before freezing the cells is about 10 mM to
about 50 mM. In some embodiments, the freezing medium before
freezing the cells contains about 10 mM to about 35 mM sodium
bicarbonate (e.g., about 10 mM, about 15 mM, about 20 mM, about 25
mM, about 30 mM, or about 35 mM, including any concentration in
between these values). In some embodiments, the freezing medium
before freezing the cells contains about 10 mM to about 50 mM HEPES
(e.g., about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30
mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM,
including any concentration in between these values). In some
embodiments, the freezing medium before freezing the cells contains
about 10 mM to about 12 mM PBS (e.g., about 10 mM, about 11 mM, or
about 12 mM, including any concentration in between these values).
In some embodiments, the freezing medium before freezing the cells
contains about 10 mM to about 20 mM MOPS (e.g., about 10 mM, about
12 mM, about 15 mM, about 18 mM, or about 20 mM, including any
concentration in between these values). In some embodiments, the
freezing medium before freezing the cells contains about 10 mM to
about 30 mM TES (e.g., about 10 mM, about 15 mM, about 20 mM, about
25 mM, or about 30 mM, including any concentration in between these
values). In some embodiments, the freezing medium before freezing
the cells contains about 10 mM to about 30 mM TRIS (e.g., about 10
mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM,
including any concentration in between these values). In some
embodiments, the freezing medium before freezing the cells contains
about 10 mM to about 30 mM TEST (e.g., about 10 mM, about 15 mM,
about 20 mM, about 25 mM, or about 30 mM, including any
concentration in between these values). In some embodiments, a
cryoprotective agent is a permeating agent or a non-permeating
agent. In some embodiments, a cryoprotective agent is an agent
selected from a group consisting of glycerol, dimethylsulfoxide
(DMSO), propanediol, ethylene glycol, and sugars. In some
embodiments, the freezing medium added into the harvested and
concentrated cells is a concentrated freezing medium. In some
embodiments, the freezing medium containing concentrated
cryoprotective agent may contain 20-30% (v/v) dimethylsulfoxide
(DMSO) or glycerol. In some embodiments, the concentrated freezing
medium is poured (e.g., 1 part freezing medium (containing
concentrated cryoprotective agent) volume:3 parts cell culture
fluid) into the harvested and concentrated cells. In some
embodiments, the freezing medium containing the cells before
freezing the cells contains about 5% to about 12.5% of DMSO or
glycerol.
[0076] In some embodiments, a freezing medium may further comprise
additional components found in cell culture medium. In some
embodiments, the freezing medium may contain Eaglel's Minimum
Essential Medium (EMEM) or Dulbecco's Modified Eagle's Medium
(DMEM).
[0077] In some embodiments, the method of preparing a cell (such as
a mammalian cell or an insect cell) for freezing may further
comprise a step of adjusting the pH of a freezing medium or a
freezing medium containing concentrated cryoprotective agent,
wherein the pH is adjusted to about 6.7 to about 8.5 before the
pelleted cells or concentrated cells are combined with the freezing
medium or the freezing medium containing concentrated
cryoprotective agent. In some embodiments, the method of preparing
cells (such as mammalian cells or insect cells) for freezing
further comprises a step of adjusting the pH of the freezing medium
containing the cells, wherein the pH of the freezing medium is
adjusted to about 6.7 to about 8.5. In some embodiments, the
adjusted pH is a target pH or a measured pH.
[0078] In certain embodiments, the cell density prior to freezing
is measured by packed cell volume (PCV). In some embodiments, the
freezing medium comprising cells to be banked has a cell density of
8% to 28% (e.g., about any of 8%, 10%, 15%, 20%, 25% or 28%) PCV
prior to freezing. In some embodiments, the cell density in the
freezing medium before freezing may be about 21% PCV.
[0079] In some embodiments, the cells in a freezing medium are
dispensed into ampoules or single-use bags prior to freezing. In an
exemplary embodiment, the process involves: dispensing the cell
suspension into autoclaved glass ampoules that are placed on wet
ice using an autoclaved self-filling syringe, sealing the ampoules,
performing an integrity test, and freezing ampoules in a
rate-controlled freezer and then transferring ampoules to a liquid
nitrogen freezer for long term storage.
