U.S. patent application number 13/561395 was filed with the patent office on 2013-04-04 for rationally designed media for cell culture.
This patent application is currently assigned to Wyeth LLC. The applicant listed for this patent is Denis Drapeau, Yen-Tung Luan, Ryan Nolan, Wenge Wang. Invention is credited to Denis Drapeau, Yen-Tung Luan, Ryan Nolan, Wenge Wang.
Application Number | 20130084593 13/561395 |
Document ID | / |
Family ID | 39430451 |
Filed Date | 2013-04-04 |
United States Patent
Application |
20130084593 |
Kind Code |
A1 |
Luan; Yen-Tung ; et
al. |
April 4, 2013 |
RATIONALLY DESIGNED MEDIA FOR CELL CULTURE
Abstract
This invention relates to methods for rationally designing cell
culture media for use in cell cultures, e.g., cell cultures
employed in polypeptide production; cell culture media designed
with the disclosed methods; methods of producing a polypeptide of
interest, e.g., an antibody, using such media; polypeptides
produced using the methods and media disclosed herein; and
pharmaceuticals compositions containing such polypeptides. The
rationally designed media contain a concentration of an amino acid
that is calculated for use in cell mass, a concentration of the
amino acid that is calculated for use in cell maintenance, and a
concentration of the amino acid that is calculated for
incorporation into the polypeptide of interest.
Inventors: |
Luan; Yen-Tung; (Chelmsford,
MA) ; Wang; Wenge; (North Chelmsford, MA) ;
Nolan; Ryan; (Stoneham, MA) ; Drapeau; Denis;
(Salem, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Luan; Yen-Tung
Wang; Wenge
Nolan; Ryan
Drapeau; Denis |
Chelmsford
North Chelmsford
Stoneham
Salem |
MA
MA
MA
NH |
US
US
US
US |
|
|
Assignee: |
Wyeth LLC
Madison
NJ
|
Family ID: |
39430451 |
Appl. No.: |
13/561395 |
Filed: |
July 30, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11936866 |
Nov 8, 2007 |
8232075 |
|
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13561395 |
|
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60858289 |
Nov 8, 2006 |
|
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Current U.S.
Class: |
435/29 ; 435/325;
435/69.1 |
Current CPC
Class: |
C12N 5/0018 20130101;
C12N 2500/32 20130101; A61P 43/00 20180101; C12Q 1/02 20130101 |
Class at
Publication: |
435/29 ;
435/69.1; 435/325 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02 |
Claims
1. A method of producing a polypeptide in a cell culture
comprising: (1) providing a cell culture, comprising: a. cells,
comprising a nucleic acid encoding a polypeptide of interest; and
b. a desired cell culture medium, comprising a concentration of an
amino acid that is calculated for use in cell mass, a concentration
of the amino acid that is calculated for use in cell maintenance,
and a concentration of the amino acid that is calculated for
incorporation into the polypeptide of interest; and (2) maintaining
the cell culture under conditions that allow expression of the
polypeptide of interest.
2. The method of claim 1, wherein both the concentration of an
amino acid that is calculated for use in cell mass and the
concentration of the amino acid that is calculated for
incorporation into the polypeptide of interest are multiplied by a
baseline factor.
3. The method of claim 1, wherein the desired cell culture medium,
comprises a baseline adjusted amino acid concentration, A,
according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a
concentration of the amino acid that is used per unit of cell mass,
P is a concentration of the amino acid that is used for
incorporation into the polypeptide of interest per unit of
polypeptide titer, M is a multiplier for a desired peak cell
density of the cell culture, N is a multiplier for a desired
concentration of the polypeptide of interest, Y is a cell
maintenance factor, and F is a baseline factor.
4-5. (canceled)
6. The method of claim 1, wherein the method comprises providing a
starting cell culture medium, wherein the volume of the starting
cell culture medium is about 60-99% of the volume of a desired cell
culture medium volume; and providing a feeding cell culture medium
to the cell culture, wherein the volume of the feeding cell culture
medium is about 1-40% of the desired cell culture medium volume,
and wherein the resulting desired cell culture medium comprises the
concentration of an amino acid that is calculated for use in cell
mass, the concentration of the amino acid that is calculated for
use in cell maintenance, and the concentration of the amino acid
that is calculated for incorporation into the polypeptide of
interest.
7. The method of claim 6, wherein the resulting desired cell
culture medium comprises a baseline-adjusted amino acid
concentration, A, according to the formula
A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a concentration of the
amino acid that is used per unit of cell mass, P is a concentration
of the amino acid that is used for incorporation into the
polypeptide of interest per unit of polypeptide titer, M is a
multiplier for a desired peak cell density of the cell culture, N
is a multiplier for a desired concentration of the polypeptide of
interest, Y is a cell maintenance factor, and F is a baseline
factor.
8. The method of claim 7, wherein the starting cell culture medium
comprises a concentration, B, of the amino acid according to the
formula B=[A-(Z*V)]/(1-V), wherein Z is a concentration of the
amino acid in the feeding cell culture medium, and V is a volume of
the feeding culture medium as a proportion of the desired cell
culture medium volume.
9. The method of claim 3, wherein Y is 0 to about 1.5.
10. The method of claim 3, wherein F is about 1 to about 1.5.
11-18. (canceled)
19. The method of claim 3, wherein the combined concentration of
leucine, lysine, threonine, and valine in the desired cell culture
medium is between about 60% and about 80% of the concentration of
the total essential amino acids in the desired cell culture
medium.
20. The method of claim 3, wherein the combined concentration of
the essential amino acids in the desired cell culture medium is
between about 30% and about 50% of the concentration of the total
amino acids in the desired cell culture medium.
21. The method of claim 3, wherein the concentration of amino acids
in the desired cell culture medium is between about 120 mM and
about 350 mM.
22. The method of claim 3, wherein the concentration of proline in
the cell culture is maintained at greater than about 1 mM.
23. The method of claim 3, wherein the concentration of proline in
the cell culture is maintained at greater than about 2 mM.
24-29. (canceled)
30. A method of cell culture comprising: (1) providing a cell
culture, comprising: a. cells; and b. a desired cell culture
medium, comprising a concentration of an amino acid that is
calculated for use in cell mass and a concentration of the amino
acid that is calculated for use in cell maintenance; and (2)
maintaining the cell culture under conditions that allow growth and
replication of the cells in the cell culture.
31. The method of claim 30, wherein the desired cell culture
medium, comprises a baseline-adjusted amino acid concentration, A',
according to the formula A'=[(M*X)+(Y*M*X)]*F, wherein X is a
concentration of the amino acid that is used per unit of cell mass,
M is a multiplier for a desired peak cell density of the cell
culture, Y is a cell maintenance factor, and F is a baseline
factor.
32-33. (canceled)
34. The cell culture medium of claim 36, comprising a total
concentration of amino acids from between about 120 mM and about
350 mM.
35. (canceled)
36. A cell culture medium for use in the production of a
polypeptide of interest, comprising a concentration of an amino
acid that is calculated for use in cell mass, a concentration of
the amino acid that is calculated for use in cell maintenance, and
a concentration of the amino acid that is calculated for
incorporation into the polypeptide of interest.
37. The cell culture medium of claim 36, comprising a
baseline-adjusted amino acid concentration, AT, according to the
formula A'=[(M*X)+(Y*M*X)]*F, wherein X is a concentration of the
amino acid that is used per unit of cell mass, M is a multiplier
for desired peak cell density of the cell culture, Y is a cell
maintenance factor, and F is a baseline factor.
38. The cell culture medium of claim 36 for use in the production
of a polypeptide of interest, comprising a baseline-adjusted amino
acid concentration, A, according to the formula
A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a concentration of the
amino acid that is used per unit of cell mass, P is a concentration
of the amino acid that is used for incorporation into the
polypeptide of interest per unit of polypeptide titer, M is a
multiplier for desired peak cell density of the cell culture, N is
a multiplier for desired concentration of the polypeptide of
interest, Y is a cell maintenance factor, and F is a baseline
factor.
39-41. (canceled)
42. A method for determining an optimized concentration of an amino
acid used in a cell culture medium for the production of a
polypeptide of interest in a cell culture, comprising: (1)
determining the amino acid concentration required for the cell mass
of the cells at a target cell density; (2) determining the amino
acid concentration required to produce the polypeptide of interest
at a target polypeptide titer; (3) determining the amino acid
concentration required for cell maintenance of the cells; and (4)
adding the concentrations obtained from step (1), step (2), and
step (3) to provide an optimized concentration of the amino acid
used in the cell culture medium for the production of the
polypeptide of interest.
43-45. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority from U.S.
Provisional Patent Application No. 60/858,289, filed Nov. 8, 2006,
the content of which is hereby incorporated by reference herein in
its entirety
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to methods for rationally designing
cell culture media for use in cell cultures employed in, e.g.,
polypeptide production; cell culture media designed with the
disclosed methods; methods of producing large quantities of a
polypeptide of interest, e.g., an antibody, using such media;
polypeptides produced using the methods and media disclosed herein;
and pharmaceuticals compositions containing such polypeptides. The
invention is particularly useful in large-scale cell cultures. The
methods and compositions disclosed herein are particularly useful
to produce significant quantities of polypeptides in batch,
fed-batch and perfusion animal cell cultures.
[0004] 2. Related Background Art
[0005] A large proportion of biotechnology products, whether
commercially available or only in development, are protein
therapeutics; thus, there is a demand for production of these
polypeptides in cell cultures. Furthermore, the cellular machinery
of an animal cell (as opposed to, e.g., a bacterial cell) is often
required to produce many forms of polypeptide therapeutics (such as
glycosylated proteins or hybridoma-produced monoclonal antibodies
(MAbs)). Consequently, there is an increasing demand for optimizing
production of these polypeptides in cell cultures, and particularly
in animal cell cultures.
[0006] As compared to bacterial cell cultures, animal cell cultures
have lower production rates and typically generate lower production
yields. Thus, a significant quantity of research focuses on animal
cell culture conditions that optimize the polypeptide output, i.e.,
conditions that support high cell density and high titer. For
example, it has been determined that maintaining glucose
concentrations in cell culture media at low concentrations and
culturing cells in a production phase at an osmolality of about 400
to 600 mOsm increases production of recombinant proteins by animal
cell cultures, wherein culturing in all phases is also at a
selected glutamine concentration (preferably between about 0.2 to
about 2 mM). It has also been determined that restricted feeding of
glucose to animal cell cultures in fed-batch processes controls
lactate production without requiring the constant-rate feeding of
glucose. Further, it is known that modification of the total
cumulative concentration of amino acids, the concentration of
individual amino acids, and the ratios of individual amino acids to
each other (e.g., glutamine to asparagine) and to total amino acids
(e.g., glutamine to total amino acids) in the media of a
large-scale cell culture can result in substantially improved
large-scale polypeptide production.
[0007] Traditionally, medium studies for animal cell cultures focus
on three techniques: 1) enriching the medium components of the
starting medium and increasing the frequency of culture feeding; 2)
applying multi-factorial design to different medium strengths and
different component concentrations; and 3) analyzing conditioned
(spent) medium for amino acids, vitamins, and other components, and
adding those components that are at low levels or are depleted.
These methods generally use cell density, viability and titer
responses as indicators of optimization.
[0008] However, the above methods only indirectly detect the
nutrient requirement for cells based on the end result, i.e., cell
density, viability, and titer, rather than detecting and providing
the cell with the actual nutrient requirement for optimized protein
production.
SUMMARY OF THE INVENTION
[0009] The present invention provides methods for rationally
designing cell culture media, e.g., large-scale cell culture media,
for use in, e.g. large-scale cell cultures employed in polypeptide
production; cell culture media, e.g., large-scale cell culture
media, designed with the disclosed methods; methods of producing
large quantities of a polypeptide of interest, e.g., an antibody,
using such media; polypeptides produced using the methods and media
disclosed herein; and pharmaceuticals compositions containing such
polypeptides. These methods and compositions are useful for
culturing, e.g., batch, fed-batch, and perfusion culturing, of
cells. These methods and compositions are particularly useful for
large-scale culturing, e.g., batch, fed-batch, and perfusion
culturing, of animal cells, e.g., mammalian cells.
[0010] A rationally designed medium of the present invention
contains a concentration of an amino acid that is calculated for
use in cell mass, a concentration of the amino acid that is
calculated for use in cell maintenance, and a concentration of the
amino acid that is calculated for incorporation into the
polypeptide of interest.
[0011] In one embodiment, the invention provides a method of
producing a polypeptide in a cell culture comprising providing a
cell culture, comprising cells, comprising a nucleic acid encoding
a polypeptide of interest, and a desired cell culture medium,
comprising a concentration of an amino acid that is calculated for
use in cell mass, a concentration of the amino acid that is
calculated for use in cell maintenance, and a concentration of the
amino acid that is calculated for incorporation into the
polypeptide of interest; and, maintaining the cell culture under
conditions that allow expression of the polypeptide of interest. In
one embodiment of the invention, the desired cell culture medium
comprises a baseline-adjusted amino acid concentration, A,
according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a
concentration of the amino acid that is used per unit of cell mass,
P is a concentration of the amino acid that is used for
incorporation into the polypeptide of interest per unit of
polypeptide titer, M is a multiplier for a desired peak cell
density of the cell culture, N is a multiplier for a desired
concentration of the polypeptide of interest, Y is a cell
maintenance factor, and F is a baseline factor.
[0012] In another embodiment, the invention provides a method of
producing a polypeptide in a cell culture, comprising providing a
cell culture, comprising cells, comprising a nucleic acid encoding
a polypeptide of interest; and a starting cell culture medium,
wherein the volume of the starting cell culture medium is about
60-99% of the volume of a desired cell culture medium volume;
providing a feeding cell culture medium to the cell culture,
wherein the volume of the feeding cell culture medium is about
1-40% of the desired cell culture medium volume, and wherein the
resulting desired cell culture medium comprises a concentration of
an amino acid that is calculated for use in cell mass, a
concentration of the amino acid that is calculated for use in cell
maintenance, and a concentration of the amino acid that is
calculated for incorporation into the polypeptide of interest; and,
maintaining the cell culture under conditions that allow expression
of the polypeptide of interest. In one embodiment of the invention,
the resulting desired cell culture medium comprises a
baseline-adjusted amino acid concentration, A, according to the
formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a concentration of
the amino acid that is used per unit of cell mass, P is a
concentration of the amino acid that is used for incorporation into
the polypeptide of interest per unit of polypeptide titer, M is a
multiplier for a desired peak cell density of the cell culture, N
is a multiplier for a desired concentration of the polypeptide of
interest, Y is a cell maintenance factor, and F is a baseline
factor; and maintaining the cell culture under conditions that
allow expression of the polypeptide of interest. In another
embodiment of the invention, the starting cell culture medium
comprises a concentration, B, of the amino acid according to the
formula B=[A-(Z*V)]/(1 V), wherein Z is a concentration of the
amino acid in the feeding cell culture medium, and V is a volume of
the feeding culture medium as a proportion of the desired cell
culture medium volume. In another embodiment of the methods
disclosed herein, Y is 0 to about 1.5. In yet another embodiment of
the methods disclosed herein, F is about 1 to about 1.5. In a
further embodiment of the methods disclosed herein, Y is 0 to about
1.5 and F is about 1 to about 1.5.
[0013] In one embodiment of the methods disclosed herein, the
desired cell culture medium comprises greater than or equal to
about 3 mM tyrosine. In another embodiments of the methods
disclosed herein, the desired cell culture medium comprises:
between about 7 mM and about 30 mM leucine; between about 7 mM and
about 30 mM lysine; between about 7 mM and about 30 mM threonine;
between about 7 mM and about 30 mM proline; and/or between about 7
mM and about 30 mM valine. In a further embodiment of the methods
disclosed herein, the combined concentration of leucine, lysine,
threonine, proline, and valine in the desired cell culture medium
is between about 35 mM and about 150 mM. In yet another embodiment,
the combined concentration of leucine, lysine, threonine, and
valine in the desired cell culture medium is between about 60% and
about 80% of the concentration of the total essential amino acids
in the desired cell culture medium.
[0014] In one embodiment of the methods disclosed herein, the
combined concentration of the essential amino acids in the desired
cell culture medium is between about 30% and about 50% of the
concentration of the total amino acids in the desired cell culture
medium. In another embodiment of the methods disclosed herein, the
concentration of amino acids in the desired cell culture medium is
between about 120 mM and about 350 mM. In a further embodiment of
the methods disclosed herein, the concentration of praline in the
cell culture is maintained at greater than about 1 mM. In yet
another embodiment of the methods disclosed herein, the
concentration of proline in the cell culture is maintained at
greater than about 2 mM. In some embodiments of the methods of
producing a polypeptide, the cell culture is a large-scale cell
culture. In other embodiments, the cells are animal cells.
[0015] A further aspect of the invention provides polypeptides
produced according to the methods disclosed herein. Another aspect
of the invention provides a pharmaceutical composition comprising a
polypeptide produced according to the methods disclosed herein and
a pharmaceutically acceptable carrier.
[0016] A further aspect of the invention provides a method of cell
culture comprising: providing a cell culture, comprising: cells;
and a desired cell culture medium, comprising a concentration of an
amino acid that is calculated for use in cell mass and a
concentration of the amino acid that is calculated for use in cell
maintenance; and maintaining the cell culture under conditions that
allow growth and replication of the cells in the cell culture. In
one embodiment of the invention, the desired cell culture medium
comprises a baseline-adjusted amino acid concentration, A',
according to the formula A'=[(M*X)+(Y*M*X)]*F, wherein X is a
concentration of the amino acid that is used per unit of cell mass,
M is a multiplier for a desired peak cell density of the cell
culture, Y is a cell maintenance factor, and F is a baseline
factor. In some embodiments of the methods of cell culture, the
cell culture is a large-scale cell culture. In other embodiments,
the cells are animal cells.
[0017] A further aspect of the invention provides a cell culture
medium, comprising a total concentration of amino acids from
between about 120 mM and about 350 mM. Another aspect of the
invention provides a cell culture medium for use in the production
of a polypeptide of interest, comprising a total concentration of
amino acids from between about 120 mM and about 350 mM.
[0018] Another aspect of the invention provides a cell culture
medium for use in the production of a polypeptide of interest,
comprising a concentration of an amino acid that is calculated for
use in cell mass, a concentration of the amino acid that is
calculated for use in cell maintenance, and a concentration of the
amino acid that is calculated for incorporation into the
polypeptide of interest. In one embodiment of the invention, the
cell culture medium for use in the production of a polypeptide of
interest comprises a baseline-adjusted amino acid concentration, A,
according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a
concentration of the amino acid that is used per unit of cell mass,
P is a concentration of the amino acid that is used for
incorporation into the polypeptide of interest per unit of
polypeptide titer, M is a multiplier for desired peak cell density
of the cell culture, N is a multiplier for desired concentration of
the polypeptide of interest, Y is a cell maintenance factor, and F
is a baseline factor.
