NZ712315B2 - Cell culture compositions with antioxidants and methods for polypeptide production - Google Patents
Cell culture compositions with antioxidants and methods for polypeptide production Download PDFInfo
- Publication number
- NZ712315B2 NZ712315B2 NZ712315A NZ71231514A NZ712315B2 NZ 712315 B2 NZ712315 B2 NZ 712315B2 NZ 712315 A NZ712315 A NZ 712315A NZ 71231514 A NZ71231514 A NZ 71231514A NZ 712315 B2 NZ712315 B2 NZ 712315B2
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- cell culture
- culture medium
- cell
- antibody
- polypeptide
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
- C12N2500/33—Amino acids other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/46—Amines, e.g. putrescine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
Abstract
Cell culture media comprising antioxidants are provided herein as are methods of using the media for cell culturing and polypeptide production from cells. Compositions comprising polypeptides, such as therapeutic polypeptides, produced by the methods herein are also provided. The invention reduces discolouration of protein compositions when produced by cells in cell culture media. Protein compositions had less intense colour when made by cells in contact with certain antioxidants. Examples used aminoguanidine, s-carboxymethylcysteine, carnosine, hypotaurine, or taurine. iscolouration of protein compositions when produced by cells in cell culture media. Protein compositions had less intense colour when made by cells in contact with certain antioxidants. Examples used aminoguanidine, s-carboxymethylcysteine, carnosine, hypotaurine, or taurine.
Description
CELL CULTURE COMPOSITIONS WITH ANTIOXIDANTS AND METHODS FOR
POLYPEPTIDE PRODUCTION
CROSS REFERENCES TO RELATED APPLICATIONS
This application claims the priority benefit of U.S. provisional application Serial
No. 61/799,602, filed March 15, 2013, the contents of which are incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
Generally described are cell culture media comprising antioxidants, methods of
using the media for cell culture and polypeptide production as well as compositions and kits
comprising the polypeptides produced by the methods described herein. The present
invention generally relates to methods of producing an antibody or fragment thereof using
cell culture methods defined herein.
BACKGROUND OF THE INVENTION
Cell culture manufacturing technology is widely used for the production of protein-
based products such as pharmaceutical formulations of therapeutic proteins. Commercial
production of protein-based products, such as an antibody product, requires optimization of
cell culture parameters in order for the cell to produce enough of the protein product to meet
manufacturing demands. However, when cell culture parameters are optimized for
improving productivity of the protein product it is also necessary to maintain the desired
quality attributes of the product such as the glycosylation profile, aggregate levels, charge
heterogeneity, and amino acid sequence integrity (Li et al., mAbs, 2010, 2(5):466-477).
Another quality attribute of concern is the color of the protein product. Regulatory
requirements regarding acceptable color levels for liquid formulations of therapeutic products
for human use must be met (United States Pharmacopoeia Inc., 2000, p. 1926-1927 and
Council of Europe. European Pharmacopoeia, 2008, 7 Ed. p. 22). Thus, producing a protein
product that has an acceptable color is an important aspect of therapeutic protein production.
Recent trends towards the subcutaneous delivery of therapeutic proteins, such as
monoclonal antibodies, has been accompanied by an increase in concentration of the
formulated protein substance, for example at concentrations about 100 mg/mL or greater
(Daugherty et al., Adv Drug Deliver Rev, 2006, 58(5-6):686-706). A correlation between
increased color intensity in compositions comprising increasing amounts of therapeutic
protein has been observed and this relationship may be due low-level protein product variants
previously unobservable by standard methods for monitoring color intensity of the
formulated product.
Oxidation is a major chemical degradation pathway for protein pharmaceuticals.
For example, methionine, cysteine, histidine, tryptophan, and tyrosine are amino acid
residues that are susceptible to oxidation due to their reactivity with reactive oxygen species
(ROS) and this oxidation is often observed in pharmaceutical protein formulations during
storage. Although it is known that cell culture conditions can impact quality attributes of the
protein product, such as production of sufficient amounts for large-scale manufacturing, the
impact of these conditions on the color intensity of the final protein product remains unclear.
There is a continuing need to provide improved and cost-effective methods of
producing proteins (e.g., antibodies) having acceptable product quality attributes such as
color intensity. Cell culture media, whether chemically undefined or chemically defined,
having components that consistently deliver protein products at lower color intensities while
maintaining a desired protein concentration (e.g., ≥ 100 mg/mL) would find use in the
development of protein products, such as antibodies. It is an object of the invention to go at
least some way toward addressing these needs; and/or to at least provide the public with a
useful choice.
BRIEF SUMMARY OF THE INVENTION
[0006A] In a first aspect, the invention relates to a method of producing an antibody or
fragment thereof comprising the step of culturing a Chinese Hamster Ovary (CHO) cell
comprising a nucleic acid encoding the antibody or fragment thereof in a cell culture medium
comprising a media component selected from the group consisting of hypotaurine at a
concentration of about 1 mM to about 50 mM, s-carboxymethylcysteine at a concentration of
about 0.5 mM to about 120 mM, and taurine at a concentration of about 2 mM to about 50
mM, and wherein the cell culture medium comprising the media component reduces the color
intensity of a composition comprising the antibody or fragment thereof produced by the cell
as compared to the color intensity of a composition comprising the polypeptide produced by
the cell cultured in a cell culture medium that does not comprise the media component.
[0006B] In a second aspect, the invention relates to a method of producing an antibody or
fragment thereof comprising the step of culturing a Chinese Hamster Ovary (CHO) cell
comprising a nucleic acid encoding the antibody or fragment thereof in a cell culture
medium, wherein the cell culture medium comprises one or more of components (a)-(e):
(a) hypotaurine at a concentration of about 1 mM to about 50 mM;
(b) s-carboxymethylcysteine at a concentration of about 0.5 mM to about 120 mM;
(c) butylated hydroxyanisole at a concentration of about 0.001 mM to about 0.2 mM;
(d) lipoic acid at a concentration of about 0.01 mM to about 1.5 mM; and
(e) quercitrin hydrate at a concentration of about 0.005 mM to about 0.04 mM;
and wherein the cell culture medium comprising one or more of components (a)-(e) reduces
the color intensity of a composition comprising the antibody or fragment thereof produced by
the cell as compared to a composition comprising the polypeptide produced by the cell
cultured in a cell culture medium that does not comprise one or more of components (a)-(e).
BRIEF DESCRIPTION
In some embodiments, described herein is a method of culturing a cell comprising a
nucleic acid encoding a polypeptide, wherein the method comprises the step of contacting the
cell with a cell culture medium comprising hypotaurine or an analog or precursor thereof,
wherein the cell culture medium comprising the hypotaurine or an analog of precursor thereof
reduces the color intensity of a composition comprising the polypeptide produced by the cell
as compared to the color intensity of a composition comprising the polypeptide produced by
the cell cultured in a cell culture medium that does not comprise the hypotaurine or an analog
or precursor thereof. In some embodiments, the cell culture medium comprising the
hypotaurine or an analog or precursor thereof reduces the color intensity of a composition
comprising the polypeptide produced by the cell by at least about 0.1% as compared to a
composition comprising the polypeptide produced by the cell cultured in a cell culture
medium that does not comprise the hypotaurine or an analog or precursor thereof. In some
embodiments, the cell culture medium comprising the hypotaurine or an analog or precursor
thereof reduces the color intensity of a composition comprising the polypeptide produced by
the cell by about 5% to about 50% as compared to a composition comprising the polypeptide
produced by the cell cultured in a cell culture medium that does not comprise the hypotaurine
or an analog or precursor thereof. In some of the embodiments herein, the cell culture
medium comprises the hypotaurine or an analog or precursor thereof at a concentration of at
least about 0.0001 mM. In some of the embodiments herein, the cell culture medium
comprises the hypotaurine or an analog or precursor thereof at a concentration from about
0.0001 mM to about 500.0 mM. In some of the embodiments herein, the cell culture medium
comprises the hypotaurine or an analog or precursor thereof at a concentration from about 1.0
mM to about 40.0 mM. In some of the embodiments herein, the cell culture medium
comprises the hypotaurine or an analog or precursor thereof at a concentration from about 1.0
mM to about 10.0 mM. In some of the embodiments herein, the hypotaurine or an analog or
precursor thereof is selected from the group consisting of hypotaurine, s-
carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine. In some of the
embodiments herein, the cell culture medium comprising the hypotaurine or an analog or
precursor thereof is a chemically defined cell culture medium. In some of the embodiments
herein, the cell culture medium comprising the hypotaurine or an analog or precursor thereof
is a chemically undefined cell culture medium. In some of the embodiments herein, the cell
culture medium comprising the hypotaurine or an analog or precursor thereof is a basal cell
culture medium. In some of the embodiments herein, the cell culture medium comprising the
hypotaurine or an analog or precursor thereof is a feed cell culture medium. In some of the
embodiments herein, the cell is contacted with the cell culture medium comprising the
hypotaurine or an analog or precursor thereof during the cell’s growth phase. In some of the
embodiments herein, the cell is contacted with the cell culture medium comprising the
hypotaurine or an analog or precursor thereof during the cell’s production phase. In some of
the embodiments herein, the hypotaurine or an analog or precursor thereof is added to the cell
culture medium on at least one day of a cell culture cycle. In some of the embodiments
herein, the hypotaurine or an analog or precursor thereof is added to the cell culture medium
on day 0 of a 14 day cell culture cycle. In any of the embodiments herein, the hypotaurine or
an analog or precursor thereof can be added to the cell culture medium on any day of a cell
culture cycle. In some of the embodiments herein, the cell is a mammalian cell. In some of
the embodiments herein, the mammalian cell is a Chinese Hamster Ovary (CHO) cell. In
some of the embodiments herein, the polypeptide is an antibody or fragment thereof.