[0080] In some embodiments, the cell viability after thawing is
improved by using the methods described herein. In some
embodiments, the cell viability is increased by at least about any
of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, or 85% as compared to the cell viability after frozen in a
freezing medium with a pH of 6.7 or lower and/or without cooling
the harvest cell culture during the harvesting process and
thawing.
[0081] pH Adjustment
[0082] According to the methods as described herein, the pH of the
freezing medium (containing or not containing the cells) may be
adjusted, for example, by adding a base to the medium. In some
embodiments, the pH of the freezing medium is adjusted to a pH that
is above about 6.7. In some embodiments, the pH of the freezing
medium is adjusted to a pH (a target pH or a measured pH) between
about 6.7 and about 8.5. In some embodiments, the pH of the
freezing media is adjusted to a pH between about 6.8 to about 8.3,
between about 6.9 to about 8.3, between about 7.0 to about 8.3,
between about 7.1 to about 8.3, between about 7.2 to about 8.3,
between about 7.3 to about 8.3, between about 7.4 to about 8.3,
between about 7.5 to about 8.3, between about 7.6 to about 8.3,
between about 7.7 to about 8.3, or between about 7.8 to about 8.3
In some embodiments, the pH of the freezing media is adjusted to a
pH (a target pH or a measured pH) between about 7.2 to about 7.8.
In some embodiments, the target pH or measured pH is about 7.2 to
about 8.3. In some embodiments, the target pH or measured pH is
about 7.2 to about 7.8 (e.g. pH of about 7.5). In some embodiments,
if the first pH adjustment is not sufficient to increase the pH to
be with the target pH range (e.g., pH of about 7.3 to about 7.7), a
second pH adjustment is performed. In some embodiments, more than
one pH adjustments may be performed.
[0083] The base added to adjust the pH may be any base that is well
known to those skilled in the art, but in exemplary embodiments,
the base is sodium carbonate, sodium bicarbonate, HEPES sodium
salt, sodium hydroxide, or potassium hydroxide.
[0084] The pH of the freezing media may be measured at any point
prior to freezing and the pH may be adjusted at any time prior to
freezing. In some embodiments, the pH of the freezing medium is
measured and/or adjusted prior to combination with the cells to be
banked. In other embodiments, the pH of the freezing medium is
measured and/or adjusted after combination with the cells to be
banked. In some embodiments, the pH of the freezing medium is
measured and/or adjusted more than once. In some embodiments, the
pH of the freezing medium is measured and/or adjusted twice, three
times, or more prior to freezing. In other embodiments, the pH of
the freezing medium is measured and/or adjusted before and after
combination with the cells to be banked. In some embodiments, the
pH is adjusted according to the following equation:
V.sub.base=C.sub.base*V.sub.p (pH.sub.t-pH.sub.i), wherein
C.sub.base is a base-specific coefficient, V.sub.base is a volume
of the base to add to the freezing medium, V.sub.p is the volume of
the freezing medium, pH.sub.t is the target pH, and pH.sub.i is the
initial pH. As used herein, the initial pH is the pH of the
freezing medium containing the cells (i.e., after it is combined
with the cells) but before the pH is adjusted for freezing.
C.sub.base represents a specific coefficient that depends on the
type and concentration of base chosen for pH adjustment. The
C.sub.base coefficient can be obtained depending on the choice of
base. In an exemplary embodiment, where the base is 1M sodium
carbonate, the pH adjustment is performed according to Equation 1,
below:
Equation 1: Calculation of Base Volume to Add for pH Adjust
[0085] V.sub.Na2CO3=0.0085V.sub.p(pH.sub.t-pH.sub.i),
wherein V.sub.Na2CO3 is a volume of 1M sodium carbonate to add into
the freezing medium, V.sub.p is the volume of the freezing medium,
pH.sub.t is the target pH, and pH.sub.i is the initial pH. The
initial pH is the pH of the freezing medium containing the cells
(i.e., after it is combined with the cells) but before the pH is
adjusted for freezing. The target pH of the freezing medium may be
a pH that is above physiological pH, such as a pH above 7.2. In
some embodiments, the target pH of the freezing medium is between
7.2 and 8.3. In some embodiments, the target pH of the freezing
medium is between 7.2 and 7.8. In some embodiments, the target pH
of the freezing medium is at any of 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
7.8, 7.9, 8.0, 8.1, 8.2, and 8.3.