[0019] Yet another aspect of the invention provides a cell culture
medium, comprising a baseline-adjusted amino acid concentration,
A', according to the formula A'=[(M*X)+(Y*M*X)]*F, wherein X is a
concentration of the amino acid that is used per unit of cell mass,
M is a multiplier for desired peak cell density of the cell
culture, Y is a cell maintenance factor, and F is a baseline
factor. In some embodiments, the cell culture medium is a
large-scale cell culture medium. In other embodiments, the cell
culture medium is an animal cell culture medium.
[0020] Yet another aspect of the invention provides a method for
determining an optimized concentration of an amino acid used in a
cell culture medium for the production of a polypeptide of interest
in a cell culture, comprising: determining the amino acid
concentration required for the cell mass of the cells in the cell
culture at a target cell density; determining the amino acid
concentration required to produce the polypeptide of interest in
the cell culture at a target polypeptide titer; determining the
amino acid concentration required for cell maintenance of the cells
in the cell culture; and adding the concentrations to provide an
optimized concentration of the amino acid used in the cell culture
medium for the production of the polypeptide of interest in the
cell culture.
[0021] A further aspect of the invention provides a method for
determining an optimized amino acid concentration, A, of an amino
acid used in a cell culture medium for the production of a
polypeptide of interest in a cell culture, comprising: determining
the amino acid concentration, X, required for cell mass of the
cells at a set cell density; determining the amino acid
concentration, P, required to produce the polypeptide of interest
at a set polypeptide titer; and determining the optimized amino
acid concentration, A, according to the formula
A=[(M*X)+(N*P)+(M*Y*X)]*F, wherein M is a multiplier for a desired
target cell density of the cell culture, N is a multiplier for a
desired target concentration of the polypeptide of interest, Y is a
cell maintenance factor; and F is a baseline factor. In some
embodiments of the methods for determining an optimized amino acid
concentration, the cell culture is a large-scale cell culture. In
other embodiments, the cells are animal cells.
[0022] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 depicts the cell density (Y-axis; "Cell Density
(10.sup.6 cells/mL)") over time (X-axis; "Days") for CHO cells
engineered to express anti-IL-22. Cells were cultured in the
rationally designed medium of Example 2.
[0024] FIG. 2 depicts the titer (Y-axis; "Titer (g/L)") of
anti-IL-22 antibody over time (X-axis; "Days") for CHO cells
engineered to express anti-IL-22. Cells were cultured in the
rationally designed medium of Example 2.
[0025] FIG. 3 depicts the cell density (Y-axis; "Cell Density
(10.sup.6 cells/mL)") over time (X-axis; "Days") for CHO cells
engineered to express anti-IL-22. Cells were cultured in the
rationally designed medium of Example 3.
[0026] FIG. 4 depicts the titer (Y-axis; "Titer (g/L)") of
anti-IL-22 antibody over time (X-axis; "Days") for CHO cells
engineered to express anti-IL-22. Cells were cultured in the
rationally designed medium of Example 3.
[0027] FIG. 5 depicts the titer (Y-axis; "Titer (g/L)") of
anti-IL-22 antibody over time (X-axis; "Days") for CHO cells
engineered to express anti-IL-22. Cells were cultured in
"Traditional Medium" (a medium based on traditional cell culture
requirements, see, e.g., U.S. Published Patent Application No.
2006/0121568), "Rational Design Medium" prepared using the methods
herein, or "Traditional Medium+Proline," which contains an
additional 3.7 mM proline added to the "Traditional Medium" (see
Example 4).
[0028] FIG. 6 depicts the cell density (Y-axis; "Cell Density
(10.sup.6 cells/mL)" over time (X-axis; "Days") for CHO cells
engineered to express anti-IL-22. Cells were cultured in
"Traditional Medium," "Rational Design Medium," or "Traditional
Medium+Proline" (see Example 4).
[0029] FIG. 7 depicts the cell viability (Y-axis; "Viability [%]")
over time (X-axis; "Days") for CHO cells engineered to express
anti-IL-22. Cells were cultured in "Traditional Medium," "Rational
Design Medium," or "Traditional Medium+Proline" (see Example
4).
[0030] FIG. 8 depicts the concentration of the designated amino
acid (Y-axis; "[.mu.M]") ((FIG. 8A) proline; (FIG. 8B) threonine;
(FIG. 8C) valine; (FIG. 8D) tryptophan; or (FIG. 8E) tyrosine) over
time (X-axis; "Days") for CHO cells for cells engineered to express
anti-IL-22. Cells were cultured in "Traditional Medium," "Rational
Design Medium," or "Traditional Medium+Proline" (see Example
4).
[0031] FIG. 9 depicts the cell density (Y-axis; "Cell Density
(10.sup.6 cells/mL)") over time (X-axis; "Days") for CHO cells
engineered to express anti-IL-22. Cells were cultured in "Rational
Design Medium" or "Rational Design Medium without maintenance and
baseline factors." The figure is representative of 5 independent
replicates (n=5) (see Example 6).
[0032] FIG. 10 depicts cell viability (Y-axis: "Viability (%)")
over time (X-axis; "Days") for CHO cells engineered to express
anti-IL-22. Cells were cultured in "Rational Design Medium" or
"Rational Design Medium without maintenance and baseline factors."
The figure is representative of 5 independent replicates (n=5) (see
Example 6).
[0033] FIG. 11 depicts antibody titer (Y-axis; "Titer (g/L)") over
time (X-axis; "Days") for CHO cells engineered to express
anti-IL-22. Cells were cultured in "Rational Design Medium" or
"Rational Design Medium without maintenance and baseline factors."
The figure is representative of 5 independent replicates (n=5) (see
Example 6).
DETAILED DESCRIPTION OF THE INVENTION
[0034] The term "batch culture" as used herein refers to a method
of culturing cells in which all the components that will ultimately
be used in culturing the cells, including the medium as well as the
cells themselves, are provided at the beginning of the culturing
process. A batch culture is typically stopped at some point and the
cells and/or components in the medium are harvested and optionally
purified.
[0035] The term "fed-batch culture" as used herein refers to a
method of culturing cells in which additional components are
provided to the culture at some time subsequent to the beginning of
the culture process. The provided components typically comprise
nutritional supplements for the cells that have been depleted
during the culturing process. A fed-batch culture is typically
stopped at some point and the cells and/or components in the medium
are harvested and optionally purified. In a preferred embodiment of
the present invention, the cell culture is an animal cell culture,
e.g., a mammalian cell culture, that is a batch or fed-batch
culture.
[0036] The term "perfusion culture" as used herein refers to a
method of culturing cells in which additional components are
provided continuously or semi-continuously to the culture
subsequent to the beginning of the culture process. The provided
components typically comprise nutritional supplements for the cells
that have been depleted during the culturing process. Portions of
the cells and/or components in the medium are typically harvested
on a continuous or semi-continuous basis and are optionally
purified.
[0037] The term "bioreactor" as used herein refers to any vessel
used for the growth of a prokaryotic or eukaryotic cell culture,
e.g., an animal cell culture (such as a mammalian cell culture).
The bioreactor can be of any size so long as it is useful for the
culturing of cells, e.g., mammalian cells. Typically, the
bioreactor will be at least 30 ml and may be 1, 10, 100, 250, 500,
1000, 2500, 5000, 8000, 10,000, 12,0000 liters or more, or any
intermediate volume. The internal conditions of the bioreactor,
including, but not limited to pH and temperature, are typically
controlled during the culturing period. The bioreactor can be
composed of any material that is suitable for holding mammalian
cell cultures suspended in media under the culture conditions of
the present invention, including glass, plastic or metal. The term
"production bioreactor" as used herein refers to the final
bioreactor used in the production of the polypeptide or protein of
interest. The volume of a large-scale cell culture production
bioreactor is generally greater than about 100 ml, typically at
least about 10 liters, and may be 500, 1000, 2500, 5000, 8000,
10,000, 12,0000 liters or more, or any intermediate volume. One of
ordinary skill in the art will be aware of, and will be able to
choose, suitable bioreactors for use in practicing the present
invention.
[0038] The terms "cell density," "cell concentration," or the like,
as used herein, refer to that number, weight, mass, etc. of cells
present in a given volume of medium. "Peak cell density" or the
like refers to the maximum number of cells that can be reached in a
given volume of medium, and "desired peak cell density" or the like
refers to the maximum number of cells that a practitioner desires
to obtain (e.g., targets) in a given cell volume. Variations of
such target value(s) will be clear to those of skill in the art,
e.g., one of skill may express a target value(s) in terms of
desired cell mass, and such target value(s) may be in one or more
appropriate units of measure (e.g., desired peak units of cell
mass).
[0039] The term "cell viability" as used herein refers to the
ability of cells in culture to survive under a given set of culture
conditions or experimental variations. The term as used herein also
refers to that portion of cells that are alive at a particular time
in relation to the total number of cells, living and dead, in the
culture at that time.
[0040] The terms "culture" and "cell culture" as used herein refer
to a cell population that is suspended in a cell culture medium
under conditions suitable to survival and/or growth of the cell
population. As used herein, these terms may refer to the
combination comprising the cell population (e.g., the animal cell
culture) and the medium in which the population is suspended.
[0041] The term "integrated viable cell density" or "IVC" as used
herein refers to the average density of viable cells over the
course of the culture multiplied by the amount of time the culture
has run. Assuming the amount of polypeptide and/or protein produced
is proportional to the number of viable cells present over the
course of the culture, integrated viable cell density is a useful
tool for estimating the amount of polypeptide and/or protein
produced over the course of the culture.
[0042] The terms "medium," "cell culture medium," and "culture
medium" as used herein refer to a solution containing nutrients
that nourish growing animal, e.g., mammalian, cells. Typically,
these solutions provide essential and nonessential amino acids,
vitamins, energy sources, lipids, and trace elements required by
the cell for minimal growth and/or survival. The solution may also
contain components that enhance growth and/or survival above the
minimal rate, including hormones and growth factors. The solution
is preferably formulated to a pH and salt concentration optimal for
cell survival and proliferation. In one embodiment, the medium is a
defined medium. Defined media are media in which all components
have a known chemical structure. In another embodiment of the
invention, the medium may contain an amino acid(s) derived from any
source or method known in the art, including, but not limited to,
an amino acid(s) derived either from single amino acid addition(s)
or from peptone or protein hydrolysate (including animal or plant
source(s)) addition(s).
[0043] The teen "seeding" as used herein refers to the process of
providing a cell culture to a bioreactor or another vessel. The
cells may have been propagated previously in another bioreactor or
vessel. Alternatively, the cells may have been frozen and thawed
prior to, e.g., immediately prior to, providing them to the
bioreactor or vessel. The term refers to any number of cells,
including a single cell.
[0044] The term "titer" as used herein refers to the total amount
of polypeptide of interest produced by an animal cell culture,
divided by a given amount of medium volume; thus "titer" refers to
a concentration. Titer is typically expressed in units of
milligrams of polypeptide per milliliter of medium.
[0045] As used herein, the term "antibody" includes a protein
comprising at least one, and typically two, VH domains or portions
thereof, and/or at least one, and typically two, VL domains or
portions thereof. In certain embodiments, the antibody is a
tetramer of two heavy immunoglobulin chains and two light
immunoglobulin chains, wherein the heavy and light immunoglobulin
chains are interconnected by, e.g., disulfide bonds. The
antibodies, or a portion thereof, can be obtained from any origin,
including, but not limited to, rodent, primate (e.g., human and
nonhuman primate), camelid, etc., or they can be recombinantly
produced, e.g., chimeric, humanized, and/or in vitro-generated,
e.g., by methods well known to those of skill in the art.
[0046] Examples of binding fragments encompassed within the term
"antigen-binding fragment" of an antibody include, but are not
limited to, (i) a Fab fragment, a monovalent fragment consisting of
the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2 fragment, a
bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment, which consists of a VH domain; (vi) a camelid or
camelized heavy chain variable domain (VHH); (vii) a single chain
Fv (scFv; see below); (viii) a bispecific antibody; and (ix) one or
more fragments of an immunoglobulin molecule fused to an Fc region.
Furthermore, although the two domains of the Fv fragment, VL and
VH, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the VL and VH regions pair
to form monovalent molecules (known as single chain Fv (scFv));
see, e.g., Bird et al. (1988) Science 242:423-26; Huston et al.
(1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-83). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding fragment" of an antibody. These fragments may be
obtained using conventional techniques known to those skilled in
the art, and the fragments are evaluated for function in the same
manner as are intact antibodies.
[0047] The "antigen-binding fragment" can, optionally, further
include a moiety that enhances one or more of, e.g., stability,
effector cell function or complement fixation. For example, the
antigen binding fragment can further include a pegylated moiety,
albumin, or a heavy and/or a light chain constant region.
[0048] Other than "bispecific" or "bifunctional" antibodies, an
antibody is understood to have each of its binding sites identical.
A "bispecific" or "bifunctional antibody" is an artificial hybrid
antibody having two different heavy/light chain pairs and two
different binding sites. Bispecific antibodies can be produced by a
variety of methods including fusion of hybridomas or linking of
Fab' fragments. See, e.g., Songsivilai and Lachmann (1990) Clin.
Exp. Immunol. 79:315-21; Kostelny et al. (1992) J. Immunol.
148:1547-53.
[0049] The phrase "protein" or "protein product" refers to one or
more chains of amino acids. As used herein, the term "protein" is
synonymous with "polypeptide" and, as is generally understood in
the art, refers to at least one chain of amino acids liked via
sequential peptide bonds. In certain embodiments, a "protein of
interest" or a "polypeptide of interest" is a protein encoded by an
exogenous nucleic acid molecule that has been transformed into a
host cell. In certain embodiments, wherein the "protein of
interest" is coded for by an exogenous DNA with which the host cell
has been transformed, the nucleic acid sequence of the exogenous
DNA determines the sequence of amino acids. In certain embodiments,
a "protein of interest" is a protein encoded by a nucleic acid
molecule that is endogenous to the host cell. In certain
embodiments, expression of such an endogenous protein of interest
is altered by transfecting a host cell with an exogenous nucleic
acid molecule that may, for example, contain one or more regulatory
sequences and/or encode a protein that enhances expression of the
protein of interest. Methods and compositions of the present
invention may be used to produce any protein of interest,
including, but not limited to proteins having pharmaceutical,
diagnostic, agricultural, and/or any of a variety of other
properties that are useful in commercial, experimental and/or other
applications. In addition, a protein of interest can be a protein
therapeutic. Namely, a protein therapeutic is a protein that has a
biological effect on a region in the body on which it acts or on a
region of the body on which it remotely acts via intermediates.
Examples of protein therapeutics are discussed in more detail
below. In certain embodiments, proteins produced using methods
and/or compositions of the present invention may be processed
and/or modified. For example, a protein to be produced in
accordance with the present invention may be glycosylated.
[0050] The present invention may be used to culture cells for the
advantageous production of any therapeutic protein, such as
pharmaceutically or commercially relevant enzymes, receptors,
antibodies (e.g., monoclonal and/or polyclonal antibodies), Fc
fusion proteins, cytokines, hormones, regulatory factors, growth
factors, coagulation/clotting factors, antigen binding agents, etc.
One of ordinary skill in the art will be aware of other proteins
that can be produced in accordance with the present invention, and
will be able to use methods disclosed herein to produce such
proteins.
Expression Constructs and Generation of Recombinant Host Cells
[0051] The present invention uses recombinant host cells, e.g.,
prokaryotic or eukaryotic host cells, i.e., cells transfected with
an expression construct containing a nucleic acid that encodes a
polypeptide of interest. The phrase "animal cells" encompasses
invertebrate, nonmammalian vertebrate (e.g., avian, reptile and
amphibian), and mammalian cells. Nonlimiting examples of
invertebrate cells include the following insect cells: Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori (silkworm/silk moth).
[0052] A number of mammalian cell lines are suitable host cells for
recombinant expression of polypeptides of interest. Mammalian host
cell lines include, for example, COS, PER.C6, TM4, VERO076, MDCK,
BRL-3A, W138, Hep G2, MMT, MRC 5, FS4, CHO, 293T, A431, 3T3, CV-1,
C3H10T1/2, Colo205, 293, HeLa, L cells, BHK, HL-60, FRhL-2, U937,
HaK, Jurkat cells, Rat2, BaF3, 32D, FDCP-1, PC12, M1x, murine
myelomas (e.g., SP2/0 and NS0) and C2C12 cells, as well as
transformed primate cell lines, hybridomas, normal diploid cells,
and cell strains derived from in vitro culture of primary tissue
and primary explants. Any eukaryotic cell that is capable of
expressing the polypeptide of interest may be used in the disclosed
media design methods. Numerous cell lines are available from
commercial sources such as the American Type Culture Collection
(ATCC). In one embodiment of the invention, the cell culture, e.g.,
the large-scale cell culture, employs hybridoma cells. The
construction of antibody-producing hybridoma cells is well known in
the art. In one embodiment of the invention, the cell culture,
e.g., the large-scale cell culture, employs CHO cells.
[0053] Alternatively, it may be possible to recombinantly produce
polypeptides of interest in lower eukaryotes such as yeast, or in
prokaryotes such as bacteria. Suitable yeast strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces
strains, Candida, or any yeast strain capable of expressing
polypeptide of interest. Suitable bacterial strains include
Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any
bacterial strain capable of expressing the polypeptide of interest.
Expression in bacteria may result in formation of inclusion bodies
incorporating the recombinant protein. Thus, refolding of the
recombinant protein may be required in order to produce active or
more active material. Several methods for obtaining correctly
folded heterologous proteins from bacterial inclusion bodies are
known in the art. These methods generally involve solubilizing the
protein from the inclusion bodies, then denaturing the protein
completely using a chaotropic agent. When cysteine residues are
present in the primary amino acid sequence of the protein, it is
often necessary to accomplish the refolding in an environment that
allows correct formation of disulfide bonds (a redox system).
General methods of refolding are disclosed in Kohno (1990) Meth.
Enzymol. 185:187-95, EP 0433225, and U.S. Pat. No. 5,399,677.
[0054] The present invention uses constructs, in the form of
plasmids, vectors, and transcription or expression cassettes,
comprised of at least one polynucleotide encoding a polypeptide of
interest. Vectors are capable of directing the expression of genes
to which they are operably linked. Such vectors are referred to
herein as "recombinant expression vectors" or "expression vectors."