In other embodiments, described herein are methods of culturing a cell comprising a
nucleic acid encoding a polypeptide, wherein the method comprises the step of contacting the
cell with a cell culture medium, wherein the cell culture medium comprises one or more of
components (a)-(h): (a) hypotaurine; (b) s-carboxymethylcysteine; (c) carnosine; (d)
anserine; (e) butylated hydroxyanisole; (f) lipoic acid; (g) quercitrin hydrate; and (h)
aminoguanidine; and wherein the cell culture medium comprising one or more of components
(a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by
the cell as compared to a composition comprising the polypeptide produced by the cell
cultured in a cell culture medium that does not comprise the one or more of components (a)-
(h). In some embodiments, the cell culture medium comprising one or more of components
(a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by
the cells by at least about 0.1% as compared to a composition comprising the polypeptide
produced by the cell cultured in a cell culture medium that does not comprise the one or more
of components (a)-(h). In some embodiments, the cell culture medium comprising one or
more of components (a)-(h) reduces the color intensity of a composition comprising the
polypeptide produced by the cells by about 5% to about 75% as compared to a composition
comprising the polypeptide produced by the cell cultured in a cell culture medium that does
not comprise the one or more of components (a)-(h). In some embodiments, the cell culture
medium comprising one or more of components (a)-(h) reduces the color intensity of a
composition comprising the polypeptide produced by the cells by about 5% to about 50% as
compared to a composition comprising the polypeptide produced by the cell cultured in a cell
culture medium that does not comprise the one or more of components (a)-(h). In some of
the embodiments herein, the cell culture medium comprising one or more of components (a)-
(h) comprises the one or more components (a)-(h) in an amount selected from: (a)
hypotaurine at a concentration from at least about 0.0001 mM; (b) s-carboxymethylcysteine
at a concentration from at least about 0.0001 mM; (c) carnosine at a concentration from at
least about 0.0001 mM; (d) anserine at a concentration from at least about 0.0001 mM; (e)
butylated hydroxyanisole at a concentration from at least about 0.0001 mM; (f) lipoic acid at
a concentration from at least about 0.0001 mM; (g) quercitrin hydrate at a concentration from
at least about 0.0001 mM; and (h) aminoguanidine at a concentration from at least about
0.0003 mM. In a further embodiment, the cell culture medium comprises hypotaurine at a
concentration from about 2.0 mM to about 50.0 mM. In some of the embodiments herein, the
cell culture medium comprises s-carboxymethylcysteine at a concentration from about 8.0
mM to about 12.0 mM. In some of the embodiments herein, the cell culture medium
comprises carnosine at a concentration from about 8.0 mM to about 12.0 mM. In some of the
embodiments herein, the cell culture medium comprises anserine at a concentration from
about 3.0 mM to about 5.0 mM. In some of the embodiments herein, the cell culture medium
comprises butylated hydroxyanisole at a concentration from about 0.025 mM to about 0.040
mM. In some of the embodiments herein, the cell culture medium comprises lipoic acid at a
concentration from about 0.040 mM to about 0.060 mM. In some of the embodiments herein,
the cell culture medium comprises quercitrin hydrate at a concentration from about 0.010 mM
to about 0.020 mM. In some embodiments, the cell culture medium comprises
aminoguanidine at a concentration from about 0.0003 mM to about 245 mM. In some
embodiments, the cell culture medium comprises aminoguanidine at a concentration from
about 0.0003 mM to about 10 mM. In some of the embodiments herein, the cell culture
medium is a chemically defined cell culture medium. In some of the embodiments herein, the
cell culture medium is a chemically undefined cell culture medium. In some of the
embodiments herein, the cell culture medium is a basal cell culture medium. In some of the
embodiments herein, the cell culture medium is a feed cell culture medium. In some of the
embodiments herein, the cell is contacted with the cell culture medium during the cell’s
growth phase. In some of the embodiments herein, the cell is contacted with the cell culture
medium during the cell’s production phase. In some of the embodiments herein, the one or
more of components (a)-(h) is added to the cell culture medium on at least one day of a cell
culture cycle. In some of the embodiments herein, the one or more of components (a)-(h) is
added to the cell culture medium on day 0 of a 14 day cell culture cycle. In any of the
embodiments herein, the one or more of components (a)-(h) can be added to the cell culture
medium on any day of a cell culture cycle. In some of the embodiments herein, wherein the
cell is a mammalian cell. In some of the embodiments herein, wherein the mammalian cell is
a Chinese Hamster Ovary (CHO) cell. In some of the embodiments herein, wherein the
polypeptide is an antibody or fragment thereof.
In some embodiments, described herein are also methods of producing a
polypeptide comprising the step of culturing a cell comprising a nucleic acid encoding the
polypeptide in a cell culture medium comprising hypotaurine or an analog or precursor
thereof, and wherein the cell culture medium comprising the hypotaurine or an analog or
precursor thereof reduces the color intensity of a composition comprising the polypeptide
produced by the cell as compared to the color intensity of a composition comprising the
polypeptide produced by the cell cultured in a cell culture medium that does not comprise the
hypotaurine or an analog or precursor thereof. In some embodiments, the cell culture medium
comprising the hypotaurine or an analog or precursor thereof reduces the color intensity of a
composition comprising the polypeptide produced by the cell by at least about 0.1% as
compared to a composition comprising the polypeptide produced by the cell cultured in a cell
culture medium that does not comprise the hypotaurine or an analog or precursor thereof. In
some embodiments, the cell culture medium comprising the hypotaurine or an analog or
precursor thereof reduces the color intensity of a composition comprising the polypeptide
produced by the cell by about 5% to about 50% as compared to a composition comprising the
polypeptide produced by the cell cultured in a cell culture medium that does not comprise the
hypotaurine or an analog or precursor thereof. In some of the embodiments herein, the cell
culture medium comprises the hypotaurine or an analog or precursor thereof at a
concentration from at least about 0.0001 mM. In some of the embodiments herein, the cell
culture medium comprising comprises the hypotaurine or an analog or precursor thereof at a
concentration from about 0.0001 mM to about 500.0 mM. In some of the embodiments
herein, the cell culture medium comprises the hypotaurine or an analog or precursor thereof
at a concentration from about 1.0 mM to about 40.0 mM. In some of the embodiments
herein, the cell culture medium comprises the hypotaurine or an analog or precursor thereof
at a concentration from about 1.0 mM to about 10.0 mM. In some of the embodiments herein,
the hypotaurine or an analog or precursor thereof is selected from the group consisting of
hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine. In
some of the embodiments herein, the cell culture medium is a chemically defined cell culture
medium. In some of the embodiments herein, the cell culture medium is a chemically
undefined cell culture medium. In some of the embodiments herein, the cell culture medium
is a basal cell culture medium. In some of the embodiments herein, the cell culture medium
is a feed cell culture medium. In some of the embodiments herein, the hypotaurine or an
analog or precursor thereof is added to the cell culture medium on at least one day of a cell
culture cycle. In some of the embodiments herein, the hypotaurine or an analog or precursor
thereof is added to the cell culture medium on day 0 of a 14 day cell culture cycle. In any of
the embodiments herein, the hypotaurine or an analog or precursor thereof can be added to
the cell culture medium on any day of a cell culture cycle. In some of the embodiments
herein, the cell is a mammalian cell. In some embodiments, the mammalian cell is a Chinese
Hamster Ovary (CHO) cell. In some of the embodiments herein, the polypeptide is an
antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments,
the antibody is secreted into the cell culture medium comprising the hypotaurine or an analog
or precursor thereof. In some embodiments, the method further comprises the step of
recovering the polypeptide from the cell culture medium comprising the hypotaurine or an
analog or precursor thereof. In some embodiments, the composition comprising the recovered
polypeptide is a liquid composition or a non-liquid composition. In some embodiments, the
composition comprising the recovered polypeptide appears as a colorless or slightly colored
liquid.