[0086] The pH of the freezing medium can be measured using methods
known in the art. For example, the pH of the medium can be measured
on BioProfile.RTM. 400 (Nova Biomedical) or BioProfile.RTM. FLEX
(Nova Biomedical) analyzers. As used herein, all references to pH
in this application, including measured pH, target pH, adjusted pH,
and initial pH refer to a pH measurement taken with the sample
temperature adjusted to about 37.degree. C. (e.g., between
36.degree. C. and 38.degree. C. or between 35.degree. C. and
39.degree. C.).
III. Freezing Media
[0087] Cell freezing media provided herein may find use in methods
(e.g., a method of freezing eukaryotic cells (e.g., mammalian
cells, insect cells, etc.); and/or a method of improving thaw
recovery of cell banks comprising eukaryotic cells (e.g., mammalian
cells or insect cells)) and in compositions (e.g., a cell pool
comprising a buffered solution, a cryoprotective agent, and
eukaryotic cells (e.g., mammalian cells or insect cells)).
[0088] In some embodiments, a freezing medium described herein
comprises a buffered solution and a cryoprotectant. In some
embodiments, the buffer comprises a zwitterionic buffer. In some
embodiments, the buffer includes PBS, HEPES, TES, TRIS, and
TEST.
[0089] The freezing medium may comprise any cryoprotectant known in
the art and described herein, such as DMSO, glycerol, ethylene
glycol, non-permeating macromolecules, sugars, etc. In some
embodiments, the concentration of DMSO or glycerol in the cell
freezing medium is 5%-12.5% by volume (v/v) after combination with
the cells to be banked. In some embodiments, the freezing medium
may be provided by adding a freezing medium containing concentrated
buffers and/or cryoprotectant into concentrated cells. In some
embodiments, the freezing medium concentrated cryoprotective agent
may contain about 20% to about 30% (v/v) DMSO or glycerol.
[0090] In some embodiments, a freezing medium may comprises
additional components found in cell culture medium. In some
embodiments, Ham's F10 (Sigma), Minimal Essential Medium ([MEM],
Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
([DMEM], Sigma) that are suitable for culturing mammalian cells may
be added into the freezing medium described herein for freezing
mammalian cells. In addition, any of the media described in Ham and
Wallace, Meth. Enz., 58:44 (1979), Barnes and Sato, Anal. Biochem.,
102:255 (1980), Vijayasankaran et al., Biomacromolecules.,
6:605:611 (2005), Patkar et al., J Biotechnology, 93:217-229
(2002), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; or
4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. No. Re. 30,985; or
U.S. Pat. No. 5,122,469, the disclosures of all of which are
incorporated herein by reference in their entirety, may be
supplemented or modified as detailed herein.
[0091] As would be understood by the skilled artisan, the cell
freezing medium detailed herein may comprise other components that
are useful for cell culture or freezing. For example, it is
understood that the media may comprise additional components such
as amino acids (e.g., glutamine, arginine, or asparagine), vitamins
(including but not limited to B vitamins such as any one or more of
vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7, vitamin
B9, or vitamin B12), transition metals (including but not limited
to nickel, iron (e.g., ferric iron or ferrous iron), or zinc), and
other media components. Any media provided herein may also be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), ions
(such as sodium, chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleosides (such as adenosine and
thymidine), trace elements (defined as inorganic compounds usually
present at final concentrations in the micromolar range), and
glucose or an equivalent energy source. In some aspects, a freezing
medium provided herein contains proteins derived from a plant or an
animal. In some embodiments, a freezing medium provided herein is
free of proteins derived from a plant or an animal. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art.
[0092] The cell freezing medium (a cell pool) as described herein
may further comprise one or more cells to be banked. In an
exemplary embodiment, these cells are mammalian cells, such as CHO
cells. Exemplary cell types that may find use in the methods
described herein include Chinese hamster ovary (CHO) cells, NS0
murine myeloma cells, PER.C6.RTM. human cells, and hybridomas. In
another exemplary embodiment, these cells are insect cells, such as
High Five.TM., S2 (Schneider 2), Sf9, and Sf21. In some
embodiments, the cells are recombinant cells comprising a
heterologous nucleic acid encoding a polypeptide (e.g., a
therapeutic protein). As one of skill in the art would appreciate,
these cells may further comprise recombinant plasmids or other
useful biological compounds. In some embodiments, the cells to be
banked may be useful for the production of therapeutic proteins and
biological products such as antibodies, antibody fragments,
enzymes, receptor fusion proteins, or fragments thereof.