In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" may be used interchangeably
as the plasmid is the most common vector form. However, the
invention is intended to include other forms of expression vectors
that serve equivalent functions, including, but not limited to,
viral vectors (e.g., replication defective retroviruses, modified
alphaviruses, adenoviruses and adeno-associated viruses).
[0055] Constructs that are suitable for expression of proteins in
animal cells are well known in the art. For example,
polynucleotides may be operably linked to an expression control
sequence such as those present in the pMT2 or pED expression
vectors disclosed in, e.g., Kaufman et al. (1991) Nuc. Acids Res.
19:4485-90. Other suitable expression control sequences are found
in vectors known in the art and include, but are not limited to:
HaloTag.TM. pHT2, pACT, pBIND, pCAT.RTM.3, pCI, phRG, phRL
(Promega, Madison, Wis.); pcDNA3.1, pcDNA3.1-E, pcDNA4/HisMAX,
pcDNA4/HisMAX-E, pcDNA3.1/Hygro, pcDNA3.1/Zeo, pZeoSV2, pRc/CMV2,
pBudCE4 pRc/RSV (Invitrogen, Carlsbad, Calif.); pCMV-3Tag Vectors,
pCMV-Script.RTM. Vector, pCMV-Tag Vectors, pSG5 Vectors
(Stratagene, La Jolla, Calif.); pDNR-Dual, pDNR-CMV (Clonetech,
Palo Alto, Calif.); and pSMEDA (Wyeth, Madison, Wis.). General
methods of expressing recombinant proteins are also known and are
exemplified in, e.g., Kaufman (1990) Meth. Enzymol. 185:537-66.
[0056] As defined herein "operably linked" means enzymatically or
chemically ligated to form a covalent bond between the
polynucleotide to be expressed and the expression control sequence
in a manner that the encoded protein is expressed by the
transfected host cell.
[0057] The recombinant expression constructs of the invention may
carry additional sequences, such as regulatory sequences (e.g.,
sequences that regulate either vector replication (e.g., origins of
replication, transcription of the nucleic acid sequence encoding
the polypeptide (or peptide) of interest) or expression of the
encoded polypeptide), tag sequences such as histidine, and
selectable marker genes. The term "regulatory sequence" is intended
to include promoters, enhancers and any other expression control
elements (e.g., polyadenylation signals, transcription splice
sites) that control transcription, replication or translation. Such
regulatory sequences are described, for example, in Goeddel, Gene
Expression Technology: Methods in Enzymology, Academic Press, San
Diego, Calif. (1990). Those skilled in the art will recognize that
the design of the expression vector, including the selection of
regulatory sequences, will depend on various factors, including
choice of the host cell and the level of protein expression
desired. Preferred regulatory sequences for expression of proteins
in mammalian host cells include viral elements that direct high
levels of protein expression, such as promoters and/or enhancers
derived from the FF-1a promoter and BGH poly A, cytomegalovirus
(CMV) (e.g., the CMV promoter/enhancer), Simian virus 40 (SV40)
(e.g., the SV40 promoter/enhancer), adenovirus (e.g., the
adenovirus major late promoter (AdMLP)), and polyoma. Viral
regulatory elements, and sequences thereof, are described in, e.g.,
U.S. Pat. Nos. 5,168,062; 4,510,245; and 4,968,615, all of which
are incorporated by reference herein in their entireties.
[0058] Suitable vectors, containing appropriate regulatory
sequences, including promoter sequences, terminator sequences,
polyadenylation sequences, enhancer sequences, marker genes and
other sequences as appropriate, may be either chosen or
constructed. Inducible expression of proteins, achieved by using
vectors with inducible promoter sequences, such as
tetracycline-inducible vectors, e.g., pTet-On.TM. and pTet-Off.TM.
(Clontech, Palo Alto, Calif.), may also be used in the disclosed
method. For further details regarding expression vectors, see, for
example, Molecular Cloning: a Laboratory Manual (2nd ed.) eds.
Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989). Many known techniques and protocols for
manipulation of nucleic acids, for example, in preparation of
nucleic acid constructs, mutagenesis, sequencing, introduction of
DNA into cells, gene expression, and analysis of proteins, are also
described in detail in Current Protocols in Molecular Biology (2nd
ed.) eds. Ausubel et al., Wiley & Sons, Alameda, Calif.
(1992).
[0059] A polynucleotide inserted into an expression construct for
producing a polypeptide of interest may encode any polypeptide that
is capable of being expressed in the host cell used in the cell
culture. Thus, the polynucleotide may encode full-length gene
products, portions of full-length genes, peptides, or fusion
proteins. Such polynucleotides may consist of genomic DNA or cDNA,
and may be derived from any animal. Polynucleotides may be isolated
from cells or organisms by methods well known in the art, e.g., PCR
or RT-PCR, or may be produced by known conventional chemical
synthesis methods. Such chemically synthetic polynucleotides may
possess biological properties in common with natural
polynucleotides, and thus may be employed as substitutes for
natural polynucleotides.
[0060] Polypeptides may also be recombinantly produced by operably
linking the polynucleotide encoding the polypeptide of interest to
suitable control sequences in one or more insect expression
vectors, such as baculovirus vectors, and employing an insect cell
expression system. Materials and methods for baculovirus/Sf9
expression systems are commercially available in kit form (e.g.,
the MAXBAC.RTM. kit, Invitrogen, Carlsbad, Calif.).
[0061] Transfection of host cells, e.g., animal cells, with the
expression construct may be achieved by numerous methods that are
well known in the art. Cells may be either transiently transfected
or stably transfected. Several different well-established methods
exist for the delivery of molecules, particularly nucleic acids,
into host cells, e.g., animal cells. Depending on the cell type,
the desired transfection (i.e., transient or stable), and the
specific experimental requirements, such as transfection of
difficult cell lines or primary cells, the type of molecule
transfected (genomic DNA, DNA, oligonucleotides), or the expression
construct chosen, each transfer method possesses advantages and
disadvantages known to those of skill in the art. Common
transfection methods include, e.g., calcium phosphate
precipitation, liposome mediated transfection, DEAE
Dextran-mediated transfection, gene guns, electroporation,
nanoparticle delivery, polyamines, episomes, and polyethylenimines.
In addition, numerous transfection kits and reagents are
commercially available from companies such as Invitrogen
(VOYAGER.TM., LIPOFECTIN.RTM.), EMD Biosciences, San Diego, Calif.
(GENEJUICE.TM.), Qiagen, Germantown, Md. (SUPERFECT.TM.), Orbigen,
San Diego, Calif. (SAPPHIRE.TM.), and many others known to those of
skill in the art. Transfection protocols may also be found in Basic
Methods in Molecular Biology (2.sup.nd ed.) eds. Davis et al.,
Appleton and Lange, CT (1994).
[0062] The present invention uses cell cultures, e.g., large-scale
animal cell cultures, to produce large quantities of the
polypeptide of interest. Methods for large-scale transient
transfections are disclosed in Large-scale Mammalian Cell Culture
Technology (Biotechnology and Bioprocessing Series) ed. Lubiniecki,
Marcel Dekker, NY (1990); Kunaparaju et al. (2005) Biotechnol.
Bioeng. 91:670-77; Maiorella et al. (1988) Bio/Technology
6:1406-10; Baldi et al., supra; Lan Pham et al., supra; Meissner et
al., supra; Durocher et al., supra). In general, large-scale
transient gene expression in mammalian cell cultures may employ any
one of several common types of transfection modes, e.g.,
polyethylenimine, electric field pulse, CALFECTION.TM. or calcium
phosphate, to achieve high transfection efficiency at desired
scales or volumes, e.g., greater that 10 liters (Derouazi et al.,
supra; Rols et al. (1992) Eur. J. Biochem. 206(1):115-21; Wunn and
Bernard (1999) Curr. Opin. Biotechnol. 10(2):156-59; Schlaeger and
Christensen (1999) Cytotechnology 30(1-3):71-83; Jordan et al.
(1998) Cytotechnology 26(1):39-47; Lindell et al. (2004) Biochim.
Biophys. Acta 1676(2):155-61). These large-scale cultures are
generally grown in bioreactors, shakers, or incubators with stir
plates, and may also be known as "spinner" or "suspension"
cultures. Thus, as opposed to traditional transfections, in which
cells are attached to plates or flasks, the disclosed methods
generally use suspension cultures. Large-scale cell cultures are
generally considered to be cell cultures that have a volume of
greater than about 100 ml.
[0063] In some instances, cell lines expressing the polypeptide of
interest may be first produced and then used to seed a large-scale
cell culture. Stable cell lines that express a protein of interest
may be produced by various well-known methods, including the
methods used for transient transfection disclosed herein. In
general, stable cell lines are produced by long-term growth and
selection in a chemically defined media. For example, cells
transfected (e.g., by calcium phosphate precipitation, or liposomal
transfection) with a nucleic acid that encodes a polypeptide of
interest may concomitantly be transfected with a vector carrying a
neomycin resistance gene, which confers resistance to
neomycin/geneticin (G418). The transfected cells are then grown in
G418-containing media, and the surviving cells clonally expanded to
produce a stably expressing cell line. Aliquots of this cell line
may then be used to seed a large-scale culture and to produce large
quantities of the protein of interest.
[0064] Transfecting cells requires the optimization of several
variables, including cell-seeding density (e.g., about
1.times.10.sup.5 to about 3.times.10.sup.6 cells/ml culture), serum
concentration (e.g., 0-10%), incubation temperature (e.g., about
20-38.degree. C.), transfection vehicle or reagent (chemical or
electric), culture volume (e.g., about 5 ml-20 liters), and
incubation time (e.g., about 24-144 hours). For each cell type,
optimal parameters will vary. However, commercial suppliers
generally provide optimization guidelines for transfecting
particular cell types, as do various references known to those of
skill in the art that utilize transfection of the host cell chosen.
These sources may be used to direct transfection of the chosen host
cell, or may be used as a starting point from which simple trial
and error may be used to provide optimum transfection
parameters.
Cell Culture
[0065] Typical procedures for producing a polypeptide of interest
include batch cultures and fed-batch cultures. Batch culture
processes traditionally comprise inoculating a large-scale
production culture with a seed culture of a particular cell
density, growing the cells under conditions conducive to cell
growth and viability, harvesting the culture when the cells reach a
specified cell density, and purifying the expressed polypeptide.
Fed-batch culture procedures include an additional step or steps of
supplementing the batch culture with nutrients and other components
that are consumed during the growth of the cells. One of ordinary
skill in the art will recognize that the present invention can be
employed in any system in which cells are cultured including, but
not limited to, batch, fed-batch and perfusion systems. In certain
preferred embodiments of the present invention, the cells are grown
in fed-batch systems.
[0066] A persistent and unsolved problem with traditional cultures,
e.g., batch and fed-batch cultures, is the production of metabolic
waste products, which have detrimental effects on cell growth,
viability, and production of expressed polypeptides. Two metabolic
waste products that have particularly detrimental effects are
lactate and ammonium, which are produced as a result of glucose and
glutamine metabolism, respectively. In addition to the enzymatic
production of ammonium as a result of glutamine metabolism,
ammonium also accumulates in cell cultures as a result of
nonmetabolic degradation over time.
[0067] Traditional media formulations, including commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ([DMEM], Sigma), contain relatively high levels of glucose
and glutamine (the latter in comparison to other amino acids).
Previously, these components were believed to be required in
abundance since they are the primary metabolic energy sources for
the cells. However, rapid consumption of these nutrients leads to
the accumulation of lactate and ammonium as described above.
Additionally, high initial levels of glucose and glutamine, and the
subsequent accumulation of lactate and ammonium, result in high
osmolarity, a condition that by itself is often detrimental to cell
growth, cell viability and the production of polypeptides. The
rationally designed medium disclosed herein may be modified to
decrease the accumulation of harmful metabolic products. Such
modifications may be found in, e.g., U.S. Published Patent
Application Nos. 2005/0070013 (restricted glucose feeding) and
2006/0121568 (modifications of amino acid content and ratios) (both
of which are hereby incorporated by reference herein in their
entireties).
Rational Media Design and Formulations
[0068] Traditional media formulations begin with a relatively low
level of total amino acids in comparison with the media
formulations of the present invention. For example, DME-F12 (a
50:50 mixture of Dulbecco's Modified Eagle's medium and Ham's F12
medium) has a total amino acid content of 7.29 mM, and the
traditional cell culture medium known as RPMI-1640 has a total
amino acid content of 6.44 mM (see e.g., Morton (1970) In Vitro
6:89-108; Ham (1965) Proc. Nat. Acad. Sci. U.S.A. 53:288-93; Moore
et al. (1967) J. Am. Medical Assn. 199:519-24, all incorporated by
reference herein). More recent media formulations (such as the
media disclosed in U.S. Published Patent Application No.
2006/0121568) contain higher levels of amino acids and nutrients.
Traditional formulations, however, are not based on actual
calculated cell requirements, which include cell growth, cell
maintenance, and, for cell cultures used to produce recombinant
polypeptides, production requirements. Using these variables,
provided herein are methods of determining media formulations with
much higher, and yet nontoxic, concentrations of total amino
acids.
[0069] The cell culture media formulations, e.g., the large-scale
cell culture media formulations, described herein, when used in
accordance with, e.g., other culturing steps described herein, and
with, e.g., modifications such as those found in, e.g., U.S.
Published Patent Application No. 2006/0121568, optimize cell
density and polypeptide titer. An amino acid concentration of the
media formulations described herein is based on the concentration
of the amino acid(s) required for: 1) cell mass; 2) cell
maintenance; and 3) polypeptide production. In one embodiment of
the invention, a cell culture medium contains a concentration of
the amino acid(s) that is calculated for use in cell mass, a
concentration of the amino acid(s) that is calculated for use in
cell maintenance, and a concentration of the amino acid(s) that is
calculated for incorporation into the polypeptide of interest. In
another embodiment of the invention, a cell culture medium contains
a concentration, A, of an amino acid that is represented by the
formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is the concentration
of the amino acid that is used per unit of cell mass, P is the
concentration of the amino acid that is used for incorporation into
the polypeptide of interest per unit of polypeptide titer, M is the
multiplier for the desired cell mass (i.e., the desired peak units
of cell mass), N is the multiplier for the desired concentration of
the polypeptide of interest (i.e., desired or target polypeptide
titer), Y is the cell maintenance factor; and F is the baseline
factor.
[0070] The concentration, P, of the amino acid that is used for
incorporation into the polypeptide of interest per unit of
polypeptide titer in the formula above is based on the primary
structure of the recombinant protein, i.e., the amino acid content
of the polypeptide. Thus, P will vary based on the polypeptide of
interest that is to be produced by the large-scale cell culture. P
may then be converted to the amino acid requirement for the target
concentration of the polypeptide of interest using N, the
multiplier for the desired concentration of the polypeptide of
interest (i.e., desired or target polypeptide titer). In some
representative examples of the invention below, the basic unit of
polypeptide titer is 1 g/L. In Table 1, below, which contains a
representative calculation using the formula supplied herein, the
amino acid concentration of the cell culture medium that is
required for the polypeptide of interest at a titer of 10 g/L
(column 4) is determined by multiplying the concentration, P, of
the amino acid required for 1 g/L (column 2) by the multiplier, N,
wherein N=10.
TABLE-US-00001 TABLE 1 Representative Determination of
Baseline-Adjusted Amino Acid Concentration Required For Target
Titer of 10 g/L Antibody at a Desired Cell Mass, Where Desired Cell
Mass Is Represented by Desired Peak Cell Density of 15 .times.
10.sup.6 cells/ml 7 8 (A) (Column 7 .times. F) Calculated Baseline-
3 (X) 4 (P .times. N) 5 (X .times. M) 6 (X .times. M .times. Y)
Total AA Adjusted AA AA Total AA Total AA Total AA Concentration
Concentration Concentration Concentration Concentration
Concentration Required for Required for 2 (P) Required for Required
for Required for Required for Target Target AA Cell Mass Target
Desired Peak Cell Antibody Titer Antibody Titer Concentration
Represented Antibody Titer Cell Density of Maintenance and Desired
and Desired Required For by Cell of 10 g/L 15 .times. 10.sup.6
cells/ml (Y = 100%) or Peak Cell Peak Cell 1 Initial Density (N =
10) (M = 15) (Y = 1) Density Density Amino Antibody of 10.sup.6
Column Column Column 5 .times. (Column (F = 1.3) Acid Titer of 1
g/L cells/ml 2 .times. 10 3 .times. 15 100% 4 + 5 + 6) Column 7
.times. 1.3 (AA) mM mM mM mM mM mM mM ALA 0.41 0.30 4.08 4.56 4.56
13.20 17.17 ARG 0.17 0.16 1.73 2.38 2.38 6.50 8.45 ASN 0.26 0.15
2.62 2.23 2.23 7.08 9.21 ASP 0.29 0.29 2.87 4.36 4.36 11.59 15.07
CYS 0.19 0.09 1.88 1.42 1.42 4.73 6.14 GLU 0.40 0.16 4.00 2.33 2.33
8.67 11.26 GLN 0.39 0.29 3.87 4.40 4.40 12.67 16.47 GLY 0.47 0.25
4.72 3.80 3.80 12.33 16.02 HIS 0.16 0.07 1.59 1.07 1.07 3.72 4.83
ILE 0.14 0.16 1.43 2.33 2.33 6.10 7.93 LEU 0.49 0.25 4.88 3.80 3.80
12.49 16.24 LYS 0.52 0.24 5.20 3.55 3.55 12.31 16.00 MET 0.10 0.06
0.97 0.86 0.86 2.69 3.50 PHE 0.23 0.12 2.34 1.78 1.78 5.89 7.65 PRO
0.59 0.16 5.94 2.33 2.33 10.61 13.79 SER 0.97 0.34 9.73 5.11 5.11
19.96 25.94 THR 0.68 0.20 6.80 3.04 3.04 12.88 16.75 TRP 0.16 0.04
1.64 0.56 0.56 2.75 3.58 TYR 0.35 0.12 3.48 1.78 1.78 7.03 9.14 VAL
0.74 0.23 7.36 3.50 3.50 14.36 18.67 Total 7.71 3.68 77.13 55.21
55.21 187.55 243.82 (mM)
[0071] The multiplier N may be calculated, e.g., by multiplying the
integrated viable cell density (IVC) by the specific productivity
(qp) of a particular cell line (N=IVC*qp). For example, if the seed
density of a particular cell line is 0.8.times.10.sup.6 cells/ml,
the cell density at day 6 and day 10 is 15.times.10.sup.6 cells/ml,
and the cell density at day 18 is 11.times.10.sup.6 cells/ml (i.e.,
73% of the value at days 6 and 15), then the IVC=211.times.10.sup.6
(cells/ml)*day (i.e., [(0.8+15)/2]*6 days+[(15+15)/2]*4
days+[(15+11)/2]*8 days). If the average specific productivity (qp)
of the chosen cell line is, e.g., 47 .mu.g/10.sup.6 cells/day, then
N=10 g/L at day 18 (i.e., 211.times.10.sup.6
(cells/ml)*day.times.47 .mu.g/10.sup.6 cells/day). One of skill in
the art will realize that these calculations may be performed with
any cell line, or that N may be estimated based on cell
characteristics and origin. Alternatively, the multiplier N need
not be calculated from IVC and qp, and may simply be a reasonable
target titer for a particular cell culture. A prophetic example,
describing the calculation of IVC and qp, and the further selection
of reasonable N and M values, is provided as Example 5 (below).