In some embodiments, described herein is a method of producing a polypeptide
comprising the step of culturing a cell comprising a nucleic acid encoding the polypeptide in
a cell culture medium, wherein the cell culture medium comprises one or more of
components (a)-(h): (a) hypotaurine; (b) s-carboxymethylcysteine; (c) carnosine; (d)
anserine; (e) butylated hydroxyanisole; (f) lipoic acid; (g) quercitrin hydrate; and (h)
aminoguanidine; and wherein the cell culture medium comprising one or more of components
(a)-(h) reduces the color intensity of a composition comprising the polypeptide produced by
the cell as compared to a composition comprising the polypeptide produced by the cell
cultured in a cell culture medium that does not comprise one or more of components (a)-(h).
In some embodiments, the cell culture medium comprising one or more of components (a)-
(h) reduces the color intensity of a composition comprising the polypeptide produced by the
cells by at least about 0.1% as compared to a composition comprising the polypeptide
produced by the cell cultured in a cell culture medium that does not comprise the one or more
of components (a)-(h). In some embodiments, the cell culture medium comprising one or
more of components (a)-(h) reduces the color intensity of a composition comprising the
polypeptide produced by the cells by about 5% to about 50% as compared to a composition
comprising the polypeptide produced by the cell cultured in a cell culture medium that does
not comprise the one or more of components (a)-(h). In some embodiments, the cell culture
medium comprising one or more of components (a)-(h) reduces the color intensity of a
composition comprising the polypeptide produced by the cells by about 5% to about 75% as
compared to a composition comprising the polypeptide produced by the cell cultured in a cell
culture medium that does not comprise the one or more of components (a)-(h). In some of
the embodiments herein, the cell culture medium comprises the one or more components (a)-
(h) in an amount selected from: (a) hypotaurine at a concentration from at least about 0.0001
mM; (b) s-carboxymethylcysteine at a concentration from at least about 0.0001 mM; (c)
carnosine at a concentration from at least about 0.0001 mM; (d) anserine at a concentration
from at least about 0.0001 mM; (e) butylated hydroxyanisole at a concentration from at least
about 0.0001 mM; (f) lipoic acid at a concentration from at least about 0.0001 mM; (g)
quercitrin hydrate at a concentration from at least about 0.0001 mM; and (h) aminoguanidine
at a concentration from at least about 0.0003 mM. In some embodiments, the cell culture
medium comprises hypotaurine at a concentration from about 2.0 mM to about 50.0 mM. In
some of the embodiments herein, the cell culture medium comprises s-carboxymethylcysteine
at a concentration from about 8.0 mM to about 12.0 mM. In some of the embodiments
herein, the cell culture medium comprises carnosine at a concentration from about 8.0 mM to
about 12.0 mM. In some of the embodiments herein, the cell culture medium comprises
anserine at a concentration from about 3.0 mM to about 5.0 mM. In some of the embodiments
herein, the cell culture medium comprises butylated hydroxyanisole at a concentration from
about 0.025 mM to about 0.040 mM. In some of the embodiments herein, the cell culture
medium comprises lipoic acid at a concentration from about 0.040 mM to about 0.060 mM.
In some of the embodiments herein, the cell culture medium comprises quercitrin hydrate at a
concentration from about 0.010 mM to about 0.020 mM. In some embodiments, the cell
culture medium comprises aminoguanidine at a concentration from about 0.0003 mM to
about 245 mM. In some embodiments, the cell culture medium comprises aminoguanidine at
a concentration from about 0.0003 mM to about 10 mM. In some of the embodiments herein,
the cell culture medium is a chemically defined cell culture medium. In some of the
embodiments herein, the cell culture medium is a chemically undefined cell culture medium.
In some of the embodiments herein, the cell culture medium is a basal cell culture medium.
In some of the embodiments herein, the cell culture medium is a feed cell culture medium. In
some of the embodiments herein, the cell is contacted with the cell culture medium during the
cell’s growth phase. In some of the embodiments herein, the cell is contacted with the cell
culture medium during the cell’s production phase. In some of the embodiments herein, the
one or more of components (a)-(h) is added to the cell culture medium on at least one day of
a cell culture cycle. In some of the embodiments herein, the one or more of components (a)-
(h) is added to the cell culture medium on day 0 of a 14 day cell culture cycle. In any of the
embodiments herein, the one or more of components (a)-(h) can be added to the cell culture
medium on any day of a cell culture cycle. In some of the embodiments herein, the cell is a
mammalian cell. In some embodiments, the mammalian cell is a Chinese Hamster Ovary
(CHO) cell. In some of the embodiments herein, the polypeptide is an antibody or fragment
thereof. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the
antibody is secreted into the cell culture medium. In some of the embodiments herein, the
method further comprises the step of recovering the polypeptide from the cell culture medium
comprising one or more of components (a)-(h). In some embodiments, a composition
comprising the recovered polypeptide is a liquid composition or a non-liquid composition. In
some embodiments, the composition comprising the recovered polypeptide appears as a
colorless or slightly colored liquid. In some of the embodiments herein, a polypeptide can be
produced by the any of the methods described herein.
In some embodiments, described herein is a pharmaceutical composition comprising
a polypeptide produced by any of the methods described herein and a pharmaceutically
acceptable carrier.
In some embodiments, described herein is a kit for supplementing a cell culture
medium with chemically defined constituents, the kit comprising hypotaurine or an analog or
precursor thereof at a concentration of at least about 0.0001 mM, and wherein the
hypotaurine or an analog or precursor is selected from the group consisting of hypotaurine, s-
carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine.
In other embodiments, also described herein is a kit for supplementing a cell culture
medium with chemically defined constituents, the kit comprising one or more of: (a)
hypotaurine in an amount to provide from at least about 0.0001 mM hypotaurine in the cell
culture medium; (b) s-carboxymethylcysteine in an amount to provide from at least about
0.0001 mM s-carboxymethylcysteine in the cell culture medium; (c) carnosine in an amount
to provide from at least about 0.0001 mM carnosine in the cell culture medium; (d) anserine
in an amount to provide from at least about 0.0001 mM anserine in the cell culture medium;
(e) butylated hydroxyanisole in an amount to provide from at least about 0.0001 mM
butylated hydroxyanisole; (f) lipoic acid in an amount to provide from at least about 0.0001
mM lipoic acid in the cell culture medium; (g) quercitrin hydrate in an amount to provide
from at least about 0.0001 mM quercitrin hydrate in the cell culture medium; and (h)
aminoguanidine in an amount to provide from at least about 0.0003 mM aminoguanidine in
the cell culture medium.
In some embodiments, described herein is a cell culture medium comprising from at
least about 0.0001 mM of hypotaurine or an analog or precursor thereof selected from the
group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic
acid, and taurine.
In other embodiments, described herein is a cell culture medium comprising one or
more of components (a)-(h): (a) from at least about 0.0001 mM hypotaurine; (b) from at least
about 0.0001 mM s-carboxymethylcysteine; (c) from at least about 0.0001 mM carnosine; (d)
from at least about 0.0001 mM anserine; (e) from at least about 0.0001 mM butylated
hydroxyanisole; (f) from at least about 0.0001 mM lipoic acid; (g) from at least about 0.0001
mM quercitrin hydrate; and (h) from at least about 0.0003 mM aminoguanidine.
In some embodiments, described herein is a composition comprising (a) a cell
comprising a nucleic acid encoding a polypeptide; and (b) any cell culture medium described
herein.
In some embodiments, described herein is a composition comprising: (a) a
polypeptide; and (b) any cell culture medium described herein. In some embodiments, the
polypeptide is secreted into the cell culture medium by a cell comprising a nucleic acid
encoding the polypeptide.
The specification is considered to be sufficient to enable one skilled in the art to
practice the invention. Various modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims. All publications, patents, and
patent applications cited herein are hereby incorporated by reference in their entirety for all
purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph of representative compounds screened for impact on color
intensity in a representative cell culture medium containing an antibody. Numerical results
were normalized to the positive control where the value for the positive control was set at 0%
change in color intensity. Values higher than 0% indicate increased color intensity. Values
lower than 0% indicate reduced color intensity.
Figure 2 is a subplot of Figure 1 showing compounds that reduced color intensity in
a representative cell culture medium containing an antibody. Numerical results were
normalized to the positive control where the value for the positive control was set at 0%
change in color intensity. Values lower than 0% indicate reduced color intensity.