IV. Eukaryotic Cells and Cell Banks
[0093] Also provided herein is a cell bank comprising a plurality
of containers and each container containing a freezing medium
containing an eukaryotic cell (e.g., a mammalian cell, an insect
cell, etc.) comprising a nucleic acid (e.g., a heterologous nucleic
acid) encoding a polypeptide, wherein the medium has a pH of about
6.7 to about 8.5 prior to freezing the cell. The cell bank may be a
Prebank, a master cell bank (MCB), or a working cell bank (WCB). A
Prebank may comprise frozen (e.g., stored in liquid nitrogen
freezer) containers (e.g., ampoules) containing cells producing a
specific polypeptide from which a MCB is prepared. A MCB may
comprise frozen (e.g., stored in liquid nitrogen freezer)
containers (e.g., ampoules) containing a cell culture derived from
the subculture of the Prebank and from which all subsequence cells
for production are derived. MCBs are produced and stored in
accordance with cGMPs and may be used for the production of
polypeptide product. A WCB may comprise frozen (e.g., stored in
liquid nitrogen freezer) containers (e.g., ampoules) containing a
cell culture derived from the subculture of the MCB. WCBs are
produced and stored in accordance with cGMPs and may be used for
the production of polypeptide product. In some embodiments, the
containers are ampoules.
[0094] Eukaryotic cells (e.g., mammalian cells, insect cells, etc.)
that can be frozen in a freezing medium and stored as described
herein may include any eukaryotic cells that can be cultured and/or
are useful for producing a polypeptide. In some embodiments, the
cell is a mammalian cell, such as a Chinese Hamster Ovary (CHO)
cell. CHO cells may include, but are not limited to, DHFR.sup.- CHO
cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)),
e.g., ATCC.RTM. CRL-9096.TM.; and CHO-K1 (ATCC.RTM.
CRL-61.TM.).
[0095] Other examples of mammalian cells include, without
limitation, monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2). Other useful mammalian host
cell lines include myeloma cell lines such as NS0 and Sp2/0. For a
review of certain mammalian host cell lines suitable for antibody
production, see, e.g., Yazaki and Wu, Methods in Molecular Biology,
Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp.
255-268.
[0096] In some embodiments, the cell is an insect cell line, such
as High Five.TM., S2 (Schneider 2), Sf9, and Sf21.
[0097] In some embodiments, the cells described herein (e.g.,
mammalian cell, or insect cells) comprise a nucleic acid (e.g., a
heterologous nucleic acid) encoding a polypeptide and the cells are
useful for producing the polypeptide. In some embodiments, the
nucleic acid is introduced into the cells. Any methods known in the
art for introducing a nucleic acid into a cell may be used. For
example, the cells may be transformed with vectors (e.g., an
expression vector) comprising one or more nucleic acids encoding
the polypeptide. In some embodiments, the cell is a stable cell
line. In some embodiments, the polypeptide is selected from the
group consisting of an antibody, an antibody fragment, an enzyme,
and a receptor fusion protein.
[0098] Examples of polypeptides include mammalian proteins, such
as, e.g., renin; a growth hormone, including human growth hormone
and bovine growth hormone; growth hormone releasing factor;
parathyroid hormone; thyroid stimulating hormone; lipoproteins;
alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;
follicle stimulating hormone; calcitonin; luteinizing hormone;
glucagon; clotting factors such as factor VIIIC, factor IX, tissue
factor, and von Willebrands factor; anti-clotting factors such as
Protein C; atrial natriuretic factor; lung surfactant; a
plasminogen activator, such as urokinase or human urine or
tissue-type plasminogen activator (t-PA); bombesin; thrombin;
hemopoietic growth factor; tumor necrosis factor-alpha and -beta;
enkephalinase; RANTES (regulated on activation normally T-cell
expressed and secreted); human macrophage inflammatory protein
(MIP-1-alpha); a serum albumin such as human serum albumin;
Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; a microbial
protein, such as beta-lactamase; DNase; IgE; a cytotoxic
T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;
activin; vascular endothelial growth factor (VEGF); receptors for
hormones or growth factors; protein A or D; rheumatoid factors; a
neurotrophic factor such as bone-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6),
or a nerve growth factor such as NGF-b; platelet-derived growth
factor (PDGF); fibroblast growth factor such as aFGF and bFGF;
epidermal growth factor (EGF); transforming growth factor (TGF)
such as TGF-alpha and TGF-beta, including TGF-.beta.1, TGF-.beta.2,
TGF-.beta., TGF-.beta.4, or TGF-.beta.5; insulin-like growth
factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I),
insulin-like growth factor binding proteins (IGFBPs); CD proteins
such as CD3, CD4, CD8, CD19 and CD20; erythropoietin;
osteoinductive factors; immunotoxins; a bone morphogenetic protein
(BMP); an interferon such as interferon-alpha, -beta, and -gamma;
colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;
interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase;
T-cell receptors; surface membrane proteins; decay accelerating
factor; viral antigen such as, for example, a portion of the AIDS
envelope; transport proteins; homing receptors; addressins;
regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18,
an ICAM, VLA-4 and VCAM; a tumor associated antigen such as CA125
(ovarian cancer antigen) or HER2, HER3 or HER4 receptor;
immunoadhesins; and fragments and/or variants of any of the
above-listed proteins as well as antibodies, including antibody
fragments, binding to a protein, including, for example, any of the
above-listed proteins.