[0072] As used herein, "cell mass," "cell density," and the like
refer to a collection of cells. For example, a cell mass can refer
to a cell pellet. As used herein, "desired cell mass" and the like
refers to a collection of cells, e.g., a cell pellet, that a
practitioner desires to obtain in a cell culture. As used herein,
"unit of cell mass" and the like reflects a number of ways of
representing cell mass, e.g., cell number, cell density, cell
volume, packed cell volume, dry cell weight, etc. One skilled in
the art will know which is the most convenient or appropriate way
of representing a unit of cell mass, etc., for a particular
experimental condition. One skilled in the art will also understand
that, depending on the unit of cell mass, M, the multiplier for
desired cell mass, i.e., the desired peak units of cell mass, will
be represented by either desired peak cell numbers, desired peak
cell density, desired peak cell volume, desired peak packed cell
volume, desired peak dry weight, etc.
[0073] In one embodiment of the invention, the unit of cell mass is
represented by cell density and the desired cell mass is
represented by desired peak cell density. In another embodiment,
the unit of cell mass is represented by dry cell weight or mass.
The dehydrated cell mass consists essentially of all proteins,
carbohydrates, lipids, and nucleic acids present in that cell mass.
Thus, the concentration, X, of an amino acid can be determined
experimentally, by first spinning a known number of cells to a cell
pellet, drying the cell pellet, and subsequently exposing the dried
pellet to acid-hydrolysis, thereby lysing the cellular proteins of
the cell pellet to individual amino acids, which may then be
quantified by an amino acid analyzer (see, e.g., Example 1). This
provides the amino acid concentration, X, of a given number of
cells, which may then be converted to the amino acid requirement
for the desired peak cell density using M, the multiplier for the
desired peak cell density. In Table 1, the total amino acid
concentration of the cell culture medium that is required for the
desired peak cell density of 15.times.10.sup.6 cells/ml (column 5)
is determined by multiplying the concentration, X, of the amino
acid required for 10.sup.6 cells/ml (column 3) by the multiplier,
M, wherein M=15. Thus, in some representative examples of the
invention, the unit of cell mass is 10.sup.6 cells/ml.
Alternatively, e.g., for an amino acid known to be susceptible to
degradation, the concentration, X, of the amino acid that is used
in the formula above may be determined from literature values,
e.g., Nyberg et al. (1999) Biotechnol. Bioeng. 62:324-35; Nadeau et
al. (2000) Metab. Eng. 2:277-92; and Bonarius (1996) Biotechnol.
Bioeng. 50:299-318, or the methods disclosed in such publications
or similar publications known in the art.
[0074] When the multiplier M for desired cell mass is represented
desired peak cell density, M may be chosen as, e.g., the peak
density of a particular cell line, by the density at which the
productivity of the cell line is maximized, or by the predicted
density for the cell line at a particular time period based on the
specific growth rate.
[0075] The amino acid concentration of the cell culture medium that
is required for cell maintenance, Y, is a percentage of the amino
acid concentration required for the desired cell mass, e.g.,
desired peak cell density. In one embodiment of the invention, the
maintenance requirement ranges from 0% to 300% of the desired cell
mass requirement, which provides sufficient nutrients for cell use
without risk of nutrient-induced toxicity. In another embodiment of
the invention, the maintenance requirement ranges from 0% to 150%
of the desired cell mass requirement, which provides sufficient
nutrients for cell use without risk of nutrient-induced toxicity.
The maintenance requirement increases as culture duration increases
(e.g., 100-150% maintenance for a 21 day culture). In Table 1, the
amino acid concentration of the cell culture medium that is
required for cell maintenance (column 6) is determined by
multiplying the amino acid concentration required for the desired
peak cell density (column 5) by the cell maintenance factor, Y,
which in this representative example is 100%, in order to allow an
extended culture period. The maintenance factor, Y, will differ for
different cells (and different cell lines) depending on the unique
metabolic demands of the cells in culture. Further, the maintenance
requirements for cells in culture will also differ due to the
variability in processes, e.g., inoculation density, culture
duration, time of temperature shift, etc. As an initial guideline,
one may provide, e.g., 0% maintenance (daily) for cultures at days
0 to 5, 3% to 5% maintenance (daily) for cultures at days 6 to 10,
and 7% to 10% maintenance (daily) for cultures at days 11 to 21.
For a process greater than 21 days, cultures may be provided 2% to
5% maintenance (daily) for those additional days. One of ordinary
skill in the art will realize that adjusting the maintenance
factor, Y, to optimize density and titer, is merely a matter of
routine trial and error. Adjusting amino acid concentration
according to the cell maintenance requirement is important for
increased viability and productivity of the cell culture, and thus
is an important aspect of the present invention. For example,
adjusting amino acid concentration according to the cell
maintenance requirement enables the cell culture to sustain greater
cell density, cell viability, and to produce higher polypeptide
titer (see, e.g., Example 6).
[0076] Once the amino acid requirements of desired: 1) cell mass
(column 5); 2) cell maintenance (column 6); and 3) polypeptide
production for the target titer (column 4) are determined, the
calculated total amino acid concentration of the target cell
culture may be obtained. In Table 1, the calculated total amino
acid concentration of the cell culture medium that is required for
the target cell culture (column 7), is determined by adding the
amino acid concentration of the cell culture medium that is
required for the polypeptide of interest at a titer of 10 g/L
(column 4), the amino acid concentration of the cell culture medium
that is required for the desired peak cell density of
15.times.10.sup.6 cells/ml (column 5), and the amino acid
concentration of the cell culture medium that is required for a
selected level of cell maintenance (column 6).
[0077] Once the calculated total amino acid concentration of the
cell culture medium that is required for the target cell culture is
obtained as described above, the value is adjusted to a desired
cell culture medium amino acid concentration, A, by a baseline
factor, F, which allows for the driving force of amino transfer,
e.g., the extra amino acids required for mass transfer, the extra
amino acids required to drive transport across cell membrane, etc.
This adjusted value, A, is referred to herein as the
"baseline-adjusted amino acid concentration" or "optimized
concentration." The baseline-adjusted amino acid concentration, A,
represents the cumulative total amount of an amino acid(s) that
will be delivered to the culture, expressed relative to the final
volume of the culture, which includes the volume of the starting
medium, plus the volume of any feeds for perfusion or fed-batch
culture(s). Adjusting the total amino acid concentration to the
baseline-adjusted amino acid concentration is an important aspect
of the invention because it allows higher cell viability, cell
density, and polypeptide titers (see, e.g., Example 6).
[0078] The baseline factor, F, which increases the calculated total
amino acid concentration of the cell culture medium by up to 200%,
ranges from 1 (0% increase) to 3 (200% increase). In one embodiment
of the invention, the range for F is between 1 and 1.5. In another
embodiment of the invention, a value of F below 1 may be offset by
modifying the calculated total amino acid concentration of the cell
culture medium (Table 1, column 7), which may be achieved by
modifying the amino acid concentration required for desired cell
mass, the amino acid concentration required for cell maintenance,
and/or the amino acid concentration required for incorporation into
the polypeptide of interest. For example, a baseline factor of 0.5
may be offset by increasing the calculated total amino acid
concentration of the cell culture medium by, e.g., a factor of two
(or more), which may be achieved by varying M, X, N, P and/or Y. In
Table 1, the baseline-adjusted amino acid concentration, A, of the
cell culture medium that is required for the target titer and
desired peak cell density (column 8), is determined by multiplying
the calculated total amino acid concentration of the cell culture
medium (column 7), by the baseline factor, F, which in the
representative example of Table 1 is 1.3 (corresponding to a 30%
increase over the calculated total amino acid concentration of the
cell culture medium).
[0079] Medium containing the baseline-adjusted concentration, A, of
an amino acid is referred to herein as the "desired cell culture
medium." Thus, the "desired cell culture medium" represents a goal
medium that contains the baseline-adjusted concentration, A. This
medium comprises at least one amino acid concentration determined
by the above formula. Preferably the desired cell culture medium
contains more than one amino acid concentration determined by the
above formula. More preferably, the desired cell culture medium
contains at least twelve adjusted amino acid concentrations, e.g.,
an adjusted concentration of arginine, histidine, isoleucine,
leucine, lysine, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine, and valine, determined by the above formula.
It will be understood by one of skill in the art that the
baseline-adjusted amino acid concentration, A, in the desired cell
culture medium may be achieved by any number of means, including,
but not limited to, individually adding the amino acid(s), adding
peptone or other protein hydrolysates, and/or by adding another
concentrated cell culture medium (e.g., a feeding cell culture
medium (or medium mix, e.g., a medium powder)) to a starting cell
culture medium (or starting cell culture medium mix, e.g., a medium
powder). One of skill in the art will understand that the addition
of peptone (or other protein hydrolysate) may be directed by the
amino acid contents of the particular peptone product of choice, or
by determining the amino acid concentration provided by a
particular peptone, e.g., in general, 5 g/L peptone provides a
concentration of about 40 mM to about 50 mM total amino acids.
[0080] The amino acid concentration in the desired cell culture
medium is based on at least one baseline-adjusted amino acid
concentration, A, determined by the inventive formula disclosed
herein; however, that concentration, as well as other
concentrations of amino acids in the desired cell culture medium,
may be varied from the baseline-adjusted amino acid
concentration(s) due to the influence of several factors. For
example, certain amino acids may be produced during culturing, and
thus may be kept at a low level. Other amino acids may be varied
based on published values (see, e.g., U.S. Published Patent
Application No. 2006/0121568). Further, some amino acids, e.g.,
methionine, may be consumed at a greater rate by particular cell
types, and thus should be added in excess. Yet other amino acids,
such as proline, provide a driving force for cell growth and
polypeptide production (see Example 4), and these amino acids
should be provided, in some cases, at a greater amount than
determined by the above inventive formula. In addition, one may
modify the baseline-adjusted amino acid concentration if the
concentration obtained using the above formula is considered to be
toxic (e.g., consideration of the levels of serine, tyrosine,
methionine and valine). It is within the knowledge of one of skill
in the art, upon obtaining the baseline-adjusted amino acid
concentration, A, of an amino acid(s) for use in the desired cell
culture medium, to vary the baseline-adjusted amino acid
concentration based on factors such as those noted herein.
[0081] Additional media components, for example, vitamins, salts,
glucose, elements, may be calculated from (or based on) various
sources, e.g., U.S. Published Patent Application Nos. 2005/0070013
and 2006/0121568. Further, the baseline-adjusted amino acid
concentration, A, of an amino acid obtained using the above formula
may be modified to provide a particular ratio in relation to
another amino acid (e.g., the ratio of glutamine to asparagine) or
to fall within a desired combined concentration (e.g., the combined
concentration of glutamine and asparagine). For example, it is
known that a high asparagine, low glutamine medium, combined with
temperature shift, enables uptake of lactate, thereby detoxifying a
cell culture (U.S. Published Patent Application No. 2006/0121568).
Thus, one may wish to modify the baseline-adjusted concentration,
A, of glutamine and/or asparagine, in order to obtain an optimum
ratio.
[0082] It will be noted from the representative example in Table 1
that the combined concentration of the baseline-adjusted amino acid
concentrations for use in the desired cell culture medium is high,
i.e., over 243 mM. Thus, disclosed herein is the finding that a
high concentration of amino acids may be used in a desired cell
culture medium without toxicity or titer detriment if that
concentration is based upon the calculated amino acid requirements
for a target cell density and polypeptide titer. In one embodiment
of the invention, the combined concentration of amino acids in the
desired cell culture medium is between about 120 mM and about 250
mM. In other embodiments, the combined concentration of amino acids
in the desired cell culture medium is greater than about 250 mM,
e.g., about 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,
500 or 510 mM, or any intermediate value.
[0083] The above-identified baseline-adjusted concentration, A, of
an amino acid that is used in a desired cell culture medium, may be
used in batch, fed-batch, and perfusion cultures. When used in
batch culture, the initial concentration of the amino acid used in
the desired cell culture medium is the baseline-adjusted amino acid
concentration, A. When used in fed-batch or perfusion cultures, the
baseline-adjusted amino acid concentration, A, represents the
cumulative total amount of an amino acid(s) that will be delivered
to the culture, which includes the volume of the starting medium
plus the volume of all feeds. Thus, for fed-batch cultures, which
use either continuous feeds (e.g., feeds on days 3-21) or periodic
feeds (e.g., feeds every 2-3 days), the starting medium is
engineered to contain a starting concentration of the amino acid,
B, according to the formula B=[A-(Z*V)]/(1-V), wherein Z is the
concentration of the amino acid in the feeding cell culture medium,
and V is the volume of the feeding culture medium as a proportion
of the desired cell culture medium volume. A representative example
of the calculations required to obtain the starting amino acid
concentration, B, is provided in Table 2, below. In Table 2, some
baseline-adjusted amino acid concentrations, A (column 2), are
converted to the starting media amino acid concentration, B (column
5), based on a 17% feed volume (V=17%), and the feeding medium
amino acid concentration, Z (column 4). In this example, several
baseline-adjusted amino acid concentrations, A (column 2), and
starting amino acid concentrations, B (column 5), are modified to
the values shown in bold in columns 3 and 6. Modification of
asparagine, aspartic acid, glutamine, and cysteine concentrations
was based on the concentrations suggested by U.S. Published Patent
Application No. 2006/0121568; methionine was adjusted by 50% to
compensate for its consumption at a higher amount than predicted;
alanine, glutamic acid and glycine are produced by the cultures
(and thus kept at a low level); and serine, tyrosine, and valine
concentrations were decreased to nontoxic levels. It will therefore
be understood that the starting amino acid concentration, B, may be
based on either the baseline-adjusted amino acid concentration, A,
or on the modified baseline-adjusted amino acid concentration.
[0084] One of skill in the art will appreciate that the feeding
medium used to obtain the desired cell culture medium during
fed-batch and perfusion cell culturing should be as highly
concentrated as possible in order to avoid overflow in the
container in which the culture is carried out (e.g., a bioreactor
or shaker flask) and to avoid diluting the media components. In the
example set forth in Table 2, a preferred feeding media is denoted
"Feed Medium," and the amino acid concentrations, Z, of the Feed
Medium are set forth in column 4. However, any highly concentrated
feeding medium, or any method of providing highly concentrated
amino acids to the starting cell culture medium may be used, as
long as the desired baseline-adjusted amino acid concentration, A,
will be achieved in the target volume. Such methods of providing
highly concentrated amino acids to a cell culture are commonly used
and well known to one of skill in the art.
[0085] It will be understood by one of skill in the art that the
starting concentration of the amino acid, B, in the starting cell
culture medium may be achieved by any number of means, including,
but not limited to, individually adding the amino acid(s), adding
peptone and/or other protein hydrolysate, and/or adding another
concentrated culture medium (or medium mix, e.g., a medium powder)
to the starting cell culture medium (or starting cell culture
medium mix, e.g., a medium powder). It will also be understood by
one of skill in the art that the concentration of the amino acid,
Z, in the feeding cell culture medium may be achieved by any number
of means, including, but not limited to, individually adding the
amino acid(s), adding peptone and/or other protein hydrolysate,
and/or adding another concentrated culture medium (or medium mix,
e.g., a medium powder) to the feeding cell culture medium (or
feeding cell culture medium mix, e.g., a medium powder).
[0086] It will be noted from the representative example in Table 2
that the combined concentration of amino acids for use in the
starting cell culture medium is high, i.e., over 125 mM. Thus,
disclosed herein is the finding that a high concentration of amino
acids may be used without toxicity or titer detriment in a starting
cell culture medium if the starting amino acid concentration is
based upon the calculated amino acid requirements for a desired
peak cell density and a desired polypeptide titer. In one
embodiment of the invention, the combined concentration of amino
acids is between about 70 mM and about 510 mM. In one embodiment of
the invention, the combined concentration of amino acids is between
about 120 mM and about 350 mM. In another embodiment of the
invention, the combined concentration of amino acids in the
starting cell culture medium is between about 70 mM and about 140
mM. In another embodiment of the invention, the combined
concentration of amino acids in the starting cell culture medium is
greater than about 140 mM.
TABLE-US-00002 TABLE 2 Representative Determination of Starting
Amino Acid Concentration For A Target Titer of 10 g/L Antibody, a
Desired Peak Cell Density of 15 .times. 10.sup.6 cells/ml, and a
17% Feed 1 2 (A) 3 4 (Z) 5 (B) 6 Modified Baseline- Starting AA
Starting AA Adjusted AA Modification of Concentration Concentration
Concentration Baseline-Adjusted Required For Required for Required
For AA Concentration Target Titer and Target Titer Target Titer and
Required for Target Feed Desired Peak and Desired Amino Desired
Peak Titer and Desired Medium AA Cell Density Peak Cell Acid (AA)
Cell Density Peak Cell Density Concentration (V = 17%) Density mM
mM mM mM mM ALA 17.17 0.20 6.4 -1.07 0.2 ARG 8.45 8.45 35.13 2.99
2.99 ASN 9.21 24.00 56 17.45 17.45 ASP 15.07 1.70 16 -1.23 1.7 CYS
6.14 0.40 0 0.48 0.4 GLU 11.26 0.20 6.4 -1.07 0.2 GLN 16.47 4.00 0
4.82 4.2 GLY 16.02 4.00 6.4 3.51 3.51 HIS 4.83 4.83 11.2 3.53 3.53
ILE 7.93 7.93 28.82 3.65 3.65 LEU 16.24 16.24 41.53 11.06 11.06 LYS
16.00 16.00 32 12.72 12.72 MET 3.50 5.25 12.8 3.71 3.71 PHE 7.65
7.65 16 5.94 5.94 PRO 13.79 13.79 19.2 12.68 12.68 SER 25.94 25.94
48.15 21.39 10.2 THR 16.75 16.75 25.6 14.94 14.94 TRP 3.58 3.58
5.11 3.27 3.27 TYR 9.14 9.14 12.75 8.40 5.2 VAL 18.67 18.67 25.6
17.25 10.2 Total 243.82 405.09 127.73 (mM )
[0087] As shown in Table 3A and Table 3B, the determination of the
baseline-adjusted amino acid concentration of an amino acid, A,
used in a desired cell culture medium, and the determination of the
starting amino acid concentration, B, used in the starting cell
culture medium, may be calculated for any desired target
polypeptide titer and desired peak (target) cell density. The
desired peak cell density of the large-scale culture ranges from
about 3 to about 40.times.10.sup.6 cells/mL for fed-batch culture.