Figure 3 is a series of graphs showing that a shaker flask cell culture model is
comparable to the corresponding larger scale 2L cell culture model. A) Cell growth in
culture over the duration of incubation as measured by viable cell density (VCC) and
expressed as number of cells per cell culture volume. B) Cell viability in cell culture over the
duration of incubation as measured by the number of viable cells as a percentage of the total
number of cells. C) Antibody production in cell culture over the duration of incubation as
measured by high performance liquid chromatography and expressed as antibody titer. SF
indicates shaker flask cell culture model. 2L indicates a larger scale cell culture model.
Figure 4 is a series of graphs showing that addition of hypotaurine to cell culture
media did not compromise cell growth, cell viability, or antibody production. A) Cell growth
in culture over the duration of incubation as measured by VCC and expressed as number of
cells per cell culture volume. B) Cell viability in cell culture over the duration of incubation
as measured by the number of viable cells as a percentage of the total number of cells. C)
Antibody production in cell culture over the duration of incubation as measured by high
performance liquid chromatography and expressed as antibody titer.
Figure 5 is a graph showing color intensity of antibody compositions isolated from
cell cultures grown in media supplemented with hypotaurine. 100%, 50%, or 25 % indicates
basal Media 1 supplemented with 9.16 mM, 4.58 mM or 2.29 mM hypotaurine, respectively.
Filled circles indicate color intensity values for cell culture experiments. Empty circles
indicate color intensity values for incubation screening experiments. Numerical results were
normalized to the positive control, where the value for the positive control was set at 0%
change in color intensity. Values lower than 0% indicate reduced color intensity.
Figure 6 is a graph showing color intensity of antibody compositions isolated from
cell cultures grown in media supplemented with hypotaurine. 3X, 2X, or 1X indicates basal
Media 3 supplemented with 38.85 mM, 25.9 mM or 12.95 mM hypotaurine, respectively.
Filled circles indicate color intensity values for cell culture experiments. Numerical results
were normalized to the positive control, where the value for the positive control was set at
0% change in color intensity. Values lower than 0% indicate reduced color intensity.
Figure 7 contains graphs showing that addition of hypotaurine, or carboxy methyl
cysteine to media did not compromise cell growth or cell viability. A) Cell growth in culture
over the duration of incubation as measured by VCC and expressed as number of cells per
cell culture volume. B) Cell viability in culture over duration of incubation expressed as
percent of total culture volume.
Figure 8 is a graph showing that addition of hypotaurine, or carboxy methyl
cysteine to media did not significantly reduce antibody production. Antibody production in
cell culture over the duration of incubation was measured by high performance liquid
chromatography and expressed as antibody titer.
Figure 9 is a graph showing color intensity of antibody compositions isolated from
cell cultures grown in media supplemented with hypotaurine or carboxy methyl cysteine. A
and B) Indicate two different color assays used to measure color intensity. Numerical results
were normalized to the positive control where the value for the positive control was set at 0%
change in color intensity. Values lower than 0% indicate reduced color intensity.
Figure 10 is a graph showing relative color intensity of antibody compositions
isolated from cell cultures in media supplemented with taurine, carnosine, aminoguanidine,
negative control, or positive control.
DETAILED DESCRIPTION
I. Definitions
The terms “medium” and “cell culture medium” refer to a nutrient source used for
growing or maintaining cells. As is understood by a person of skill in the art, the nutrient
source may contain components required by the cell for growth and/or survival or may
contain components that aid in cell growth and/or survival. Vitamins, essential or non-
essential amino acids, and trace elements are examples of medium components.
A “chemically defined cell culture medium” or “CDM” is a medium with a
specified composition that is free of products derived from animal or plant such as for
example animal serum and plant peptone. As would be understood by a person of skill in the
art, a CDM may be used in a process of polypeptide production whereby a cell is in contact
with, and secretes a polypeptide into, the CDM. Thus, it is understood that a composition
may contain a CDM and a polypeptide product and that the presence of the polypeptide
product does not render the CDM chemically undefined.
A “chemically undefined cell culture medium” refers to a medium whose chemical
composition cannot be specified and which may contain one or more products derived from
animal or plant such as for example animal serum and plant peptone. As would be
understood by a person of skill in the art, a chemically undefined cell culture medium may
contain a product derived from an animal or a plant as a nutrient source.
“Culturing” a cell refers to contacting a cell with a cell culture medium under
conditions suitable to the survival and/or growth and/or proliferation of the cell.
“Batch culture” refers to a culture in which all components for cell culturing
(including the cells and all culture nutrients) are supplied to the culturing vessel at the start of
the culturing process.
The phrase “fed batch cell culture,” as used herein refers to a batch culture wherein
the cells and culture medium are supplied to the culturing vessel initially, and additional
culture nutrients are fed, continuously or in discrete increments, to the culture during the
culturing process, with or without periodic cell and/or product harvest before termination of
culture.
“Perfusion culture” is a culture by which the cells are restrained in the culture by,
e.g., filtration, encapsulation, anchoring to microcarriers, etc., and the culture medium is
continuously or intermittently introduced and removed from the culturing vessel.
“Culturing vessel” refers to a container used for culturing a cell. The culturing
vessel can be of any size so long as it is useful for the culturing of cells.
As used herein, “hypotaurine analog” refers to a chemical compound that is
structurally similar to hypotaurine, but differs from hypotaurine in chemical composition
(e.g., differs by the number, location or chemical nature of functional groups or substituents
on the hypotaurine core). The hypotaurine analog may or may not have different chemical or
physical properties than hypotaurine and may or may not have improved activity in cell
culture media as compared to hypotaurine, e.g., further reducing the color intensity of a
polypeptide (e.g., an antibody) produced in the cell culture media as compared to
hypotaurine. For example, the hypotaurine analog may be more hydrophilic or it may have
altered reactivity as compared to hypotaurine. The hypotaurine analog may mimic the
chemical and/or biologically activity of hypotaurine (i.e., it may have similar or identical
activity), or, in some cases, may have increased or decreased activity as compared to
hypotaurine.
The term “titer” as used herein refers to the total amount of recombinantly
expressed polypeptide produced by a cell culture divided by a given amount of medium
volume. Titer is typically expressed in units of milligrams of polypeptide per milliliter of
medium.
A “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of
any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can
be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A
polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their
analogs. If present, modification to the nucleotide structure may be imparted before or after
assembly of the polymer.
An “isolated nucleic acid” means and encompasses a non-naturally occurring,
recombinant or a naturally occurring sequence outside of or separated from its usual context.
An isolated nucleic acid molecule is other than in the form or setting in which it is found in
nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid
molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a
nucleic acid molecule contained in cells that ordinarily express the protein where, for
example, the nucleic acid molecule is in a chromosomal location different from that of
natural cells.
An “isolated” protein (e.g., an isolated antibody) is one which has been identified
and separated and/or recovered from a component of its natural environment. Contaminant
components of its natural environment are materials which would interfere with research,
diagnostic or therapeutic uses for the protein, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. Isolated protein includes the protein in situ
within recombinant cells since at least one component of the protein's natural environment
will not be present. Ordinarily, however, isolated protein will be prepared by at least one
purification step.
A “purified” polypeptide means that the polypeptide has been increased in purity,
such that it exists in a form that is more pure than it exists in its natural environment and/or
when initially produced and/or synthesized and/or amplified under laboratory conditions.
Purity is a relative term and does not necessarily mean absolute purity.
“Contaminants” refer to materials that are different from the desired polypeptide
product. The contaminant includes, without limitation: host cell materials, such as CHOP;
leached Protein A; nucleic acid; a variant, fragment, aggregate or derivative of the desired
polypeptide; another polypeptide; endotoxin; viral contaminant; cell culture media
component, etc.
The terms “polypeptide” and “protein” are used interchangeably herein to refer to
polymers of amino acids of any length. The polymer may be linear or branched, it may
comprise modified amino acids, and it may be interrupted by non-amino acids. The terms
also encompass an amino acid polymer that has been modified naturally or by intervention;
for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with a labeling component.
Also included within the definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as
other modifications known in the art. Examples of polypeptides encompassed within the
definition herein include mammalian proteins, such as, e.g., renin; a growth hormone,
including human growth hormone and bovine growth hormone; growth hormone releasing
factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alphaantitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin;
luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor,
and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor;
lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type
plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor
necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally
T-cell expressed and secreted); human macrophage inflammatory protein (MIPalpha); a
serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-
chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial
protein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen
(CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF);
receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic
factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3,
NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-b; platelet-derived growth
factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor
(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-
β1, TGF- β2, TGF- β3, TGF- β4, or TGF- β5; insulin-like growth factor-I and -II (IGF-I and
IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins (IGFBPs);
CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive
factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as
interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-
CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell
receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for
example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins;
regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and
VCAM; a tumor associated antigen such as CA125 (ovarian cancer antigen) or HER2, HER3
or HER4 receptor; immunoadhesins; and fragments and/or variants of any of the above-listed
proteins as well as antibodies, including antibody fragments, binding to a protein, including,
for example, any of the above-listed proteins.