[0099] In some embodiments, the cells described herein (e.g.,
mammalian cells, or insect cells) comprises a nucleic acid encoding
an antibody. In some embodiments, the antibody is a monoclonal
antibody. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),
phage-display technologies (see, e.g., Clackson et al., Nature,
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132 (2004), and technologies for
producing human or human-like antibodies in animals that have parts
or all of the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad.
Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258
(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg
et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813
(1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996);
Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and
Huszar, Intern. Rev. Immunol. 13: 65-93 (1995). In some
embodiments, the antibody is a humanized antibody, a chimeric
antibody, a human antibody, a library-derived antibody, or a
multispecific antibody. In some embodiments, the antibody is an
antigen-binding fragment thereof. Examples of antigen-binding
fragment include Fab, Fab', F(ab').sub.2, and Fv fragments;
diabodies; linear antibodies; single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments. The Fab
fragment contains the heavy- and light-chain variable domains and
also contains the constant domain of the light chain and the first
constant domain (CH1) of the heavy chain. Fab' fragments differ
from Fab fragments by the addition of a few residues at the carboxy
terminus of the heavy chain CH1 domain including one or more
cysteines from the antibody hinge region. Fab'-SH is the
designation herein for Fab' in which the cysteine residue(s) of the
constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known. "Fv" is the minimum antibody
fragment which contains a complete antigen-binding site.
"Single-chain Fv" or "scFv" antibody fragments comprise the VH and
VL domains of antibody, wherein these domains are present in a
single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York, 1994), pp. 269-315. Many of the methods
for purifying an antibody described above may be suitably adapted
for purifying an antigen-binding antibody fragment.
[0100] In some embodiments, the antibodies encoded by the nucleic
acid in the cells (e.g., mammalian cells, or insect cells)
including therapeutic and diagnostic antibodies. Antibodies within
the scope of the present invention include, but are not limited to:
anti-HER2 antibodies including Trastuzumab (HERCEPTIN.RTM.) (Carter
et al., Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992), U.S. Pat.
No. 5,725,856); anti-CD20 antibodies such as chimeric anti-CD20
"C2B8" as in U.S. Pat. No. 5,736,137 (RITUXAN.RTM.), a chimeric or
humanized variant of the 2H7 antibody as in U.S. Pat. No.
5,721,108B1, or Tositumomab (BEXXAR.RTM.); anti-IL-8 (St John et
al., Chest, 103:932 (1993), and International Publication No. WO
95/23865); anti-VEGF antibodies including humanized and/or affinity
matured anti-VEGF antibodies such as the humanized anti-VEGF
antibody huA4.6.1 AVASTIN.RTM. (Kim et al., Growth Factors, 7:53-64
(1992), International Publication No. WO 96/30046, and WO 98/45331,
published Oct. 15, 1998); anti-PSCA antibodies (WO01/40309);
anti-CD40 antibodies, including S2C6 and humanized variants thereof
(WO00/75348); anti-CD11a (U.S. Pat. No. 5,622,700, WO 98/23761,
Steppe et al., Transplant Intl. 4:3-7 (1991), and Hourmant et al.,
Transplantation 58:377-380 (1994)); anti-IgE (Presta et al., J.