In one embodiment of the invention, the desired peak cell density
ranges from about 5 to about 20.times.10.sup.6 cells/mL. The target
titer of the large-scale culture ranges from about 3 to about 25
g/L. In yet another embodiment of the invention, the target titer
of the large-scale culture ranges from about 5 to about 20 g/L. For
example, in Table 3, desired peak cell density ranges from 10 to
15.times.10.sup.6 cells/mL, and target titer varies from 3 to 10
g/L.
TABLE-US-00003 TABLE 3A Representative Examples of
Baseline-Adjusted Amino Acid Concentrations, A, for Various Target
Titers (As Represented by a Multiplier for Target Polypeptide
Titer, N) and Desired Peak Cell Densities (As Represented by a
Multiplier for Desired Peak Cell Density, M). A N = 10 N = 5 N = 3
M = 15 M = 12.5 M = 10 Amino Acid Concentration mM mM mM ALA 17.2
12.5 9.50 ARG 8.5 6.3 4.81 ASN 9.2 6.5 4.89 ASP 15.1 11.3 8.68 CYS
6.1 4.3 3.20 GLU 11.3 7.7 5.60 GLN 16.5 12.1 9.14 GLY 16.0 11.3
8.43 HIS 4.8 3.3 2.46 ILE 7.9 6.0 4.60 LEU 16.2 11.4 8.50 LYS 16.0
11.1 8.18 MET 3.5 2.5 1.87 PHE 7.7 5.4 3.99 PRO 13.8 8.9 6.36 SER
25.9 17.4 12.66 THR 16.8 11.0 7.93 TRP 3.6 2.3 1.61 TYR 9.1 6.1
4.43 VAL 18.7 12.4 8.94 Total 243.8 169.8 125.8
TABLE-US-00004 TABLE 3B Representative Examples of Starting Media
Amino Acid Concentrations, B, for Various Target Titers (As
Represented by a Multiplier for Target Polypeptide Titer, N) and
Desired Peak Cell Densities (As Represented by a Multiplier for
Desired Peak Cell Density, M); Starting Media Amino Acid
Concentrations Were Determined by Subtracting Feed and Other
Modifications from Baseline-Adjusted Amino Acid Concentrations B N
= 10 N = 5 N = 3 M = 15 M = 12.5 M = 10 Amino Acid Concentration mM
mM mM ALA 0.2 0.2 0.20 ARG 3.0 1.9 1.68 ASN 17.4 14.6 10.77 ASP 1.7
1.7 1.70 CYS 0.4 0.4 0.40 GLU 0.2 0.2 0.20 GLN 4.0 4.0 4.00 GLY 3.5
3.6 3.75 HIS 3.5 2.2 1.56 ILE 3.6 2.5 2.10 LEU 11.1 6.9 5.09 LYS
12.7 7.9 5.73 MET 3.7 2.4 1.78 PHE 5.9 3.8 2.75 PRO 12.7 7.4 5.04
SER 10.2 10.2 9.00 THR 14.9 8.8 6.10 TRP 3.3 1.8 1.24 TYR 5.2 5.1
3.58 VAL 10.2 10.4 7.22 Total 127.5 95.9 73.89
[0088] Using the formula to determine baseline-adjusted amino acid
concentration(s) and to develop desired cell culture media
formulations for large-scale polypeptide production, the inventors
have identified several criteria that result in high titer and high
cell density cultures. The criteria for producing a titer higher
than 5 g/L, which are represented by the values shown in Table 4,
include, but are not limited to, one or more of the following:
greater than or equal to about 3 mM tyrosine; between about 7 mM
and about 30 mM proline; between about 7 mM and about 30 mM valine;
between about 7 mM and about 30 mM leucine; between about 7 mM and
about 30 mM threonine; between about 7 mM and about 30 mM lysine; a
combined concentration of leucine, lysine, proline, threonine and
valine that is between about 35 mM and about 150 mM; a combined
concentration of leucine, lysine, threonine and valine that is
between about 60% to about 80% of the total essential amino acids
in the desired cell culture medium; a combined concentration of the
essential amino acids in the desired cell culture medium that is
between about 30% to about 50% of the total amino acids in the
desired cell culture medium; and/or a combined concentration of
total amino acids between about 75 mM and about 510 mM.
TABLE-US-00005 TABLE 4 Representative Examples of the Amino Acid
Content and Relationships For Rationally Designed Media for Various
Target Titers and Desired Peak Cell Densities (as noted above, the
basic unit of polypeptide titer is 1 g/L, and the basic unit of
cell mass is 10.sup.6 cells/ml). N = 5 N = 10 N = 15 N = 15 N = 15
M = 10 M = 10 M = 10 M = 20 M = 30 Y = 0 Y = 1 Y = 1 Y = 1 Y = 1.5
Amino F = 1 F = 1.3 F = 1.3 F = 1.3 F = 1.3 Acid (mM) (mM) (mM)
(mM) (mM) ALA 5.08 13.21 15.86 23.77 37.62 ARG 2.46 6.39 7.51 11.65
18.88 ASN 2.80 7.28 8.98 12.85 19.62 ASP 4.34 11.29 13.15 20.71
33.94 CYS 1.89 4.91 6.14 8.60 12.91 GLU 3.55 9.24 11.84 15.89 22.96
GLN 4.87 12.66 15.18 22.80 36.15 GLY 4.90 12.73 15.79 22.39 33.93
HIS 1.50 3.91 4.94 6.78 10.01 ILE 2.27 5.91 6.84 10.88 17.96 LEU
4.98 12.94 16.12 22.71 34.25 LYS 4.97 12.92 16.30 22.46 33.23 MET
1.06 2.76 3.39 4.88 7.50 PHE 2.35 6.11 7.63 10.71 16.09 PRO 4.53
11.77 15.63 19.67 26.75 SER 8.27 21.51 27.83 36.70 52.21 THR 5.43
14.11 18.53 23.81 33.04 TRP 1.19 3.10 4.16 5.13 6.82 TYR 2.92 7.60
9.87 12.94 18.33 VAL 6.01 15.63 20.42 26.48 37.10 Total 75.37
195.97 246.11 341.81 509.28 Concentration Total Essential 32.22
83.78 105.84 145.49 214.87 Total Bold 21.39 55.61 71.37 95.46
137.61 Bold/Essential 66% 66% 67% 66% 64% Bold/Total 28% 28% 29%
28% 27% Essential/Total 43% 43% 43% 43% 42%
[0089] In Table 4, the bold amino acids are valine, threonine,
leucine and lysine, the italicized amino acids are the essential
amino acids (e.g., arginine, histidine, leucine, isoleucine,
lysine, methionine, tryptophan, threonine, and valine; and for CHO
cell cultures, proline is additionally an essential amino acid; one
of skill in the art is aware of variations in the definition of
essential amino acids as it applies to different cells, etc.).
Table 4 provides representative desired cell culture media for CHO
cells at a variety of target cell densities (M), target titers (N),
cell maintenance requirements (Y), and baseline adjustments
(F).
[0090] One of skill in the art will recognize that the rationally
designed media of the invention may be used to either produce a
polypeptide of interest, or may be used in cell culturing that is
not designed for polypeptide production. Accordingly, a rationally
designed media may be used in the disclosed methods of cell
culturing, e.g., for the efficient growth, replication and/or
maintenance of cell cultures, e.g., large-scale cell cultures, that
do not contain host cells engineered to produce an exogenous
polypeptide of interest, or which are not cultured to produce an
endogenous polypeptide of interest. In such an instance, the
desired cell culture medium need not account for the amino acid(s)
required for incorporation into the polypeptide of interest, and
instead contains an amino acid concentration(s) based on the
concentration of the amino acid(s) required for: 1) desired cell
mass, and 2) cell maintenance. Such a desired cell culture medium
used in the disclosed methods of cell culturing contains a
baseline-adjusted amino acid concentration, A', according to the
formula A'=[(M*X)+(Y*M*X)]*F, wherein X is a concentration of the
amino acid that is used per unit of cell mass, M is a multiplier
for a desired peak cell mass (e.g., desired peak cell density,
etc.) of the cell culture, Y is a cell maintenance factor, and F is
a baseline factor. A desired cell culture medium produced according
to this formula is then provided to a cell culture under conditions
that allow growth and replication of the cells in the cell culture.
In one embodiment of the invention, the method of cell culturing
uses a large-scale cell culture. In another embodiment of the
invention, the method of cell culturing uses animal cells.
Proline Addition to Cell Culture Media
[0091] Using the rational media design methods disclosed herein, it
has been determined that maintaining high proline levels throughout
the culture period of cell culture, e.g., large-scale cell culture,
results in increased polypeptide titer and increased cell density.
This proline "threshold" ranges from about 1 mM to about 2 mM, and
a level of proline in the cell culture maintained above this
threshold appears to be a driving force for producing high cell
density, high titer large-scale cell cultures. Interestingly, the
proline-driven increased polypeptide titer and cell viability
concomitantly increases the culture's requirement for additional
amino acids (in order to satisfy the increased consumption rate),
which at least partially explains why the rationally designed media
formulations herein contain high amino acid concentrations.
Providing a Cell Culture
[0092] Various methods of preparing mammalian cells for production
of polypeptides by batch and fed-batch culture are well known in
the art. As described above, a nucleic acid sufficient to achieve
expression (typically a vector containing the nucleic acid encoding
the polypeptide of interest and any operably linked genetic control
elements) may be introduced into the host cell line by any number
of well-known techniques. Typically, cells are screened to
determine which of the host cells have actually taken up the vector
and express the polypeptide or protein of interest. Traditional
methods of detecting a particular polypeptide or protein of
interest expressed by mammalian cells include, but are not limited
to, immunohistochemistry, immunoprecipitation, flow cytometry,
immunofluorescence microscopy, SDS-PAGE, Western blots,
enzyme-linked immunosorbent assay (ELISA), high performance liquid
chromatography (HPLC) techniques, biological activity assays and
affinity chromatography. One of ordinary skill in the art will be
aware of other appropriate techniques for detecting expressed
polypeptides or proteins. If multiple host cells express the
polypeptide or protein of interest, some or all of the listed
techniques can be used to determine which of the cells expresses
that polypeptide or protein at the highest levels.
[0093] Once a cell that expresses the polypeptide or protein of
interest has been identified, the cell is propagated in culture by
any of the variety of methods well known to one of ordinary skill
in the art. The cell expressing the polypeptide or protein of
interest is typically propagated by growing it at a temperature and
in a medium that is conducive to the survival, growth and viability
of the cell. The initial culture can be of any volume, but is often
of lower volume than the culture volume of the production
bioreactor used in the final production of the polypeptide or
protein of interest, and frequently cells are passaged several
times in bioreactors of increasing volume prior to seeding the
production bioreactor. The cell culture can be agitated or shaken
to increase oxygenation of the medium and dispersion of nutrients
to the cells. Alternatively or additionally, special sparging
devices that are well known in the art can be used to increase and
control oxygenation of the culture. In accordance with the present
invention, one of ordinary skill in the art will understand that it
can be beneficial to control or regulate certain internal
conditions of a cell culture, including but not limited to pH
(e.g., 6.6 to 7.6), temperature (e.g., 25.degree. C. to 42.degree.
C.), levels of oxygen and carbon dioxide (e.g., O.sub.2: 10% to 80%
and CO.sub.2: 7% to 15%, throughout culture), and osmolality (e.g.,
a starting osmolality of 260 to 340 mOsm/kg), etc.
[0094] As used herein, the teem "inoculum" is used to refer to a
volume of cells containing the nucleic acid that expresses the
polypeptide of interest, which is used to seed the production
vessel in which the large-scale animal culture will occur, e.g.,
the production bioreactor. In one embodiment of the invention, the
inoculum volume is about 60 to 80% of the final volume.
[0095] The starting cell density in the production bioreactor can
be chosen by one of ordinary skill in the art. In accordance with
the present invention, the starting cell density in the production
bioreactor can be as low as a single cell per culture volume. In
preferred embodiments of the present invention, starting cell
densities in the production bioreactor can range from about
0.1.times.10.sup.6 viable cells per mL to about 10.times.10.sup.6
viable cells per mL and higher.
[0096] Initial and intermediate cell cultures may be grown to any
desired density before seeding the next intermediate or final
production bioreactor. In one embodiment of the invention, the
inoculum cell density is about 0.5-1.times.10.sup.6 cells/ml. It is
preferred that most of the cells remain alive prior to seeding,
although total or near total viability is not required. In one
embodiment of the present invention, the cells may be removed from
the supernatant, for example, by low-speed centrifugation. It may
also be desirable to wash the removed cells with a medium before
seeding the next bioreactor to remove any unwanted metabolic waste
products or medium components. The medium may be the medium in
which the cells were previously grown or it may be a different
medium or a washing solution selected by the practitioner of the
present invention.
[0097] The cells may then be diluted to an appropriate density for
seeding the production bioreactor. The cells can be diluted into
another medium or solution, e.g., the starting cell culture medium
or the desired cell culture medium, depending on the needs and
desires of the practitioner of the present invention or to
accommodate particular requirements of the cells themselves (e.g.,
if the cells are to be stored for a short period of time prior to
seeding the production bioreactor).
Initial Growth Phase
[0098] Once the production vessel is seeded as described above, the
animal cell culture may be maintained in the initial growth phase
using the desired cell culture medium obtained by the inventive
formula disclosed herein, and under conditions conducive to the
survival, growth and viability of the cell culture. The precise
conditions will vary depending on the cell type, the organism from
which the cell was derived, and the nature and character of the
expressed polypeptide or protein.
[0099] A production bioreactor can be any volume that is
appropriate for large-scale production of polypeptides or proteins.
In a preferred embodiment, the volume of the production bioreactor
is at least 500 liters. In other preferred embodiments, the volume
of the production bioreactor is 1000, 2500, 5000, 8000, 10,000,
12,000 liters or more, or any intermediate volume. One of ordinary
skill in the art will be aware of, and will be able to choose, a
suitable bioreactor for use in practicing the present invention.
The production bioreactor may be constructed of any material that
is conducive to cell growth and viability that does not interfere
with expression or stability of the produced polypeptide or
protein.
[0100] The temperature of the cell culture in the initial growth
phase will be selected based primarily on the range of temperatures
at which the cell culture remains viable. For example, during the
initial growth phase, CHO cells grow well at 37.degree. C. In
general, most mammalian cells grow well within a range of about
35.degree. C. to 39.degree. C. In one embodiment of the invention,
the temperature for growth phase (day 0 to day 3) is 37.degree. C.,
and the temperature for production phase (after day 3) is
31.degree. C. Those of ordinary skill in the art will be able to
select appropriate temperature or temperatures in which to grow
cells, depending on the needs of the cells and the production
requirements of the practitioner.
[0101] In one embodiment of the present invention, the temperature
of the initial growth phase is maintained at a single, constant
temperature. In another embodiment, the temperature of the initial
growth phase is maintained within a range of temperatures. For
example, the temperature may be steadily increased or decreased
during the initial growth phase. Alternatively, the temperature may
be increased or decreased by discrete amounts at various times
during the initial growth phase. One of ordinary skill in the art
will be able to determine whether a single or multiple temperatures
should be used, and whether the temperature should be adjusted
steadily or by discrete amounts.
[0102] The cells may be grown during the initial growth phase for a
greater or lesser amount of time, depending on the needs of the
practitioner and the requirement of the cells themselves. In one
embodiment, the cells are grown for a period of time sufficient to
achieve a viable cell density that is a given percentage of the
maximal viable cell density that the cells would eventually reach
if allowed to grow undisturbed. For example, the cells may be grown
for a period of time sufficient to achieve a desired viable cell
density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95 or 99 percent or more of maximal viable cell
density.
[0103] In another embodiment the cells are allowed to grow for a
defined period of time. For example, depending on the starting
concentration of the cell culture, the temperature at which the
cells are grown, and the intrinsic growth rate of the cells, the
cells may be grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20 or more days. In some cases, the
cells may be allowed to grow for a month or more. The cells would
be grown for 0 days in the production bioreactor if their growth in
a seed bioreactor, at the initial growth phase temperature, was
sufficient that the viable cell density in the production
bioreactor at the time of its inoculation is already at the desired
percentage of the maximal viable cell density. The practitioner of
the present invention will be able to choose the duration of the
initial growth phase depending on polypeptide or protein production
requirements and the needs of the cells themselves.
[0104] The cell culture may be agitated or shaken during the
initial culture phase in order to increase oxygenation and
dispersion of nutrients to the cells. In accordance with the
present invention, one of ordinary skill in the art will understand
that it can be beneficial to control or regulate certain internal
conditions of the bioreactor during the initial growth phase,
including but not limited to pH, temperature, oxygenation, etc. For
example, pH can be controlled by supplying an appropriate amount of
acid or base, and oxygenation can be controlled with sparging
devices that are well known in the art.
Shifting Culture Conditions
[0105] At the end of the initial growth phase, a culture
condition(s) may be shifted so that a second set of culture
conditions is applied and a metabolic shift occurs in the culture.
The accumulation of inhibitory metabolites, most notably lactate
and ammonia, inhibits growth. A metabolic shift, accomplished by,
e.g., a change in the temperature, pH, osmolality or chemical
inductant level of the cell culture, may be characterized by, e.g.,
a reduction in the ratio of a specific lactate production rate to a
specific glucose consumption rate. In one nonlimiting embodiment,
the culture conditions are shifted by changing the temperature of
the culture. In another embodiment of the invention, the
temperature shift occurs on day 1-7. In another embodiment of the
invention, the temperature is shifted to 29.degree. C.-32.degree.
C. In another embodiment of the invention, the temperature shift
occurs at day 3, and the temperature is shifted to 31.degree. C.
Teachings regarding temperature shift and metabolic shift may be
found in the art (see, e.g., U.S. Published Patent Application No.
US 2006/0121568).
Subsequent Production Phase
[0106] Once the conditions of the cell culture have been shifted as
discussed above, the cell culture may be maintained for a
subsequent production phase under a second set of culture
conditions conducive to the survival and viability of the cell
culture and appropriate for expression of the desired polypeptide
or protein at adequate, e.g., commercially adequate, levels.