The term “antibody” herein is used in the broadest sense and specifically covers
monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they
exhibit the desired biological activity. An antibody can be human, humanized and/or affinity
matured.
The term “monoclonal antibody” as used herein refers to an antibody obtained from
a population of substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population are identical except for possible naturally occurring mutations that
can be present in minor amounts. Monoclonal antibodies are highly specific, being directed
against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations
which include different antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the antigen. In addition to
their specificity, the monoclonal antibodies are advantageous in that they can be synthesized
uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as
requiring production of the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the description may be made by a variety of
techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier,
N.Y., 1981)), recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see,
e.g., U.S. Pat. No. 4,816,567); phage-display technologies (see, e.g., Clackson et al., Nature,
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol.
Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,
Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
Methods 284(1-2): 119-132 (2004) and technologies for producing human or human-like
antibodies in animals that have parts or all of the human immunoglobulin loci or genes
encoding human immunoglobulin sequences (see, e.g., ; ;
; ; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551
(1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol.
7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368:
856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol.
14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar,
Intern. Rev. Immunol. 13: 65-93 (1995).
The term “pharmaceutical formulation” refers to a preparation which is in such form
as to permit the biological activity of the active ingredient to be effective, and which contains
no additional components which are unacceptably toxic to a subject to which the formulation
would be administered. Such formulations are sterile.
“Pharmaceutically acceptable” carriers, excipients, or stabilizers are ones which are
nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations
employed (Remington's Pharmaceutical Sciences (20 edition), ed. A. Gennaro, 2000,
Lippincott, Williams & Wilkins, Philadelphia, PA). Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers
include buffers such as phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such
as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-
forming counterions such as sodium; and/or nonionic surfactants such as Tween™,
polyethylene glycol (PEG), and Pluronics™.
A “sterile” formulation is aseptic or free or essentially free from all living
microorganisms and their spores.
A “colorless or slightly colored” liquid refers to a liquid composition comprising a
polypeptide that is measured by quantitative and/or qualitative analysis. Qualitative analysis
includes visual inspection such as comparison of the composition comprising the polypeptide
to a reference standard.
As used in this specification and the appended claims, the singular forms “a”, “an”
and “the” include plural referents unless the content clearly dictates otherwise. Thus, for
example, reference to “a compound” optionally includes a combination of two or more such
compounds, and the like.
[0051A] The term “comprising” as used in this specification and claims means “consisting at
least in part of”. When interpreting statements in this specification, and claims which include
the term “comprising”, it is to be understood that other features that are additional to the
features prefaced by this term in each statement or claim may also be present. Related terms
such as “comprise” and “comprised” are to be interpreted in similar manner.
It is understood that aspect and embodiments described herein include
“comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.
Reference to “about” a value or parameter herein includes (and describes)
embodiments that are directed to that value or parameter per se. For example, description
referring to “about X” includes description of “X.” Numeric ranges are inclusive of the
numbers defining the range.
Where aspects or embodiments are described herein in terms of a Markush group or
other grouping of alternatives, the present description encompasses not only the entire group
listed as a whole, but each member of the group individually and all possible subgroups of
the main group, but also the main group absent one or more of the group members. The
present description also envisages the explicit exclusion of one or more of any of the group
members.
II. Cell Culture Media
Cell culture media described herein may find use in methods (e.g., of culturing cells
and producing polypeptides) and in compositions (e.g., pharmaceutical formulations) as
detailed herein. Media components have been identified as capable of providing a
polypeptide product (e.g., a therapeutic protein) with acceptable quality attributes, such as an
acceptable color intensity. One or more of these identified media components can be used to
provide a polypeptide product with an acceptable color intensity. As used herein, “an
acceptable color intensity” of a polypeptide product (e.g., composition comprising the
polypeptide) can refer to the color intensity required for regulatory approval of the
polypeptide product or the color intensity desired for use in assessing consistency in lot to lot
batches of the polypeptide product. In some embodiments, the one or more media component
is an antioxidant. In some embodiments, the one or more media component is selected from
the group consisting of hypotaurine, s-carboxymethylcysteine, anserine, butylated
hydroxyanisole, carnosine, lipoic acid, quercitrin hydrate, and aminoguanidine. In some
embodiments, the one or more media component is hypotaurine or an analog or precursor
thereof. In some embodiments, the hypotaurine or an analog or precursor thereof is selected
from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine,
cysteinesulphinic acid, and taurine. In some embodiments, the one or more media component
is taurine, lipoic acid reduced, or carvedilol.
Media components may be added to the cell culture media in forms that are known
in the art. For example, hypotaurine may be provided as a compound identified by CAS
number 3005, s-carboxymethylcysteine may be provided as a compound identified by
CAS number 6383, anserine may be provided as a compound identified by CAS number
100301, butylated hydroxyanisole may be provided as a compound identified by CAS
number 250135, carnosine may be provided as a compound identified by CAS number
3050, lipoic acid may be provided as a compound identified by CAS number 12002,
quercitrin hydrate may be provided as a compound identified by CAS number 5223. As
another example, analogs or precursors of hypotaurine may be provided such as s-
carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and/or taurine. In some
embodiments, s-carboxymethylcysteine is provided as a compound identified by CAS
number 6383, cysteamine is provided as a compound identified by CAS number 601,
cysteinesulphinic acid is provided as a compound identified by CAS number 11157, and
taurine is provided as a compound identified by CAS number 1077. In some
embodiments, a compound listed in Table 4 is provided such as lipoic acid reduced identified
by CAS number 4624 or carvedilol identified by CAS number 729563. In some
embodiments, aminoguanidine is provided as aminoguanidine hydrochloride identified by
CAS number 19375. The media components described herein can be provided to the cell
culture medium as a salt, a hydrate, or a salt hydrate or any other form known to one of skill
in the art. The media components can also be provided to cell culture media as a solution, an
extract, or in solid form. In some embodiments herein, the cell culture medium is a
chemically defined medium. In other embodiments herein, the cell culture medium is a
chemically undefined medium.
In some embodiments, described herein is a cell culture medium comprising one or
more of the following components: (a) hypotaurine; (b) s-carboxymethylcysteine; (c)
carnosine; (d) anserine; (e) butylated hydroxyanisole; (f) lipoic acid; (g) quercitrin hydrate;
and (h) aminoguanidine. In some embodiments, the cell culture medium comprises 2 or 3 or
4 or 5 or 6 or each of components (a), (b), (c), (d), (e), (f), (g) and (h). It is understood that
the cell culture medium described herein may contain any combination of components (a),
(b), (c), (d), (e), (f), (g), and (h) the same as if each and every combination were specifically
and individually listed. For example, it is understood that a cell culture medium comprising
four of components (a), (b), (c), (d), (e), (f), (g), and (h) may comprise any combination of
the components so long as at least four of the components are present.
In some embodiments, a cell culture medium as described herein contains one or
more media components selected from the group consisting of (a) hypotaurine; (b) s-
carboxymethylcysteine; (c) carnosine; (d) anserine; (e) butylated hydroxyanisole; (f) lipoic
acid; (g) quercitrin hydrate; and (h) aminoguanidine in amounts as described in Table 1. It is
understood that a medium may comprise any one or more of the medium components of
Table 1 (e.g., any one or more of components (a)-(h), such as a medium comprising
components (a), (b), (c), (d) and (e) or a medium comprising components (a), (b) and (g) or a
medium comprising only one of components (a)-(h)) in any of the amounts listed in Table 1,
the same as if each and every combination of components and amounts were specifically and
individually listed. In one embodiment, the cell culture medium is a chemically defined
medium. In another embodiment, the cell culture medium is a chemically undefined medium.
In some embodiments, a cell culture medium comprises one or more of components (a)-(h),
wherein (a) is from at least about 0.0001 mM hypotaurine, (b) is from at least about 0.0001
mM s-carboxymethylcysteine, (c) is f from at least about 0.0001 mM carnosine, (d) is from at
least about 0.0001 mM anserine, (e) is from at least about 0.0001 mM butylated
hydroxyanisole, (f) is from at least about 0.0001 mM lipoic acid, (g) is from at least about
0.0001 mM quercitrin hydrate, and (h) is from at least about 0.0003 mM aminoguanidine. In
some embodiments, a cell culture medium comprises one or more of components (a)-(h),
wherein (a) is from about 2.0 mM to about 50.0 mM hypotaurine, (b) is from about 8.0 mM
to about 12.0 mM s-carboxymethylcysteine, (c) is from about 8.0 mM to about 12.0 mM
carnosine, (d) is from about 3.0 mM to about 5.0 mM anserine, (e) is from about 0.025 mM
to about 0.040 mM butylated hydroxyanisole, (f) is from about 0.040 mM to about 0.060 mM
lipoic acid, (g) is from about 0.010 mM to about 0.020 mM quercitrin hydrate, and (h) is
from about 0.0003 mM to about 10 mM aminoguanidine.