Immunol. 151:2623-2632 (1993), and International Publication No. WO
95/19181); anti-CD18 (U.S. Pat. No. 5,622,700, issued Apr. 22,
1997, or as in WO 97/26912, published Jul. 31, 1997); anti-IgE
(including E25, E26 and E27; U.S. Pat. No. 5,714,338, issued Feb.
3, 1998 or U.S. Pat. No. 5,091,313, issued Feb. 25, 1992, WO
93/04173 published Mar. 4, 1993, or International Application No.
PCT/US98/13410 filed Jun. 30, 1998, U.S. Pat. No. 5,714,338);
anti-Apo-2 receptor antibody (WO 98/51793 published Nov. 19, 1998);
anti-TNF-.alpha. antibodies including cA2 (REMICADE.RTM.), CDP571
and MAK-195 (See, U.S. Pat. No. 5,672,347 issued Sep. 30, 1997,
Lorenz et al., J. Immunol. 156(4):1646-1653 (1996), and Dhainaut et
al., Crit. Care Med. 23(9):1461-1469 (1995)); anti-Tissue Factor
(TF) (European Patent No. 0 420 937 B1 granted Nov. 9, 1994);
anti-human .alpha..sub.4.beta..sub.7 integrin (WO 98/06248
published Feb. 19, 1998); anti-EGFR (chimerized or humanized 225
antibody as in WO 96/40210 published Dec. 19, 1996); anti-CD3
antibodies such as OKT3 (U.S. Pat. No. 4,515,893 issued May 7,
1985); anti-CD25 or anti-tac antibodies such as CHI-621
(SIMULECT.RTM.) and (ZENAPAX.RTM.) (See U.S. Pat. No. 5,693,762
issued Dec. 2, 1997); anti-CD4 antibodies such as the cM-7412
antibody (Choy et al., Arthritis Rheum 39(1):52-56 (1996));
anti-CD52 antibodies such as CAMPATH-1H (Riechmann et al., Nature
332:323-337 (1988)); anti-Fc receptor antibodies such as the M22
antibody directed against Fc.gamma.RI as in Graziano et al., J.
Immunol. 155(10):4996-5002 (1995); anti-carcinoembryonic antigen
(CEA) antibodies such as hMN-14 (Sharkey et al., Cancer Res. 55(23
Suppl): 5935s-5945s (1995); antibodies directed against breast
epithelial cells including huBrE-3, hu-Mc 3 and CHL6 (Ceriani et
al., Cancer Res. 55(23): 5852s-5856s (1995); and Richman et al.,
Cancer Res. 55(23 Supp): 5916s-5920s (1995)); antibodies that bind
to colon carcinoma cells such as C242 (Litton et al., Eur J.
Immunol. 26(1): 1-9 (1996)); anti-CD38 antibodies, e.g. AT 13/5
(Ellis et al., J. Immunol. 155(2):925-937 (1995)); anti-CD33
antibodies such as Hu M195 (Jurcic et al., Cancer Res 55(23
Suppl):5908s-5910s (1995) and CMA-676 or CDP771; anti-CD22
antibodies such as LL2 or LymphoCide (Juweid et al., Cancer Res
55(23 Suppl):5899s-5907s (1995)); anti-EpCAM antibodies such as
17-1A (PANOREX.RTM.); anti-GpIIb/IIIa antibodies such as abciximab
or c7E3 Fab (REOPRO.RTM.); anti-RSV antibodies such as MEDI-493
(SYNAGIS.RTM.); anti-CMV antibodies such as PROTOVIR.RTM.; anti-HIV
antibodies such as PRO542; anti-hepatitis antibodies such as the
anti-Hep B antibody OSTAVIR.RTM.; anti-CA 125 antibody OvaRex;
anti-idiotypic GD3 epitope antibody BEC2; anti-.alpha.v.beta.3
antibody VITAXIN.RTM.; anti-human renal cell carcinoma antibody
such as ch-G250; ING-1; anti-human 17-1A antibody (3622W94);
anti-human colorectal tumor antibody (A33); anti-human melanoma
antibody R24 directed against GD3 ganglioside; anti-human
squamous-cell carcinoma (SF-25); and anti-human leukocyte antigen
(HLA) antibodies such as Smart ID10 and the anti-HLA DR antibody
Oncolym (Lym-1). The preferred target antigens for the antibody
herein are: HER2 receptor, VEGF, IgE, CD20, CD11a, and CD40.