[0107] As discussed above, the culture may be shifted by shifting
one or more of a number of culture conditions including, but not
limited to, temperature, pH, osmolality, and sodium butyrate
levels. In one embodiment, the temperature of the culture is
shifted. According to this embodiment, during the subsequent
production phase, the culture is maintained at a temperature or
temperature range that is lower than the temperature or temperature
range of the initial growth phase. For example, during the
subsequent production phase, CHO cells express recombinant
polypeptides and proteins well within a range of 25.degree. C. to
35.degree. C. In one embodiment of the invention, the production
phase begins after day 3. In another embodiment of the invention,
the production phase is carried out at 31.degree. C. As discussed
in U.S. Published Patent Application No. US 2006/0121568, multiple
discrete temperature shifts may be employed to increase cell
density or viability or to increase expression of the recombinant
polypeptide or protein.
[0108] In accordance with the formula of the present invention, a
desired cell mass (e.g., cell density) and production titer (e.g.,
target titer) are selected in order to establish the
baseline-adjusted amino acid concentration, A, and the starting
amino acid concentration, B. Thus, generally the cells are
maintained in the subsequent production phase until the desired
cell density or production titer, or a value(s) near the desired
cell density or production titer is reached. In one embodiment, the
cells are maintained in the subsequent production phase until the
titer of the recombinant polypeptide or protein reaches a maximum.
In other embodiments, the culture may be harvested prior to this
point, depending on the production requirement of the practitioner
or the needs or viability of the cells themselves. For example, the
cells may be maintained for a period of time sufficient to achieve
a viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent or more of maximal
viable cell density. In some cases, it may be desirable to allow
the viable cell density to reach a maximum, and then allow the
viable cell density to decline to some level before harvesting the
culture. In an extreme example, it may be desirable to allow the
viable cell density to approach or reach zero before harvesting the
culture.
[0109] In another embodiment of the present invention, the cells
are allowed to grow for a defined period of time during the
subsequent production phase. For example, depending on the
concentration of the cell culture at the start of the subsequent
growth phase, the temperature at which the cells are grown, and the
intrinsic growth rate of the cells, the cells may be grown for 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
or more days. In some cases, the cells may be allowed to grow for a
month or more. The practitioner of the present invention will be
able to choose the duration of the subsequent production phase
depending on polypeptide or protein production requirements and the
needs of the cells themselves. The duration of culture will help
determine the amino acid concentration required for cell
maintenance, which may range in the present invention from, e.g.,
0% to 150% of the amino acid concentration required for cell
mass.
[0110] In certain cases, it may be beneficial or necessary to
supplement the cell culture, i.e., feed the cell culture, during
the subsequent production phase with nutrients or other medium
components that have been depleted or metabolized by the cells. For
example, it might be advantageous to supplement the cell culture
with nutrients or other medium components observed to have been
depleted during monitoring of the cell culture (see "Monitoring
Cell Culture Conditions" section, below). Alternatively or
additionally, it may be beneficial or necessary to supplement the
cell culture prior to the subsequent production phase. As
nonlimiting examples, it may be beneficial or necessary to
supplement the cell culture with hormones and/or other growth
factors, particular ions (such as sodium, chloride, calcium,
magnesium, and phosphate), buffers, vitamins, nucleosides or
nucleotides, trace elements (inorganic compounds usually present at
very low final concentrations), lipids, amino acids, or glucose (or
another energy source).
[0111] These supplementary components may all be added, i.e., fed,
to the cell culture at one time, or they may be provided to the
cell culture in a series of additions. In one embodiment of the
present invention, the supplementary components are provided to the
cell culture at multiple times in proportional amounts. In another
embodiment, it may be desirable to provide only certain of the
supplementary components initially, and provide the remaining
components at a later time. In yet another embodiment of the
present invention, the cell culture is fed continually with these
supplementary components.
[0112] In accordance with the present invention, the total volume
added to the cell culture should optimally be kept to a minimal
amount. For example, the total volume of the feeding medium, or
solution containing the supplementary components, added to the cell
culture may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,
40, 45 or 50% of the volume of the cell culture prior to providing
the supplementary components. Thus, the feeding medium should be
concentrated in order to avoid bioreactor overflow or medium
component dilution. The feeding medium is preferably provided to
the main culture with the same pH, temperature, etc., but with high
concentrations of nutrients relative to the starting medium. In one
embodiment of the invention, the feeding medium is the feeding
medium designated "Feed Medium" in column 4 of Table 2.
[0113] In one embodiment of the invention, a cell culture with a
starting amino acid concentration, B, is supplemented with an
additional amino acid(s) in order to achieve a baseline-adjusted
amino acid concentration, A. In another embodiment of the
invention, the cell culture is provided with a continuous feed from
about days 3-21, or periodic feeds every 2-3 days. In another
embodiment of the invention, the feeding occurs from about day 3 to
about day 20 (for a 21-day culture) as bolus feeds. In another
embodiment of the invention, the feeding occurs as periodic feeds
about every 2-3 days. In yet another embodiment of the invention,
the feeding volume is about 1% to about 40% of the total cell
culture volume.
[0114] The cell culture may be agitated or shaken during the
subsequent production phase in order to increase oxygenation and
dispersion of nutrients to the cells. In accordance with the
present invention, one of ordinary skill in the art will understand
that it can be beneficial to control or regulate certain internal
conditions of the bioreactor during the subsequent growth phase,
including but not limited to pH, temperature, oxygenation, etc. For
example, pH can be controlled by supplying an appropriate amount of
acid or base, and oxygenation can be controlled with sparging
devices that are well known in the art.
Monitoring Cell Culture Conditions
[0115] In certain embodiments of the present invention, the
practitioner may find it beneficial or necessary to periodically
monitor particular conditions of the growing cell culture.
Monitoring cell culture conditions allows the practitioner to
determine whether the cell culture is producing the recombinant
polypeptide of interest at suboptimal levels or whether the culture
is about to enter into a suboptimal production phase. In order to
monitor certain cell culture conditions, it may be necessary to
remove small aliquots of the culture for analysis. One of ordinary
skill in the art will understand that such removal may potentially
introduce contamination into the cell culture, and will take
appropriate care to minimize the risk of such contamination.
[0116] As nonlimiting examples, it may be beneficial or necessary
to monitor temperature, pH, cell density, cell viability,
integrated viable cell density, lactate levels, ammonium levels,
osmolarity, or titer of the expressed polypeptide. Numerous
techniques are well known in the art that will allow one of
ordinary skill in the art to measure these conditions. For example,
cell density may be measured using a hemacytometer, a Coulter
counter, or cell density examination (CEDEX.RTM., Innovatis,
Malvern, Pa.). Viable cell density may be determined by staining a
culture sample with Trypan blue. Since only dead cells take up the
Trypan blue (i.e., viable cells exclude the dye), viable cell
density can be determined by counting the total number of cells,
dividing the number of cells that take up the dye by the total
number of cells, and taking the reciprocal. HPLC can be used to
determine the levels of lactate, ammonium or the expressed
polypeptide or protein. Alternatively, the level of the expressed
polypeptide or protein can be determined by standard molecular
biology techniques such as Coomassie staining of SDS-PAGE gels,
Western blotting, Bradford assays, Lowry assays, biuret assays, and
UV absorbance. It may also be beneficial or necessary to monitor
the post-translational modifications of the expressed polypeptide
or protein, including phosphorylation and glycosylation.
Harvesting Polypeptides Produced by Cell Culture
[0117] The polypeptide of interest that is produced by the cell
culture may then be purified from the culture medium or from cell
extracts for use in various applications. Soluble forms of the
polypeptide can be purified from conditioned media. Membrane-bound
forms of the polypeptide can be purified by preparing a total
membrane fraction from the expressing cell and extracting the
membranes with a nonionic detergent such as TRITON.RTM. X-100 (EMD
Biosciences, San Diego, Calif.). Cytosolic or nuclear proteins may
be prepared by lysing the host cells (via mechanical force,
Parr-bomb, sonication, detergent, etc.), removing the cell membrane
fraction by centrifugation, and retaining the supernatant.
[0118] The polypeptide can be purified using other methods known to
those skilled in the art. For example, a polypeptide produced by
the disclosed methods can be concentrated using a commercially
available protein concentration filter, for example, an AMICON.RTM.
or PELLICON.RTM. ultrafiltration unit (Millipore, Billerica,
Mass.). Following the concentration step, the concentrate can be
applied to a purification matrix such as a gel filtration medium.
Alternatively, an anion exchange resin (e.g., a MonoQ column,
Amersham Biosciences, Piscataway, N.J.) may be employed; such resin
contains a matrix or substrate having pendant diethylaminoethyl
(DEAE) or polyethylenimine (PEI) groups. The matrices used for
purification can be acrylamide, agarose, dextran, cellulose or
other types commonly employed in protein purification.
Alternatively, a cation exchange step may be used for purification
of proteins. Suitable cation exchangers include various insoluble
matrices comprising sulfopropyl or carboxymethyl groups (e.g.,
S-SEPHAROSE.RTM. columns, Sigma-Aldrich, St. Louis, Mo.).
[0119] The purification of the polypeptide from culture supernatant
may also include one or more column steps over affinity resins,
such as concanavalin A-agarose, AF-HEPARIN650,
heparin-TOYOPEARL.RTM. or Cibacron blue 3GA SEPHAROSE.RTM. (Tosoh
Biosciences, San Francisco, Calif.); hydrophobic interaction
chromatography columns using such resins as phenyl ether, butyl
ether, or propyl ether; or immunoaffinity columns using antibodies
to the labeled protein. Finally, one or more high performance
liquid chromatography (HPLC) steps employing hydrophobic HPLC
media, e.g., silica gel having pendant methyl or other aliphatic
groups (e.g., Ni-NTA columns), can be employed to further purify
the protein. Alternatively, the polypeptides may be recombinantly
expressed in a form that facilitates purification. For example, the
polypeptides may be expressed as a fusion with proteins such as
maltose-binding protein (MBP), glutathione-S-transferase (GST), or
thioredoxin (TRX). Kits for expression and purification of fusion
proteins are commercially available from New England BioLabs
(Beverly, Mass.), Pharmacia (Piscataway, N.J.), and Invitrogen
(Carlsbad, Calif.), respectively. The proteins can also be tagged
with a small epitope (e.g., His, myc or Flag tags) and subsequently
identified or purified using a specific antibody to the chosen
epitope. Antibodies to common epitopes are available from numerous
commercial sources.
[0120] As an alternative to traditional chromatography purification
modes (e.g., flow-through and bind-elute chromatography
purification modes), the polypeptides produced by the methods of
the present invention may be purified by operating a chromatography
purification column in a weak partitioning mode, a technique in
which at least one product contained in the preparation, and at
least one contaminant or impurity, both bind to a chromatographic
resin or medium. In the weak partitioning mode, the at least one
impurity binds more tightly to the medium than the polypeptide
product; and as loading (of the load fluid) continues, unbound
polypeptide product selectively passes through the medium and is
recovered from the column effluent. Such purification results in a
high degree of impurity reduction, as well as high product
recovery. Such purification can be achieved on media and resins
known in the art, including but not limited to, charged ion
exchange medium, hydrophobic interaction chromatography resin,
immobilized metal affinity chromatography resin, and hydroxyapatite
resin. In at least one embodiment, impurity/contaminant removal
under weak partitioning conditions occurs as the load fluid passes
through a medium/resin that binds at least 2.8 mg of product per ml
of medium/resin. In at least one other embodiment,
impurity/contaminant removal under weak partitioning conditions
occurs as the load fluid passes through a medium/resin at operating
conditions defined by a partition coefficient of at least 0.1. The
purified product is recovered from the effluent of the column
containing the medium/resin.
[0121] Table 5 summarizes the differences in the characteristics
between flow-through (FT), bind-elute (B-E), and the weak
partitioning (WP) modes.
TABLE-US-00006 TABLE 5 Characteristics of FT / WP / B-E Modes FT WP
B-E Kp <0.1 0.1-20 >20 Load Impurities Impurities Product +
impurities challenge 10-50 mg Prod/mL 50-500 mg Prod/mL <100 mg
Prod/mL limitation (typical) but actually (typical) but actually
dependent on load purity dependent on load purity Load Vol.
Moderate, for dilute Very high, for dilute Lower, as the product
binds impurities 10-20 CVs impurities up to 50 CVs in addition to
impurities 5-20 CVs [Product] Equal to load Initial lag, then equal
to <5% of load concentration in load concentration through load
concentration eluate much of load through much of load Residual Low
Very low Dependent on elution [Impurity] conditions, pool volume
and capacity Product <1 mg/mL <10-20 mg/mL >10-20 mg/mL
bound (Q) Operating Relatively broad Modest window Stringent
binding conditions region range of conditions of operation between
for load, broad range of FT and B-E modes elution conditions Mobile
Isocratic Isocratic Change in buffer phase(s) composition after
load which causes elution
[0122] The partition coefficient (Kp) is the ratio of the
concentration of the adsorbed product (Q) to the concentration of
the product in solution (C); thus the Kp for the weak partitioning
mode is intermediate between the Kp for the flow-through and
bind-elute modes, e.g., between about 0.1 and 20. In order to
determine the proper conditions, e.g., salt, buffer, pH, etc., for
a weak partitioning mode of purification, a high-throughput screen
or a batch purification screening study can be performed. Thus, one
skilled in the art can determine product partition coefficients
K.sub.p as a function of operating conditions (see Example
7.1).
[0123] Purification methods using a weak partitioning mode are
described in detail in U.S. patent application Ser. Nos. 11/372,054
and 11/510,634, both of which are incorporated by reference herein
in their entireties.
[0124] Some or all of the foregoing purification steps in various
combinations or with other known methods, can be employed to purify
a polypeptide of interest produced by the large-scale animal cell
culture methods and media described herein.
Pharmaceutical Compositions Containing Polypeptides Produced by
Cell Culture
[0125] The foregoing cell culture media, e.g., large-scale cell
culture media, and methods of culturing cells provides polypeptides
of interest, e.g., antibodies, soluble receptors, fusion proteins,
etc. The polypeptides produced by the disclosed cell culturing
methods, and with the novel media and related methods disclosed
herein, including antibodies and fragments thereof, may be used in
vitro, ex vivo, or incorporated into pharmaceutical compositions
and administered to individuals (e.g., human subjects) in need
thereof. Several pharmacogenomic approaches to consider in
determining whether to administer a polypeptide of the invention
are well known to one of skill in the art and include genome-wide
association, candidate gene approach, and gene expression
profiling. A pharmaceutical composition of the invention is
formulated to be compatible with its intended route of
administration (e.g., oral compositions generally include an inert
diluent or an edible carrier). Other nonlimiting examples of routes
of administration include parenteral (e.g., intravenous),
intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(topical), transmucosal, and rectal administration. The
pharmaceutical compositions compatible with each intended route are
well known in the art.
[0126] A polypeptide of the invention may be used as a
pharmaceutical composition when combined with a pharmaceutically
acceptable carrier. Such a composition may contain, in addition to
a polypeptide of the invention, carriers, various diluents,
fillers, salts, buffers, stabilizers, solubilizers, and other
materials well known in the art. The term "pharmaceutically
acceptable" means a nontoxic material that does not interfere with
the effectiveness of the biological activity of the active
ingredient(s). The characteristics of the carrier will depend on
the route of administration.
[0127] The pharmaceutical composition of the invention may also
contain additional therapeutic factors or agents for treatment of
the particular targeted disorder. For example, a pharmaceutical
composition for treatment of type 2 diabetes may also include an
oral antidiabetic agent. The pharmaceutical composition may contain
thrombolytic or antithrombotic factors such as plasminogen
activator and Factor VIII. The pharmaceutical composition may
further contain anti-inflammatory agents. Such additional factors
and/or agents may be included in the pharmaceutical composition to
produce a synergistic effect with a polypeptide of the invention,
or to minimize side effects caused by the polypeptides of the
invention.
[0128] The pharmaceutical composition of the invention may be in
the form of a liposome in which a polypeptide of the invention is
combined, in addition to other pharmaceutically acceptable
carriers, with amphipathic agents such as lipids that exist in
aggregated form as micelles, insoluble monolayers, liquid crystals,
or lamellar layers in aqueous solution. Suitable lipids for
liposomal formulation include, without limitation, monoglycerides,
diglycerides, sulfatides, lysolecithin, phospholipids, saponin,
bile acids, etc.
[0129] As used herein, the term "therapeutically effective amount"
means the total amount of each active component of the
pharmaceutical composition or method that is sufficient to show a
meaningful patient benefit, e.g., amelioration of symptoms of,
healing of, or increase in rate of healing of such conditions. When
applied to an individual active ingredient, administered alone, the
term refers to that ingredient alone. When applied to a
combination, the term refers to combined amounts of the active
ingredients that result in the therapeutic effect, whether
administered in combination, serially or simultaneously.
[0130] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of a polypeptide of
the invention is administered to a subject, e.g., a mammal (e.g., a
human). A polypeptide of the invention may be administered in
accordance with the method of the invention either alone or in
combination with other therapies. When coadministered with one or
more agents, a polypeptide of the invention may be administered
either simultaneously with the second agent, or sequentially. If
administered sequentially, the attending physician will decide on
the appropriate sequence of administering the polypeptides of the
invention in combination with other agents.
[0131] When a therapeutically effective amount of a polypeptide of
the invention is administered orally, the binding agent will be in
the form of a tablet, capsule, powder, solution or elixir. When
administered in tablet form, the pharmaceutical composition of the
invention may additionally contain a solid carrier such as a
gelatin or an adjuvant. The tablet, capsule, and powder contain
from about 5 to 95% binding agent, and preferably from about 25 to
90% binding agent. When administered in liquid form, a liquid
carrier such as water, petroleum, oils of animal or plant origin,
such as peanut oil (exercising caution in relation to peanut
allergies), mineral oil, soybean oil, or sesame oil, or synthetic
oils may be added. The liquid form of the pharmaceutical
composition may further contain physiological saline solution,
dextrose or other saccharide solution, or glycols such as ethylene
glycol, propylene glycol, or polyethylene glycol. When administered
in liquid form, the pharmaceutical composition contains from about
0.5% to about 90% by weight of the binding agent, and preferably
from about 1% to about 50% by weight of the binding agent.