Table 1. Exemplary Amounts of Media Components
Component Amount of Component in Medium
(a) Hypotaurine from about 0.0001 mM to about 920 mM; from about 0.001 mM to
about 920 mM; from about 0.01 mM to about 920 mM; from about
0.1 mM to about 920 mM; from about 0.5 mM to about 920 mM;
from about 0.0001 mM to about 820 mM; from about 0.0001 mM to
about 720 mM; from about 0.0001 mM to about 620 mM; from about
0.0001 mM to about 520 mM; from about 0.0001 mM to about 420
mM; from about 0.0001 mM to about 320 mM; from about 0.0001
mM to about 220 mM; from about 0.0001 mM to about 120 mM;
from about 0.0001 mM to about 20 mM; from about 1.0 mM to about
920 mM; from about 10.0 mM to about 920 mM; from about 20.0
mM to about 920 mM; from about 40.0 mM to about 920 mM; from
about 80.0 mM to about 920 mM; from about 160.0 mM to about 920
mM; from about 320 mM to about 920 mM; from about 640 mM to
about 920 mM; from about 800 mM to about 920 mM; from about
0.75 mM to about 700 mM; from about 1.0 mM to about 500 mM;
from about 1.25 mM to about 300 mM; from about 1.5 mM to 100
mM; from about 1.6 mM to about 90 mM; from about 1.7 mM to
about 80 mM; from about 1.8 mM to about 70 mM; from about 1.8
mM to about 60 mM; from about 1.8 mM to about 50 mM; from
about 2 mM to about 50 mM; from about 5 mM to about 50 mM;
from about 10 mM to about 50 mM; from about 15 mM to about 50
mM; from about 20 mM to about 50 mM; from about 30 mM to about
50 mM; from about 40 mM to about 50 mM; about any of 0.0001 or
0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 9.0 or 12 or 25
or 38 or 45 or 50 mM; at least about any of 0.0001 or 0.001 or 0.01 or
0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 9.0 or 12 mM and no more
than about 60 or 55 or 50 or 45 or 40 mM.
(b) s- from about 0.0001 mM to about 120 mM; from about 0.001 mM to
carboxymethyl about 120 mM; from about 0.01 mM to about 120 mM; from about
cysteine 0.1 mM to about 120 mM; from about 0.5 mM to about 120 mM;
from about 0.0001 mM to 100 mM; from about 0.0001 mM to about
80 mM; from about 0.0001 mM to about 60 mM; from about 0.0001
mM to about 40 mM; from about 0.0001 mM to about 20 mM; from
about 0.0001 mM to about 10 mM; from about 0.0001 mM to about
120 mM; from about 10 mM to about 120 mM; from about 20 mM to
about 120 mM; from about 40 mM to about 120 mM; from about 60
mM to about 120 mM; from about 80 mM to about 120 mM; from
about 100 mM to about 120 mM; from about 1.0 mM to about 100
mM; from about 2.0 mM to about 75 mM; from about 3.0 mM to
about 50 mM; from about 4.0 mM to about 25 mM; from about 5.0
mM to about 15 mM; from about 6.0 mM to about 14 mM; from
about 7.0 mM to about 13 mM; from 8.0 mM to about 12 mM; about
any of 0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0
or 10 or 15 or 20 mM; at least about any of 0.0001 or 0.001 or 0.01 or
0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 8.0 or 10 or 12 mM and no
more than about 25 or 20 or 15 mM.
(c) Carnosine from about 0.0001 mM to about 20 mM; from about 0.001 mM to
about 20 mM; from about 0.01 mM to about 20 mM; from about 0.1
mM to about 20 mM; from about 0.5 mM to about 20 mM; from
about 0.0001 mM to about 15 mM; from about 0.0001 mM to about
mM; from about 0.0001 mM to about 5.0 mM; from about 1.0 mM
to about 20 mM; from about 5.0 mM to about 20 mM; from about 10
mM to about 20 mM; from about 15 mM to about 20 mM; from about
2.0 mM to about 18 mM; from about 4.0 mM to about 16 mM; from
about 6.0 mM to about 14 mM; from about 8.0 mM to about 12 mM;
about any of 0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0
or 5.0 or 6.0 or 7.0 or 8.0 or 9.0 or 10 or 11 or 12 or 13 or 14 mM; at
least about any of 0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or
4.0 or 5.0 or 6.0 or 7.0 or 8.0 or 9.0 or 10 or 11 and no more than 15
or 14 or 13 mM.
(d) Anserine from about 0.0001 mM to about 20 mM; from about 0.001 mM to
about 20 mM; from about 0.01 mM to about 20 mM; from about 0.1
mM to about 20 mM; from about 0.5 mM to about 20 mM; from
about 0.0001 mM to about 15 mM; from about 0.0001 mM to about
mM; from about 0.0001 mM to about 5.0 mM; from about 1.0 mM
to about 20 mM; from about 5.0 mM to about 20 mM; from about 10
mM to about 20 mM; from about 15 mM to about 20 mM; from about
1.0 mM to about 15 mM; from about 2.0 mM to about 10 mM; from
about 3.0 mM to about 5.0 mM; from about 3.2 mM to about 5.0 mM;
about any of 0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0
or 5.0 or 6.0 or 7.0 or 8.0 mM; at least about any of 0.0001 or 0.001
or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 mM and no more than
9.0 or 8.0 or 7.0 or 6.0 mM.
(e) Butylated from about 0.0001 mM to about 0.2 mM; from about 0.001 mM to
hydroxyanisole about 0.2 mM; from about 0.005 mM to about 0.2 mM; from about
0.0001 mM to about 0.15 mM; from about 0.0001 mM to about 0.1
mM; from about 0.0001 mM to about 0.05 mM; from about 0.0001
mM to about 0.04 mM; from about 0.01 mM to about 0.2 mM; from
about 0.05 mM to about 0.2 mM; from about 0.1 mM to about 0.2
mM; from about 0.15 mM to about 0.2 mM; from about 0.01 mM to
about 0.15 mM; from about 0.015 mM to about 0.1 mM; from about
0.02 mM to about 0.05 mM; from about 0.025 mM to about 0.04 mM;
from about 0.03 mM to about 0.04 mM; about any of 0.0001 or 0.001
or 0.01 or 0.015 or 0.02 or 0.025 or 0.03 or 0.035 or 0.04 or 0.045 or
0.05 or 0.055 or 0.06 mM; at least about any of 0.0001 or 0.001 or
0.01 or 0.015 or 0.02 or 0.025 or 0.03 or 0.035 or 0.04 mM and no
more than 0.06 or 0.055 or 0.05 mM.
(f) Lipoic acid from about 0.0001 mM to about 1.5 mM; from about 0.001 mM to
about 1.5 mM; from about 0.01 mM to about 1.5 mM; from about
0.0001 mM to about 1.25 mM; from about 0.0001 mM to about 1.0
mM; from about 0.0001 mM to about 0.75 mM; from about 0.0001
mM to about 0.5 mM; from about 0.0001 mM to about 0.25 mM;
from about 0.05 mM to about 1.5 mM; from about 0.1 mM to about
1.5 mM; from about 0.25 mM to about 1.5 mM; from about 0.5 mM
to about 1.5 mM; from about 0.75 mM to about 1.5 mM; from about
1.0 mM to about 1.5 mM; from about 1.25 mM to about 1.5 mM;
from about 0.02 mM to about 1.25 mM; from about 0.03 mM to about
1.0 mM; from about 0.032 mM to about 0.1 mM; from about 0.034
mM to about 0.09 mM; from about 0.036 mM to about 0.08 mM;
from about 0.038 mM to about 0.07 mM; from about 0.04 mM to
about 0.06 mM; about any of 0.0001 or 0.001 or 0.01 or 0.02 or 0.03
or 0.04 or 0.05 or 0.06 or 0.07 or 0.08 or 0.09 mM; at least about any
of 0.0001 or 0.001 or 0.01 or 0.02 or 0.03 or 0.04 or 0.05 mM and no
more than 0.09 or 0.08 or 0.07 mM.