[0101] The following Examples are provided to illustrate but not to
limit the invention.
EXAMPLES
Example 1: Effect of Pool pH, Hold Temperature and Hold Time on
Thawed Cell Viability
[0102] Base Addition Method to Control Banking pH (Small Scale
Study #1)
[0103] A study was executed to gauge the feasibility of performing
a simple pH adjustment from worst case and typical pool conditions
(targeted initial pH of 6.2 and 6.7 respectively) to enhance thaw
recovery. Several pH adjustment targets were studied to determine
optimal banking pH. The pH was measured using the on
BioProfile.RTM. 400 (Nova Biomedical) instrument with the
temperature set to 37.degree. C. Similarly, pH calculations using
Equation 1 are based upon a sample temperature of 37.degree. C.
[0104] Cells were pelleted via centrifugation. Cell pools were
generated for each test condition by resuspending cells to
100.times.10.sup.6 cells/mL in spent media. The cell pools were
agitated at 37.degree. C. with or without gas permeable membranes
for CO.sub.2/O.sub.2 exchange to drive pH to initial pH conditions
(either 6.2 or 6.7). Upon reaching initial pH targets, pools were
quickly chilled to <10.degree. C. and 20% (v/v) DMSO freeze
media was added (1:3 parts cell culture fluid). pH adjustments were
performed using Equation 1 to calculate required volumes of 1M
sodium carbonate addition to target pH of 6.9, 7.1, and 7.3 and a
final offline pH measurement was taken to confirm an appropriate
increase in pH prior to mock bank generation (actual banking pH was
6.9, 7.1/7.2 and 7.5).
[0105] Thaw results showed a dramatic improvement in day 1
viability of up to 68% (from 24% to 92% for the adjustment from pH
6.2 to 7.5) (FIG. 2A), and improvement in day 4 PCV of up to 0.64%
(from 0.17% to 0.81% for the adjustment from pH 6.2 to 7.5) (FIG.
2B).
Equation 1: Calculation of Base Volume to Add for pH Adjust
[0106] V.sub.Na2CO3=0.0085V.sub.p(pH.sub.t-pH.sub.i), [0107] where
V.sub.Na2CO3=Volume of 1M Sodium Carbonate to add, V.sub.p=Volume
of Pool, pH.sub.t=Target pH, pH.sub.i=Initial pH,
[0108] Base Addition Method to Control Banking pH (Additional Small
Scale pH Studies)
[0109] Following similar procedures described above, several
studies were executed to study the effect of pH adjustment on
additional cell lines.
[0110] A total of nine CHO cell lines covering various cell types
(DP12, CHO-K1) were selected. In the days prior to banking, cells
were maintained according to standard protocols. Cells were
pelleted via centrifugation and mock pools of approximately 60-90
mL were generated for each test condition by re-suspending in spent
media to 28% packed cell volume (PCV). The pools were agitated at
37.degree. C. with or without gas permeable membranes for
CO.sub.2/O.sub.2 exchange to drive pH to initial pH conditions
(either 6.2 or 6.7). These two initial pH set points were chosen to
simulate potential worst case and typical conditions experienced
during and after hollow-fiber filter concentration. Upon reaching
initial pH targets, pools were quickly chilled to <10.degree. C.
and 20% (v/v) DMSO freeze media was added (1:3 parts cell culture
fluid) to reach a final PCV of 21%. pH adjustments were performed
after using Equation 1 to calculate the required volume of 1M
sodium carbonate addition for adjustment to pH 7.0, 7.3, 7.6, or
8.0. A final offline pH measurement was taken to confirm an
appropriate increase in pH prior to cell bank generation.
[0111] All cases were thawed in duplicate with one ampoule thawing
into each vessel. Results demonstrated that all cell types
previously exposed to lower pH could recover when adjusted to pH
values above 7.3 prior to banking. Each cell line demonstrated
different sensitivity to banking pH. Adjustment to a pH of
approximately 7.5 resulted in day 1 thaw viabilities above 80% in
most cases (FIGS. 3A-3F and FIGS. 4A-4B). Growth rates after thaw
were only impacted at the lower end of the pH range tested.