[0132] When a therapeutically effective amount of a polypeptide of
the invention is administered by intravenous, cutaneous or
subcutaneous injection, the polypeptide of the invention will be in
the form of a pyrogen-free, parenterally acceptable aqueous
solution. The preparation of such parenterally acceptable protein
solutions, having due regard to pH, isotonicity, stability, and the
like, is within the skill of those in the art. A preferred
pharmaceutical composition for intravenous, cutaneous, or
subcutaneous injection should contain, in addition to the
polypeptide of the invention, an isotonic vehicle such as sodium
chloride injection, Ringer's injection, dextrose injection,
dextrose and sodium chloride injection, lactated Ringer's
injection, or other vehicle as known in the art. The pharmaceutical
composition of the present invention may also contain stabilizers,
preservatives, buffers, antioxidants, or any other additive(s)
known to those of skill in the art.
[0133] The amount of a polypeptide of the invention in the
pharmaceutical composition of the present invention will depend
upon the nature and severity of the condition being treated, and on
the nature of prior treatments that the patient has undergone.
Ultimately, the attending physician will decide the amount of a
pharmaceutical composition or polypeptide of the invention with
which to treat each individual patient. Initially, the attending
physician will administer low doses of a pharmaceutical composition
or polypeptide of the invention and observe the patient's response.
Larger doses of a pharmaceutical composition or polypeptide of the
invention may be administered until the optimal therapeutic effect
is obtained for the patient, and at that point the dosage is not
generally increased further. It is contemplated that the various
pharmaceutical compositions used to treat a subject in need thereof
should contain about 0.1 .mu.g to about 100 mg of a polypeptide of
the invention per kg body weight.
[0134] The duration of intravenous (i.v.) therapy using a
pharmaceutical composition of the present invention will vary,
depending on the severity of the disease being treated and the
condition and potential idiosyncratic response of each individual
patient. It is contemplated that the duration of each application
of a pharmaceutical composition or a polypeptide of the present
invention may be within the range of, e.g., 1-12, 6-18, or 12-24
hrs of continuous or intermittent i.v. administration. Also
contemplated is subcutaneous (s.c.) therapy using a pharmaceutical
composition of the present invention. These therapies can be
administered daily, weekly, or, more preferably, biweekly, or
monthly. Ultimately the attending physician will decide on the
appropriate duration of i.v. or s.c. therapy, or therapy with a
small molecule, and the timing of administration of the therapy,
using the pharmaceutical composition of the present invention.
[0135] All of the references, patents, patent applications, and
publications cited in this application are hereby incorporated by
reference herein in their entireties.
EXAMPLES
[0136] The Examples which follow are set forth to aid in the
understanding of the invention but are not intended to, and should
not be construed to, limit the scope of the invention in any way.
The Examples do not include detailed descriptions of conventional
methods, such as recombinant DNA techniques. Such methods are well
known to those of ordinary skill in the art.
Example 1
Quantifying the Amino Acid Composition of CHO Cells
[0137] To quantify the amino acid composition of
antibody-expressing CHO cells and recombinant protein-expressing
CHO cells, i.e., the molar percentage of each amino acid relative
to the amino acid total of cell mass (biomass), the following
procedure was performed. Briefly, three CHO cell lines
overexpressing antibodies or recombinant protein, more specifically
anti-IL-22 antibody (recombinant human anti-IL-22 antibody),
Myo-029 antibody (anti-GDF8 IgG1 monoclonal antibody), and
recombinant human BMP-2, were grown in a shake flask for one
(BMP-2) or three (Myo-029 and anti-IL-22) days. On the final day,
the cultures were harvested and the cells spun to a concentration
of 10.sup.6 cells/mL. A 10.sup.6 cell-containing pellet was washed
twice with 1.times.PBS and the pellet resuspended in 500 .mu.L of 5
N HCl. The cell-containing suspension was heated at 100.degree. C.
for 24 hours, at which point the suspension was vacuum centrifuged.
The resulting pellet was resuspended in 500 .mu.L PBS, and amino
acid analysis was performed using gas or liquid chromatography.
[0138] Acid hydrolysis determined that methionine and tryptophan
both degrade during acid hydrolysis; thus, the concentrations of
these amino acids in Examples 2 and 3 were based on literature
values for CHO cells. In addition, it was determined that glutamine
and asparagine were converted to their acidic forms, glutamic acid
and aspartic acid, respectively, during hydrolysis; thus, the
concentrations for these four amino acids in Examples 2 and 3 were
adjusted based on the glutamine/glutamic acid and
asparagine/aspartic acid ratios reported in literature. Otherwise,
it was determined that these CHO cell lines possess similar amino
acid compositions, and closely matched reported values for other
mammalian cells (data not shown) (see Bonarius (1996) Biotechnol.
Bioeng. 50:299-318).
Example 2
Desired Peak Cell Density of 15.times.10.sup.6 cells/ml, and Target
Anti-IL-22 Antibody Titer of 9 g/L in CHO Cells (Cell Line 1) with
32% Volume Feed Using Rationally Designed Medium
Example 2.1
Rationally Designed Medium
[0139] Using the formula disclosed herein, the baseline-adjusted
amino acid concentration, A, of amino acids required for
15.times.10.sup.6 cells/ml producing 9 g/L anti-IL-22 antibody in
CHO Cell Line 1 were determined (Table 6, column 2) (cell
maintenance was set at 50%, i.e., Y=0.5). The baseline-adjusted
amino acid concentration, A (Table 6, column 2), of a variety of
amino acids were adjusted to obtain the modified baseline-adjusted
total amino acid concentrations shown in column 3 of Table 6. The
following adjustments were made: levels of Asn, Asp, and Gln were
set according to U.S. Published Patent Application No.
US2006/0121568A1; 2) because Ala, Glu, Gly were produced by the
cell cultures, their levels were adjusted to lower values; 3) the
level of Cys was adjusted due to the fact that cystine is also used
in the feeding medium, and the medium value of cystine is set to
that in U.S. Published Patent Application No. US2006/0121568A1; 4)
levels of Arg, His, Ile, Leu, Lys, Met, Phe are 20% to 100% higher
than the baseline-adjusted amino acid concentration, A, due to the
fact that the feed powder, which contains a set amino acid
composition, was used to make the desired cell culture medium, such
that an exact match was not possible. The starting medium amino
acid concentration, B (Table 6, column 4), was calculated from the
modified baseline-adjusted total amino acid concentrations shown in
column 3 of Table 6.
TABLE-US-00007 TABLE 6 Desired Cell Culture Medium Formulation
Baseline-Adjusted Modified Baseline- Starting Amino Acid Adjusted
Amino Acid Concentration Amino Acid Concentration Amino Acid (mM)
(A) Concentration (mM) (mM) (B) ALA 13.67 2.18 0.20 ARG 6.68 12.56
1.94 ASN 7.42 27.82 14.56 ASP 11.86 6.28 1.70 CYS 4.97 0.27 0.40
GLU 9.23 2.18 0.20 GLN 13.11 2.72 4.00 GLY 12.94 4.52 3.63 HIS 3.93
5.05 2.15 ILE 6.23 10.95 2.54 LEU 13.13 17.96 6.87 LYS 13.01 15.62
7.91 MET 2.82 5.71 2.38 PHE 6.19 7.67 3.76 PRO 11.50 11.15 7.36 SER
21.35 22.34 10.20 THR 13.89 14.18 8.80 TRP 3.00 2.89 1.84 TYR 7.53
7.55 5.10 VAL 15.44 15.24 10.36 Total 197.91 194.85 95.91
Example 2.2
Cell Density and Antibody Titer in Response to Rationally Designed
Medium
[0140] Cell line 1 cells (anti-IL-22-expressing CHO cells) were
obtained from shake flasks containing day 3 cultures, and were
inoculated at 0.7.times.10.sup.6 cells/ml on day 0 into the
starting cell culture medium in a 1 L bioreactor (Applikon 2 L,
(Applikon Inc, Foster City, Calif.)). On day 3 (about 80 hours),
temperature was shifted from 37.degree. C. to 31.degree. C. and
Feed Medium (see column 4 of Table 2) was added at 3.75%, 4%, 4%,
9%, 2%, 1%, 1%, 1%, 3%, 2% and 1% by volume on days 3, 5, 6, 7, 10,
11, 12, 13, 14, 17, 20, respectively, to obtain the desired cell
culture medium, which contains the amino acid concentration shown
in Table 6, column 3. Cell cultures were maintained at pH 7.0, in a
dissolved oxygen level of 30%, and with agitation at 200 rpm.
Samples were taken daily to test for cell density (CEDEX.RTM. cell
counting instrument (Innovatis, Malvern, Pa.)), viability
(CEDEX.RTM.) and certain metabolite levels (Nova BioProfile
Analyzer, Nova Biomedical Cooperation, Waltham, Mass.). Spun-down
media were saved at -80.degree. C. for antibody titer analysis
using Protein A HPLC.
[0141] The results are shown in FIGS. 1 and 2. As can be seen from
FIG. 1, the highest cell density (about 11.times.10.sup.6 cells/mL)
was achieved on day 11 of culture, with cell density decreasing
thereafter. The highest antibody titer (about 7 g/L) was obtained
on day 21 of culture. Thus, the rationally designed medium may be
used to produce high cell density and high antibody titer.
Example 3
Desired Peak Density of 15.times.10.sup.6 cells/ml, and Target
Anti-IL-22 Antibody Titer of 10 g/L in CHO Cells (Cell Line 2) with
33% Volume Feed Using Rationally Designed Medium
Example 3.1
Rationally Designed Medium
[0142] Using the formula disclosed herein, the baseline-adjusted
amino acid concentration, A, of amino acids required for
15.times.10.sup.6 cells/ml and 10 g/L anti-IL-22 antibody
(described above) were determined (cell maintenance was set at 50%,
i.e., Y=0.5). The baseline-adjusted amino acid concentration, A
(Table 7, column 2) of a variety of amino acids were adjusted as
described in Example 2 to obtain the modified baseline-adjusted
total amino acid concentrations shown in column 3 of Table 7. In
addition, for this medium formulation both the starting cell
culture medium and the feed medium were prepared from existing
powders with fixed compositions, and thus an exact match was not
possible. The starting medium amino acid concentration, B (Table 7,
column 4), was calculated from the modified baseline-adjusted total
amino acid concentrations shown in column 3 of Table 7.
TABLE-US-00008 TABLE 7 Desired Cell Culture Medium Formulation
Baseline-Adjusted Modified Baseline- Starting Amino Acid Adjusted
Amino Amino Acid Amino Concentration Acid Concentration Acid (mM)
(A) Concentration (mM) (mM) (B) ALA 14.2 2.4 0.4 ARG 6.9 15.2 5.3
ASN 7.8 32.6 21.1 ASP 12.2 6.8 2.3 CYS 5.2 0.3 0.4 GLU 9.7 2.4 0.4
GLN 13.6 2.7 4.0 GLY 13.6 4.5 3.6 HIS 4.1 5.5 2.7 ILE 6.4 13.2 5.4
LEU 13.8 20.0 9.4 LYS 13.7 16.5 8.9 MET 2.9 6.3 3.1 PHE 6.5 8.3 4.5
PRO 12.3 12.5 9.1 SER 22.6 23.8 11.8 THR 14.8 15.7 10.8 TRP 3.2 3.2
2.3 TYR 8.0 7.6 5.1 VAL 16.4 15.4 10.3 Total 207.93 214.8 121.1
Example 3.2
Cell Density and Antibody Titer in Response to Rationally Designed
Medium
[0143] Cell line 2 cells (anti-IL-22-expressing CHO cells) were
obtained from shake flasks containing day 3 cultures, and the cells
were inoculated at 0.7.times.10.sup.6 cells/ml on day 0 into the
starting cell culture medium in a 1 L bioreactor (Applikon 2 L). On
day 3 (about 80 hours), temperature was shifted from 37.degree. C.
to 31.degree. C. and feed was added continuously (1.8% by volume
per day) with an automatic feeding pump, to obtain the desired cell
culture medium, which contains the amino acid concentration shown
in Table 7, column 3. Cell cultures were maintained at pH 7.0, in a
dissolved oxygen level of 30%, and with agitation at 200 rpm.
Samples were taken daily to test for cell density (CEDEX.RTM. cell
counting instrument), viability (CEDEX.RTM.) and certain
metabolites levels (Nova Enzymatic analyzer). Spun-down media were
saved at -80.degree. C. for antibody titer analysis using Protein A
HPLC.
[0144] The results are shown in FIGS. 3 and 4. As can be seen from
FIG. 3, the highest cell density (over 12.times.10.sup.6 cells/mL)
was achieved on day 10 of culture, with cell density decreasing
thereafter. The highest antibody titer (over 10 g/L) was obtained
on day 19 of culture. Thus, the rationally designed medium may be
used to produce high cell density and high antibody titer.
Example 4
The Effect of Proline Addition on Cell Culture Performance
[0145] CHO cells producing the antibody Anti-IL-22 were cultured in
pH-controlled shake flasks (500 mL flasks with 100 mL working
volume), and maintained on a shaker at .about.100 rpm in a
temperature-controlled, 7% CO.sub.2 incubator. Cells were seeded at
0.7.times.10.sup.6 cells/mL. The culture duration was 18 days. The
first 3 days cells were maintained at 37.degree. C., after which
the temperature was shifted to 31.degree. C. for the remainder of
the culture. pH was controlled for the first 3 days with 1 M sodium
bicarbonate. Cell culture medium consisted of a medium based on
traditional cell culture requirements, termed "Traditional Medium,"
and rationally designed medium, i.e., medium formulated using the
rational design disclosed herein, termed "Rational Design Medium,"
to achieve 10.times.10.sup.6 cells/mL and 10 g/L antibody. A major
difference of note between these two formulations is that several
amino acids (PRO, THR, GLY, TYR, TRP, VAL, and PHE) are at higher
concentrations in the "Rational Design Medium" compared to the
"Traditional Medium." A third condition presented is the
traditional medium with an additional 3.7 mM proline added to
obtain the level of proline in the "Rational Design Medium", termed
"Traditional Medium+Proline." These formulations are presented in
Table 8, below. Wyeth in-house feed medium ("Feed Medium," see
Table 2) was added to the culture at 23% total by volume,
comprising daily feeds of 2% on days 3-4, 9-14, 17, 1% on days 5-6,
and 3% on day 7.
TABLE-US-00009 TABLE 8 Media Formulations for Proline Studies 1 2 3
4 Rational Traditional Traditional Design Medium Medium Medium +
Proline Amino Acid [mM] [mM] [mM] alanine 0.44 0.44 0.44
arginine.cndot.HCl 5.32 5.32 5.32 asparagine.cndot.H.sub.2O 21.08
21.08 21.08 aspartic acid 2.25 2.25 2.25 glutamine 4.00 4.00 4.00
glutamate 0.24 0.24 0.24 glycine 1.78 3.59 1.78 histidine 2.68 2.68
2.68 isoleucine 5.44 5.44 5.44 leucine 9.43 9.43 9.43
lysine.cndot.HCl 8.90 8.90 8.90 methionine 3.08 3.08 3.08
phenylalanine 3.67 4.48 3.67 proline 5.41 9.13 9.13 serine 11.82
11.82 11.82 threonine 5.71 10.85 5.71 tryptophan 1.54 2.32 1.54
tyrosine.cndot.2Na 3.34 5.13 3.34 valine 7.36 10.30 7.36
[0146] The results from these experiments are shown in FIGS. 5-7.
Addition of proline to the traditional medium, i.e. "Traditional
Medium+Proline" (FIG. 5), resulted in an antibody production
equivalent to the "Rational Design Medium" through day 14. As shown
in FIG. 6, all three media maintained a high cell density, with the
highest density displayed at day 12. As shown in FIG. 7, all three
media maintained high cell viability, with the "Rational Design
Medium" cultures maintaining greater viability on days 15-18 in
comparison to the cultures containing "Traditional Medium" and
"Traditional Medium+Proline" media. As a variety of other media,
which each contained one rationally designed concentration of
either glycine, phenylalanine, threonine, tryptophan, tyrosine or
valine did not result in cell cultures producing a higher antibody
titer than the "Traditional Medium" (data not shown), proline
serves as the rate-limiting amino acid required to achieve high
titer. This is exemplified in FIGS. 8A-E, which show that by day 14
many of the amino acids in the "Traditional Medium+Praline" reached
extremely low levels (note tyrosine was depleted), thereby
preventing further incorporation of these amino acids into
antibody. This result is also observed in the titer graph (FIG. 5),
as the slope for the "Traditional Medium+Proline" is reduced after
day 14, while antibody production is maintained through day 18 for
the "Rational Design Medium," which contains higher levels of other
amino acids.
[0147] Interestingly, the proline concentration in the "Traditional
Medium" never dropped below 1 mM (FIG. 8A); however, the effect of
proline on overall amino acid incorporation into antibody was
reduced after day 11. This finding suggests that a proline
threshold exists, i.e., the concentration of proline must be
maintained above 1 mM throughout the culture.
Example 5
Prophetic Example
Optimized Cell Culture Media for a Novel Cell Line
[0148] The methods of media design disclosed herein may be used for
any cell culture, including cell cultures that use novel cells/cell
lines. The optimized media for use with a novel cell line would
contain at least one baseline-adjusted amino acid concentration, A,
of an amino acid according to the formula
A=[(M*X)+(N*P)+(Y*M*X)]*F.
[0149] A multiplier, M, when applying the above equation to a novel
cell culture, may be selected, e.g., from 1 to 20.times.10.sup.6
cells/mL. One of skill in the art would be able to calculate a
useful M value by doubling the maximum cell density during the
growth phase, which can be calculated based on the growth rate.
[0150] A multiplier, N, when applying the above equation to a novel
cell culture, may be calculated, e.g., by multiplying the IVC by
the cell qp. One of skill in the art would be able to calculate the
IVC by estimating the growth profile, following the methods
described in the section entitled "Rational Media Design and
Formulations." One of skill in the art would be able to calculate
the qp by measuring the antibody or recombinant protein production
on a per cell basis.
[0151] A cell maintenance factor, Y, when applying the above
equation to a novel cell culture, may be estimated by using Y=1
(i.e., 100% of the amino acid(s) required for desired cell mass)
initially, and then refining the value of Y (higher or lower).
[0152] A baseline factor, F, when applying the above equation to a
novel cell culture, may be estimated by using F=1.3 (i.e., 30%
additional amino acid(s)) initially, and then refining the value of
F (higher or lower).