(g) Quercitrin from about 0.0001 mM to about 0.04 mM; from about 0.001 mM to
hydrate about 0.04 mM; from about 0.005 mM to about 0.04 mM; from about
0.001 mM to about 0.035 mM; from about 0.001 mM to about 0.03
mM; from about 0.001 mM to about 0.025 mM; from about 0.001mM
to about 0.02 mM; from about 0.001 mM to about 0.015 mM; from
about 0.001 mM to about 0.01 mM; from about 0.01 mM to about
0.04 mM; from about 0.015 mM to about 0.04 mM; from about 0.02
mM to about 0.04 mM; from about 0.025 mM to about 0.04 mM;
from about 0.03 mM to about 0.04 mM; from about 0.035 mM to
about 0.04 mM; from about 0.0075 mM to about 0.035 mM; from
about 0.01 mM to about 0.03 mM; from about 0.015 mM to about
0.025 mM; from about 0.01 mM to about 0.02 mM; about any of
0.0001 or 0.001 or 0.01 or 0.011 or 0.012 or 0.013 or 0.014 or 0.015
or 0.016 mM; at least about any of 0.0001 or 0.001 or 0.011 or 0.012
or 0.013 or 0.014 mM and no more than 0.02 or 0.019 or 0.018 mM.
(h) from about 0.0003 to about 245 mM; from about 0.0003 to about 200
Aminoguanidine mM; from about 0.0003 to about 150 mM; from about 0.0003 to
about 125 mM; from about 0.0003 to about 100 mM; from about
0.0003 to about 75 mM; from about 0.0003 to about 50 mM; from
about 0.0003 to about 40 mM; from about 0.0003 to about 30 mM;
from about 0.0003 to about 25 mM; from about 0.0003 to about 20
mM; from about 0.0003 to about 15 mM; from about 0.0003 to about
mM; from about 0.0003 to about 7.5 mM; from about 0.0003 to
about 5 mM; from about 0.0003 to about 2.5 mM; from about 0.0003
to about 1 mM; from about 0.003 to about 100 mM; from about 0.03
to about 100 mM; from about 0.3 to about 100 mM; from about 0.003
to about 10 mM; from about 0.03 to about 10 mM; from about 0.3 to
about 10 mM; about of any of 0.0003, 0.003, 0.03, 0.3, 1.0, 1.5, 2.0,
3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10 mM.
In some embodiments, described herein is a cell culture medium comprising
hypotaurine or an analog or precursor thereof selected from the group consisting of
hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine. In
some aspects, the cell culture medium comprises one or more of the following components:
(a) hypotaurine; (b) s-carboxymethylcysteine; (c) cysteamine; (d) cysteinesulphinic acid; and
(e) taurine. In some embodiments, the cell culture medium comprises 2 or 3 or 4 or each of
components (a), (b), (c), (d), and (e). It is understood that the cell culture medium described
herein may contain any combination of components (a), (b), (c), (d), and (e) the same as if
each and every combination were specifically and individually listed. For example, it is
understood that a cell culture medium comprising three of components (a), (b), (c), (d), and
(e) may comprise any combination of the components so long as at least three of the
components are present. Hypotaurine analogs include for example s-carboxymethylcysteine,
cysteamine, cysteinesulphinic acid, and taurine. Examples of hypotaurine precursors are well
known to one of skill in the art and in some embodiments a hypotaurine precursor can be a
hypotaurine analog.
In some embodiments, a cell culture medium as described herein contains
hypotaurine or an analog or precursor thereof in amounts as described in Table 2. It is
understood that a medium may comprise any one or more of the medium components of
Table 2 (e.g., any one or more of components (a)-(e), such as a medium comprising
components (a), (b), (c), and (d) or a medium comprising components (a), (b) and (c) or a
medium comprising only one of components (a)-(e)) in any of the amounts listed in Table 2,
the same as if each and every combination of components and amounts were specifically and
individually listed. In some embodiments, a cell culture medium comprises hypotaurine or
an analog or precursor thereof such as hypotaurine, s-carboxymethylcysteine, cysteamine,
cysteinesulphinic acid, and/or taurine at a concentration from at least about 0.0001 mM. In
some embodiments, a cell culture medium comprises hypotaurine or an analog or precursor
thereof such as hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid,
and/or taurine at a concentration from about 0.5 mM to about 500.0 mM.
Table 2. Exemplary Amounts of Media Components
Component Amount of Component in Medium
(a) Hypotaurine from about 0.0001 mM to about 920 mM; from about 0.001 mM to
about 920 mM; from about 0.01 mM to about 920 mM; from about
0.1 mM to about 920 mM; from about 0.5 mM to about 920 mM;
from about 0.0001 mM to about 820 mM; from about 0.0001 mM to
about 720 mM; from about 0.0001 mM to about 620 mM; from about
0.0001 mM to about 520 mM; from about 0.0001 mM to about 420
mM; from about 0.0001 mM to about 320 mM; from about 0.0001
mM to about 220 mM; from about 0.0001 mM to about 120 mM;
from about 0.0001 mM to about 20 mM; from about 1.0 mM to about
920 mM; from about 10.0 mM to about 920 mM; from about 20.0
mM to about 920 mM; from about 40.0 mM to about 920 mM; from
about 80.0 mM to about 920 mM; from about 160.0 mM to about 920
mM; from about 320 mM to about 920 mM; from about 640 mM to
about 920 mM; from about 800 mM to about 920 mM; from about
0.75 mM to about 700 mM; from about 1.0 mM to about 500 mM;
from about 1.25 mM to about 300 mM; from about 1.5 mM to 100
mM; from about 1.6 mM to about 90 mM; from about 1.7 mM to
about 80 mM; from about 1.8 mM to about 70 mM; from about 1.8
mM to about 60 mM; from about 1.8 mM to about 50 mM; from
about 2 mM to about 50 mM; from about 5 mM to about 50 mM;
from about 10 mM to about 50 mM; from about 15 mM to about 50
mM; from about 20 mM to about 50 mM; from about 30 mM to about
50 mM; from about 40 mM to about 50 mM; about any of 0.0001 or
0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 9.0 or 12 or 25
or 38 or 45 or 50 mM; at least about any of 0.0001 or 0.001 or 0.01 or
0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 9.0 or 12 mM and no more
than about 60 or 55 or 50 or 45 or 40 mM.
(b) s- from about 0.0001 mM to about 120 mM; from about 0.001 mM to
carboxymethyl about 120 mM; from about 0.01 mM to about 120 mM; from about
cysteine 0.1 mM to about 120 mM; from about 0.5 mM to about 120 mM;
from about 0.0001 mM to 100 mM; from about 0.0001 mM to about
80 mM; from about 0.0001 mM to about 60 mM; from about 0.0001
mM to about 40 mM; from about 0.0001 mM to about 20 mM; from
about 0.0001 mM to about 10 mM; from about 0.0001 mM to about
120 mM; from about 10 mM to about 120 mM; from about 20 mM to
about 120 mM; from about 40 mM to about 120 mM; from about 60
mM to about 120 mM; from about 80 mM to about 120 mM; from
about 100 mM to about 120 mM; from about 1.0 mM to about 100
mM; from about 2.0 mM to about 75 mM; from about 3.0 mM to
about 50 mM; from about 4.0 mM to about 25 mM; from about 5.0
mM to about 15 mM; from about 6.0 mM to about 14 mM; from
about 7.0 mM to about 13 mM; from 8.0 mM to about 12 mM; about
any of 0.0001 or 0.001 or 0.01 or 0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0
or 10 or 15 or 20 mM; at least about any of 0.0001 or 0.001 or 0.01 or
0.1 or 1.0 or 2.0 or 3.0 or 4.0 or 5.0 or 8.0 or 10 or 12 mM and no
more than about 25 or 20 or 15 mM.
(c) cysteamine from about 0.0001 mM to about 300 mM; from about 0.001 mM to
about 300 mM; from about 0.01 mM to about 300 mM; from about
0.0001 mM to about 250 mM; from about 0.0001 mM to about 200
mM; from about 0.0001 mM to about 150 mM; from about 0.0001
mM to about 100 mM; from about 0.0001 mM to about 50 mM; from
about 0.0001 mM to about 1 mM; from about 1 mM to about 300
mM; from about 50 mM to about 300 mM; from about 100 mM to
about 300 mM; from about 150 mM to about 300 mM; from about
200 mM to about 300 mM; from about 250 mM to about 300 mM;
from about 0.02 mM to about 300 mM; from about 0.03 mM to about
200 mM; from about 0.04 mM to about 100 mM; from about 0.05
mM to about 50 mM; from about 0.02 mM to about 1 mM; from
about 0.04 mM to about 0.8 mM; from about 0.06 mM to about 0.6
mM; from about 0.08 mM to about 0.4 mM; from about 0.1 mM to
about 0.2 mM; about any of 0.0001 or 0.001 or 0.01 or 0.02 or 0.05 or
0.1 or 0.25 or 0.5 or 1 or 5 or 10 or 25 or 50 or 100 or 200 or 300
mM; at least about 0.0001 or 0.001 or 0.01 or 0.02 or 0.05 or 0.1 or
0.25 mM and no more than about 50 or 40 or 30 mM.