Interestingly, the upper end of the pH range tested (near 8.0-8.2)
did not negatively impact thaw recovery despite being significantly
greater than normal physiological pH.
[0112] The studies (testing the CHO-K1 cell types) were expanded to
capture more information about long term exposure to high pH. These
studies tested mock banks frozen immediately after (t0) the pH
adjustment (similar to all previous small scale test cases) and at
a second time point two hours later (t2). During the 2 hour hold,
the miniature pools (approximately 10 mL per case) were held in
Falcon.RTM. tubes submerged in wet ice. There was a slight pH drift
over the hold step that resulted in a change of up to 0.2 pH units.
Results from these studies indicated that a two hour exposure to
the pH ranges tested in this study had no impact (an example is
shown in FIGS. 6A-6B).
[0113] Methods to Chill Cell Culture Fluid During Harvest
[0114] A "slow pump harvest" process in combination with a cooling
heat exchanger was adopted to introduce chilling capability into
the traditional cell banking process. Specifically, the "slow pump
harvest" process required reducing the typical harvest flow rate
from approximately 4 L/min to 600 mL/min to match harvest rates
recorded for traditional cell banking production runs. A cooling
heat exchanger line, 15' length of size 15 platinum-cured silicone
tubing fully submerged in wet ice, was inserted into the harvest
flowpath. Temperature trends obtained during a mock run with water
demonstrated that the "chilled harvest process" was capable of
reducing culture temperatures to approximately 12.degree. C. within
the first 30 minutes prior to hollow-fiber filter concentration.
Based on data at 10.degree. C., this chilling capability would
likely suspend cell metabolism and prevent a low pH drift.
[0115] Process Confirmation Study Results
[0116] Cell Banking Process.
[0117] Cell banks were produced by first accumulating
suspension-adapted CHO cells in a batch/perfusion cell culture
process and then harvesting cells for banking. The initial source
of cells was from an MCB (for WCB generation). The process involved
three stages: cell accumulation, harvest and cell concentration,
and cell banking. The purpose of the cell accumulation step was to
generate the number of cells required for production of a full-size
MCB or WCB (420.times.1 mL or 10 mL ampoules respectively) in a
single batch. Cells were cultured in selective seed train media
during initial scale-up and during a rocking bioreactor process. A
harvest process step served to concentrate the final cell culture
fluid via a hollow fiber filter (HFF) to cell densities required
for banking. A subsequent pooling and filling process served to
prepare cell bank ampoules for long-term storage. The pooling
process was held on wet ice such that a certain temperature range
(about 5-10.degree. C.) was maintained. The process flow for the
process is shown in FIG. 1.
[0118] Harvest and cell concentration processes were executed using
a hollow-fiber filter cartridge. Offline pH trends showed that the
"chilled harvest process" was effective in reducing the pH drop
during cell concentration by approximately 0.35 pH units (from
.about.6.30 to 6.66 for the original and "chilled" processes
respectively). After pH adjustment, chilled cell banks were created
at early (0 min) and late (120 min) timepoints to check for
transient impact of cell pool hold pH.
[0119] Resulting cell banks were assessed for performance in
primary culture. Cell bank ampoules were thawed into shake flasks
rather than bioreactors using a serial 1:250 dilution (1:10, then
1:25). Thaw results successfully demonstrated that the current
process (chilled harvest and pH adjustment) was capable in
delivering banks with acceptable thaw performance. Whereas, cell
bank generated from the original process experienced a decline in
viability, these new cell banks were consistent and ranged from
79.1-85.3% in day 1 viability (approximately 65% improvement). No
adverse impact was observed as cells were held at elevated pH over
time.
[0120] Analysis showed cells from pools adjusted to higher pHs were
smaller in size (FIG. 5). This observed shift in size could
indicate that higher pH causes cell dehydration (possibly from
increased osmotic pressure or enhanced cell membrane elasticity
allowing cell shrinkage or both).
CONCLUSIONS
[0121] Banking pH was determined to be most critical to post thaw
performance, impacting day 1 thaw viabilities by up to 65%. As
such, process enhancements made to the harvest and banking
procedure were designed to mitigate cell exposure to low pH and to
improve control over banking pH. Ultimately, two enhancements were
introduced to the process including: 1) chilling cell culture fluid
during harvest with silicone tubing heat exchanger; and 2) using a
pH adjustment step to increase banking pH to 7.5.
* * * * *