Example 6
Effect of Maintenance and Baseline Factors on Cell Culture
Performance
[0153] To demonstrate that the maintenance factor, Y, and the
baseline factor, F, are important/essential for cell culture
performance, anti-IL-22-expressing CHO cells were seeded at
0.7.times.10.sup.6 cells/mL and cultured for 21 days in 2 L
bioreactors with a pH set point of 7.0 and a dissolved oxygen (DO)
setpoint of 30%. The pH was controlled by 2N
Na.sub.2CO.sub.3/NaHCO.sub.3, and DO was controlled by air
(containing 7% CO.sub.2) sparge. The temperature of the culture was
37.degree. C. for the first 3 days and shifted to 31.degree. C.
after day 3, and remained at 31.degree. C. until the end of the
culture. Cells were cultured for 21 days in either (1) media
containing amino acids required for both target protein titer and
desired peak cell density (but not accounting for the maintenance
factor or baseline factor), i.e., "Rational Design Medium without
maintenance and baseline factors," or (2) "Rational Design
Medium."
[0154] As exemplified in FIGS. 9 and 10, the medium that does not
account for maintenance and baseline factors displayed a
significant decrease in viability, nearly approaching 0% at day 21
of the cell culture, and a significant decrease in cell density.
Moreover, FIG. 11 demonstrates that at day 21 of the cell culture,
this same medium was only able to support an antibody titer of 5
g/L.
[0155] In contrast, Rational Design Medium (including maintenance
and baseline factors) displayed higher viability, cell density and
antibody titers (FIGS. 9-11). At day 21 of the cell culture, the
Rational Design Medium was able to support an antibody titer of 10
g/L.
[0156] These findings suggest that accounting for maintenance and
baseline factors in determination of the amino acid concentration
in the desired cell culture medium improves cell performance as
measured by cell viability, cell density, and polypeptide
titer.
Example 7
Polypeptide Purification Using Anion Exchange Chromatography in a
Weak Partitioning Mode
Example 7.1
High-Throughput Screen to Establish Weak Partitioning and
Flow-Through Conditions
[0157] An initial screening study was performed first, which
determined the partition coefficient and/or the concentration of
product bound to the resin under various solution conditions, thus
defining the operating regions of weak partitioning (WP) and
flow-through (FT) modes for Mab-AAB, the polypeptide of interest,
and TMAE-HiCap (M) Medium. This screen varied the concentration of
sodium chloride and pH to determine their effects on the extent of
binding of Mab-AAB and process-related impurities (Protein A and
HCP) to the TMAE medium.
[0158] The levels of Protein A residuals in the test samples were
measured using a Protein A enzyme-linked immunosorbent assay
(ELISA). The amount of high molecular weight aggregate was measured
using an analytical size exclusion chromatography (SEC) assay. The
levels of host cell proteins (HCPs) were measured using a HCP
ELISA. All screening and column studies were conducted at room
temperature.
[0159] Fifty .mu.L of TMAE HiCap medium was added to each well of a
96-well filter plate. Each well was equilibrated in solutions made
up 50 mM glycine and a variable amount of Tris buffer (depending
upon the amount needed for neutralization to the pH specified in
Table 9) and sodium chloride (specified in Table 10). The pH ranged
from 7.6 to 9.0, and the sodium chloride ranged from 0 mM to 80
mM.
[0160] The buffer solutions used in each row were diluted on an
automated pipetting system (Tecan 100 RST). The stock solution for
the buffers was made from 500 mM glycine acidified with HCl to pH
3.0, and subsequently neutralized with 2 M Tris Base to the pH
levels indicated in Table 9. This titration resulted in a level of
Tris that depended upon the pH of the buffer. The buffer pH was
measured at a 1 to 10 dilution of the stock buffer concentration,
which corresponded to the dilution made by the automated pipetting
system. As a result of the glycine acidification to pH 3.0, the
buffer contributes about 10 mM of ionic strength to the final
solution. Two load (load fluid) challenges were made to the resin:
5 mg/mL to measure the partition coefficient, Kp, and 122 mg/mL to
measure the capacity of the resin for removal of impurities and the
bound product, Q, in equilibrium with a protein solution at a
concentration approximately equal to the column load
concentration.
TABLE-US-00010 TABLE 9 Buffer type and pH Target in Each Well All
columns A 50 mM Glycine, 8.8 mM Tris, pH 7.6 B 50 mM Glycine, 13.6
mM Tris, pH 7.8 C 50 mM Glycine, 16.0 mM Tris, pH 8.0 D 50 mM
Glycine, 19.6 mM Tris, pH 8.2 E 50 mM Glycine, 28.4 mM Tris, pH 8.4
F 50 mM Glycine, 37.2 mM Tris, pH 8.6 G 50 mM Glycine, 64.0 mM
Tris, pH 8.8 H 50 mM Glycine, 100 mM Tris, pH 9.0
TABLE-US-00011 TABLE 10 NaCl levels (in mM) and Protein Challenges
(mg/mL) in Each Well All Rows 1 2 3 4 5 6 7 8 9 10 11 12 NaCl (mM)
0 10 20 40 60 80 0 10 20 40 60 80 MAb-AAB 5 5 5 5 5 5 132 132 132
132 132 132 (mg/mL)
[0161] In the first stage of the high-throughput screen, each well
was equilibrated in the conditions of NaCl and pH as described in
Tables 9 and 10 in a phase volume ratio of 6:1 (300 uL solution:50
uL resin). The plate was shaken for 20 minutes, allowing
equilibrium to be reached. The solution was then removed by
centrifuging the filter plate. This equilibration cycle was
repeated three times.
[0162] In the second stage, the resin in each well was challenged
with a concentrated MAb-AAB solution to 5 mg/mL of resin with a
volume ratio of 6:1 (300 uL solution:50 uL resin) at the
appropriate NaCl concentration and pH. A 36 mg/mL solution of
Mab-AAB in 1 mM HEPES, 10 mM NaCl, pH 7.0 spiked with 300 ppm of
Protein A was used as stock solution. The loaded plate was shaken
for 20 minutes, allowing the resin and solution to equilibrate. The
supernatant was removed from the filter plate by centrifugation and
collected into a collection plate. The protein concentration in the
supernatant in each well was determined by absorbance at A280
nm.
[0163] In the third stage, resin was washed by adding solutions of
the specified NaCl and pH conditions listed in Table 10. The
supernatant was removed after shaking for 20 minutes. In the fourth
stage, 2 M NaCl was added to remove the remaining protein that was
bound to the resin. The partition coefficients were calculated for
each well using the mass eluted from stages 3 and 4 and the product
concentration from stage 2, and are shown in Table 11.
TABLE-US-00012 TABLE 11 Partition Coefficients (Kp) for the 96-well
HTS Screen for MAb-AAB 1 2 3 4 5 6 7 8 9 10 11 12 A 0.22 0.32 0.35
0.17 0.24 0.23 0.21 0.24 0.21 0.19 0.17 0.16 B 0.37 0.36 0.38 0.25
0.24 0.08 0.28 0.26 0.22 0.24 0.18 0.16 C 0.63 0.48 0.47 0.27 0.15
0.20 0.31 0.28 0.26 0.20 0.23 0.16 D 1.24 1.12 0.68 0.36 0.30 0.17
0.42 0.39 0.34 0.23 0.23 0.18 E 3.24 1.89 1.05 0.59 0.35 0.15 0.68
0.58 0.41 0.29 0.21 0.18 F 8.37 3.37 1.56 0.61 0.31 0.32 0.87 0.74
0.51 0.32 0.25 0.21 G 18.36 9.49 3.16 0.82 0.49 0.34 0.91 0.88 0.69
0.39 0.24 0.20 H 125.73 23.79 6.58 1.23 0.58 0.43 1.18 1.02 0.78
0.42 0.27 0.24
[0164] As shown in Table 11, the Kp value can be used to describe
regions where MAb-AAB binds to the TMAE medium with different
strengths. The strength of MAb-AAB binding to the TMAE medium can
be manipulated by varying conditions of pH and chloride
concentration into flow-through (K.ltoreq.0.1), weak partitioning
(0.1<K<20), and binding zones (K.gtoreq.20).
[0165] The supernatant from the load stage of all wells from each
zone were sampled and submitted for Protein A analysis. The assay
results of these samples are summarized in Table 12. There is a
region of pH and conductivity in which the TMAE chromatography step
provides very significant removal of Protein A with limited protein
loss to the resin. This region was found to be closely correlated
to the partition coefficient value, Kp, and not any specific pH or
chloride concentration.
TABLE-US-00013 TABLE 12 Protein A Log Removal Values (LRV) for
MAb-AAB binding data from HTS Screen 1 2 3 4 5 6 7 8 9 10 11 12 A
2.11 1.89 2.12 1.85 1.22 1.00 1.63 1.02 1.00 0.92 0.85 1.02 B 2.79
2.37 2.42 1.96 1.23 1.13 1.77 1.81 1.22 0.85 0.94 1.52 C >3.05
>3.03 2.74 2.16 1.37 1.11 2.25 2.15 1.96 1.16 1.06 0.95 D
>3.41 >2.98 >3.06 2.50 1.94 1.18 3.39 3.11 2.57 1.41 1.02
0.89 E >2.87 >2.93 >3.01 >2.95 2.13 1.75 >3.09 3.27
3.09 1.66 1.89 0.99 F >2.64 >2.89 >2.99 >3.11 2.29 1.82
>3.07 >3.11 >3.15 2.19 1.24 0.84 G >2.33 >2.58
>2.89 >3.07 2.41 2.14 >3.09 >3.11 >3.14 2.80 1.46
0.85 H >1.63 >2.36 >2.76 >3.01 2.86 2.37 >2.98
>3.05 >3.15 3.16 3.45 0.85
Example 7.2
Column Runs Under Flow-Through Conditions
[0166] The following experiment was performed in the flow-through
(FT) mode, where the MAb-AAB interacts only very weakly with the
column. Two runs were performed with load challenges of 110 mg/ml
and 200 mg/ml of resin.
[0167] For all TMAE (HiCapM) anion exchange chromatography runs
described, the following conditions were used (exceptions are noted
in the individual experimental descriptions). [0168] Operational
flow rate--150-300 cm/hr [0169] Equilibration 1--50 mM Tris, 2.0 M
NaCl, pH 7.5 (5 column volumes) [0170] Equilibration 2--as
specified, approximately equivalent to the load pH and chloride
content [0171] Post-load wash--as specified, approximately
equivalent to the load pH and chloride content [0172] Strip
buffer--50 mM Tris, 2.0 M NaCl, pH 7.5 (5 column volumes)
Mabselect Protein A Chromatography
[0173] The culture containing the monoclonal antibody was purified
at Pilot scale using a MabSelect column (2,389 mL) connected to a
Millipore K-prime 400 chromatography system. A Mabselect Protein A
column was equilibrated with 5 column volumes of 50 mM Tris/150 mM
NaCl, pH 7.5 at a flow rate of 300 cm/hr. The column was then
loaded at a load of approximately 40 mg product/ml resin. This was
followed by a 5 column volume (CV) wash in 1 M NaCl, 50 mM Tris, pH
7.5, and a 5CV wash containing 10 mM Tris, 75 mM NaCl, pH 7.5 wash.
The column was then eluted using 50 mM glycine, 75 mM NaCl, pH 3.0.
The product pool was neutralized to pH 7.6 using 2 M Tris, pH 8.5.
The neutralized peak had a chloride concentration of approximately
90 mM.
TMAE HiCap (M) Chromatography
[0174] The neutralized Protein A pool was further purified over the
TMAE step with the equilibration, load, and wash solutions at pH
7.5 with 50 mM Tris and 75 mM sodium chloride. Five column volumes
of wash were used. The column dimensions and load challenges for
these two studies were: Run 1: 7.0 cm diameter.times.20.6 cm bed
height (volume--793 mL) with a load concentration of 11.9 mg/mL;
and Run 2: 7.0 cm diameter.times.13 cm bed height (volume--500 mL)
with a load concentration of 17.6 mg/mL.
[0175] These load conditions were in the flow-through (FT) region
(Table 13). Batch binding studies were used to measure the
partition coefficient (Kp), and the bound product was determined by
protein in the column strip by using UV absorbance. This method of
determining the bound product typically underestimates the amount
of product bound during the load due to isocratic elution of the
product during the wash. The levels of Protein A, HCP and high
molecular weight aggregates (HMW) in the load and product pool were
measured, and the extent of removal calculated. The results are
presented in Table 13. There is poor removal of Protein A and HMW,
and modest reduction in HCP levels.
TABLE-US-00014 TABLE 13 Removal of HCP, Protein A, and HMW under FT
Conditions Par- tition Bound Re- Load Coef- Product Protein cov-
Challenge ficient (mg/mL HCP A HMW ery Run (mg/mL) (Kp) resin)
(LRV) (LRV) (Fold) (%) 1 110 0.17 1.4 2.3 0.1 -- 96 2 200 0.17 3.3
2.0 <0.1 1.5 96 *Impurity levels were 38.5 ppm ProA and 51,943
ppm HCP (Run 1), 8.8 ppm ProA and 25,398 ppm HCP (Run 2).
Example 7.3
Column Runs Under Weak Partitioning Conditions (High Product
Challenge)
TMAE (HiCap M) Anion Exchange Chromatography
[0176] Several Mabselect Protein A runs were performed essentially
as described in Example 7.2 to generate the load material for these
runs. The partially purified antibody pool from the Protein A step
was further purified over the TMAE column. The load to the TMAE
column was in 50 mM Tris, pH 8.2. The column diameter was 0.5 cm
and the bed height was 10 cm bed height (volume--2.0 mL). The
column was challenged to a load of 500 mg/mL resin, with a load
concentration of 27.7 mg/mL.
[0177] The column was equilibrated with 5CV of a solution
containing 50 mM Tris, 2M NaCl, pH 7.5 followed by another
equilibration step comprising a 50 mM Tris, pH 8.2 solution. The
column was then loaded to 500 mg product/ml resin with the
neutralized Protein A peak from the previous step and the product
was recovered in the column effluent during the load cycle and some
column volumes of the wash fraction.
[0178] These load conditions are in the weak partitioning region.
Batch binding studies were used to measure the partition
coefficient (Kp), and product binding at high protein
concentrations. At pH 8.2, and an approximate chloride content of
12 mM, the partition coefficient, Kp, is estimated to be 1.9 (from
interpolation of the dataset from the HTS screen).
[0179] The levels of HCP and Protein A were measured in three
fractions during the loading stage representing load challenges of
approximately 250, 375, and 500 mg/ml of resin. The results from
example 7.3 are presented in Table 14. These results demonstrate
that very high product challenges can be achieved in weak
partitioning mode, without breakthrough of impurities. Excellent
reduction in both HCP and Protein A was achieved, along with a 50%
reduction in HMW content. In comparison to the results for
operation in the flow-through mode in Table 13, the removal of
impurities was much better in the weak partitioning mode.
TABLE-US-00015 TABLE 14 Removal of HCP, Protein A and HMW for a 500
mg/ml TMAE load challenge Early Middle Final fraction fraction Late
fraction product (250 mg/ml) (375 mg/ml) (500 mg/ml) pool (ppm)
Residual HCP <7.6 <7.6 <7.6 <7.6 ppm (ng/mg product)
HCP Log >3.5 >3.5 >3.5 >3.5 Removal Value (LRV)
Residual 0.3 Not 0.1 0.6 Protein A determined ppm (ng/mg product)
ProA Log 2.9 Not 2.3 2.5 Removal Value (LRV) determined HMW Not Not
Not 2 fold determined determined determined removal * The
impurities in the load were 25,398 ppm of HCP, 99.5 ppm of Protein
A, and 2.3% HMW
Example 7.4
Column Runs Under Weak Partitioning Conditions (Robustness
Studies)
[0180] To further confirm the performance of the TMAE column in the
region of weak partitioning, several runs were designed varying the
pH and NaCl concentration in the load to test process robustness.
All runs were performed at a load challenge of 250 mg/ml resin.
Several Mabselect Protein A runs were performed essentially as
described in Example 7.2 to generate the load material for these
runs. The only factor varied in those runs was the sodium chloride
concentration in the Protein A elution, which was varied to match
the NaCl concentration in the TMAE load for a particular
experiment. The columns were equilibrated with Equil 2 buffers and
washed with Wash buffers which had approximately the same pH and
sodium chloride content of the load.
[0181] These load conditions are in the weak partitioning region.
Batch binding studies were used to measure the partition
coefficient (Kp). The runs are ranked by the partition coefficients
listed in Table 15. The bound product was determined by measuring
the protein in the column strip using UV absorbance, and ranges
from 7.8-25.3 mg/mL. Protein A, HCP and HMW results from these
experiments are also presented in Table 15. The removal of all
impurities was found to be robust in operating ranges which cover
13.5-38.8 mM total chloride and pH 7.8-8.4.
TABLE-US-00016 TABLE 15 Process Robustness Studies on Removal of
HCP, Protein A, and HMW in WP Mode HCP Protein NaCl Bound in A in
Concentration Product Load load HCP Protein A HMW Recovery (mM) Kp
(mg/mL) pH (ppm) (ppm) (LRV) (LRV) (Fold) (%) 38.8 0.26 9.4 7.8
26,391 493.5 3.7 1.8 2.0 92 13.5 0.41 7.9 7.8 12,821 69.2 3.3
>1.9 1.8 87 27.4 0.50 8 8.0 23,465 252 3.6 2.2 3.2 91 18.5 0.73
7.8 8.0 21,626 308 3.7 >3.2 2.9 90 23.5 0.80 9.5 8.1 18,004 343
3.2 >3.2 3.5 94 27.7 0.86 9.5 8.2 24,821 280 3.6 >3.2 2.6 99
18.5 1.48 10 8.2 17,669 252 3.7 >3.1 3.9 95 22.0 5.35 25.3 8.4
29,293 533 3.6 >2.9 2.3 90 * Impurity levels were 38.5 ppm ProA
and 51,943 ppm HCP (Run 1), 8.8 ppm ProA and 25,398 ppm HCP (Run
2). +includes the Cl-- ion contribution from NaCl, buffers and
titrants
Example 7.5
Summary
[0182] From these studies, it can be seen that Protein A removal
(LRV) varies strongly with Kp, while HCP LRV is excellent at all
the values of Kp at or above 0.26, but much reduced at Kp=0.17
(under flow-through conditions). Host cell protein removal is over
one log lower for flow-through conditions compared to weak
partitioning conditions, even for a reduced load challenge. The
bound product ranges from 7.8-25 mg/ml for these weak partitioning
conditions on this combination of resin and monoclonal antibody.
The partition coefficient appears to be optimal between
0.41<Kp<5.4. It does not appear to be optimal at Kp=0.17 and
a bound product of 1.4-3.3 mg/mL, the conditions of Example
7.2.
[0183] These studies suggest an alternative mode of purifying a
polypeptide produced using rational design media cell culture,
which will significantly reduce the presence of impurities, high
molecular weight aggregates, DNA, host cell proteins, etc.
* * * * *