(d) from about 0.0001 mM to 100 mM; from about 0.001 mM to 100
cysteinesulphinic mM; from about 0.01 mM to 100 mM; from about 0.1 mM to 100
acid mM; from about 0.0001 mM to about 80 mM; from about 0.0001 mM
to about 60 mM; from about 0.0001 mM to about 40 mM; from about
0.0001 mM to about 20 mM; from about 0.0001 mM to about 1 mM;
from about 1 to 100 mM; from about 20 mM to about 100 mM; from
about 40 mM to about 100 mM; from about 60 mM to about 100 mM;
from about 80 mM to about 100 mM; from about 0.1 mM to about 50
mM; from about 0.2 mM to about 10 mM; from about 0.3 mM to
about 1 mM; from about 0.1 mM to about 1 mM; from about 0.2 mM
to about 0.8 mM; from about 0.3 mM to about 0.6 mM; about any of
0.0001 or 0.001 or 0.01 or 0.1 or 0.2 or 0.3 or 0.4 or 0.5 or 0.6 or 0.7
or 1 or 10 or 25 or 50 or 100 mM; at least about 0.0001 or 0.001 or
0.01 or 0.1 or 0.1 or 0.2 or 0.3 or 0.4 mM and no more than 20 or 10
or 15 mM.
(e) taurine from about 0.0001 mM to 500 mM; from about 0.001 mM to 500
mM; from about 0.01 mM to 500 mM; from about 0.1 mM to 500
mM; from about 0.5 mM to 500 mM; from about 0.0001 mM to about
450 mM; from about 0.0001 mM to about 400 mM; from about
0.0001 mM to about 350 mM; from about 0.0001 mM to about 300
mM; from about 0.0001 mM to 250 mM; from about 0.0001 mM to
200 mM; from about 0.0001 mM to 150 mM; from about 0.0001 mM
to 100 mM; from about 0.0001 mM to about 50 mM; from about 1
mM to about 500 mM; from about 50 mM to about 500 mM; from
about 100 mM to about 500 mM; from about 150 mM to about 500
mM; from about 200 mM to about 500 mM; from about 250 mM to
about 500 mM; from about 300 mM to about 500 mM; from about
350 mM to about 500 mM; from about 400 mM to about 500 mM;
from about 450 mM to about 500 mM; from about 1.0 mM to about
400 mM; from about 2.0 mM to about 300 mM; from about 3.0 mM
to about 200 mM; from about 4.0 mM to about 100 mM; from about
1.0 mM to about 10 mM; about any of 0.0001 or 0.001 or 0.01 or 0.1
or 1or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 mM; at least about any of
0.0001 or 0.001 or 0.01 or 0.1 or 1or 2 or 3 or 4 or 5 or 6 and no more
than 13 or 12 or 11 mM.
In some embodiments, a cell culture medium described herein comprises lipoic acid
reduced at a concentration of about 0.0001 mM to about 0.5 mM. In some embodiments, a
cell culture medium described herein comprises carvedilol at a concentration of about 0.0001
mM to about 1.5 mM.
Individual media components described herein may be present in amounts that
result in one or more advantageous properties for culturing cells and/or polypeptide
production from cell culture. Advantageous properties include, but are not limited to,
reduced oxidation of polypeptides in cell culture and/or reduced color intensity of a
composition comprising a polypeptide produced by a cell cultured in a cell culture media
described herein. Advantageous properties of the cell culture media described herein also
include reduction of color intensity of a composition comprising a polypeptide produced by a
cell cultured in the cell culture media without affecting one or more product attributes such as
the amount of the polypeptide produced by the cells (e.g., antibody titer), the glycosylation
(e.g., N-glycosylation) profile of the polypeptide, the polypeptide charge heterogeneity in the
composition, or the amino acid sequence integrity of the polypeptide. In some embodiments,
a one or more advantageous property for culturing a cell in a cell culture media described
herein is reduced color intensity of a composition comprising a polypeptide produced by the
cell without affecting cell viability, the amount of the polypeptide produced by the cells, the
glycosylation (e.g., N-glycosylation) profile of the polypeptide, the polypeptide charge
heterogeneity in the composition, and/or the amino acid sequence integrity of the
polypeptide. In some embodiments, a one or more advantageous property for culturing a cell
in a cell culture media described herein is reduced color intensity of a composition
comprising a polypeptide produced by the cell and reduced oxidation of the polypeptide in
cell culture. These advantageous properties are applicable to methods of culturing a cell
comprising a nucleic acid encoding a polypeptide of interest and methods of producing a
polypeptide of interest in cell culture as described herein.
In some embodiments, one more media component selected from the group
consisting of hypotaurine, s-carboxymethylcysteine, anserine, butylated hydroxyanisole,
carnosine, lipoic acid, quercitrin hydrate, and aminoguanidine is provided herein in an
amount that results in one or more advantageous property for culturing cells and/or
polypeptide production from cell culture. In some embodiments, an amount of hypotaurine
in cell culture media that results in one or more advantageous property is from about 0.5 mM
to about 100 mM, from about 1.6 mM to about 90 mM, from about 1.7 mM to about 80 mM,
from about 1.8 mM to about 70 mM, from about 1.9 mM to about 60 mM, from about 2.0
mM to about 50 mM, or from about 1.75 mM to about 50 mM. In some embodiments, an
amount of s-carboxymethylcysteine in cell culture media that results in one or more
advantageous property is from about 0.5 mM to about 120 mM, from about 5.0 mM to about
mM, from about 6.0 mM to about 14 mM, from about 7.0 mM to about 13 mM, or from
8.0 mM to about 12 mM. In some embodiments, an amount of anserine in cell culture media
that results in one or more advantageous property is from about 0.5 mM to about 20 mM,
from about 2.0 mM to about 10 mM, or from about 3.0 mM to about 5.0 mM. In some
embodiments, an amount of butylated hydroxyanisole in cell culture media that results in one
or more advantageous property is from about 0.005 mM to about 0.2 mM, from about 0.02
mM to about 0.05 mM, or from about 0.025 mM to about 0.04 mM. In some embodiments,
an amount of carnosine in cell culture media that results in one or more advantageous
property is from about 0.5 mM to about 20 mM, from about 6.0 mM to about 14 mM, or from
about 8.0 mM to about 12 mM. In some embodiments, an amount of lipoic acid in cell
culture media that results in one or more advantageous property is from about 0.01 mM to
about 1.5 mM lipoic acid, from about 0.036 mM to about 0.08 mM, from about 0.038 mM to
about 0.07 mM or from about 0.04 mM to about 0.06 mM. In some embodiments, an amount
of quercitrin hydrate in cell culture media that results in one or more advantageous property
is from about 0.005 mM to about 0.04 mM, from about 0.01 mM to about 0.03 mM, from
about 0.015 mM to about 0.025 mM or from about 0.01 mM to about 0.02 mM. In some
embodiments, an amount of aminoguanidine in cell culture media that results in one or more
advantageous property is from about 0.0003 mM to about 245 mM, from about 0.003 mM to
about 150 mM, from about 0.03 mM to about 100 mM, from about 0.03 mM to about 50
mM, from about 0.03 mM to about 25 mM, from about 0.03 to about 10 mM. In some
embodiments, an amount of one more media component selected from the group consisting
of hypotaurine, s-carboxymethylcysteine, anserine, butylated hydroxyanisole, carnosine,
lipoic acid, quercitrin hydrate, and aminoguanidine in cell culture media that results in one or
more advantageous property is provided in Table 1.
In some embodiments, one more media component selected from the group
consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and
taurine is provided herein in an amount that results in one or more advantageous property for
culturing cells and/or polypeptide production from cell culture. In some embodiments, an
amount of hypotaurine in cell culture media that results in one or more advantageous property
is from about 0.5 mM to about 100 mM, from about 1.6 mM to about 90 mM, from about 1.7
mM to about 80 mM, from about 1.8 mM to about 70 mM, from about 1.9 mM to about 60
mM, from about 2.0 mM to about 50 mM, or from about 1.75 mM to about 50 mM. In some
embodiments, an amount of s-carboxymethylcysteine in cell culture media that results in one
or more advantageous property is from about 0.5 mM to about 120 mM, from about 5.0 mM
to about 15 mM, from about 6.0 mM to about 14 mM, from about 7.0 mM to about 13 mM,
or from about 8.0 mM to about 12 mM. In some embodiments, an amount of cysteamine in
cell culture media that results in one or more advantageous property is from about 0.01 mM
to about 300 mM, from about 0.02 mM to about 1 mM, from about 0.04 mM to about 0.8
mM, from about 0.06 mM to about 0.6 mM, from about 0.08 mM to about 0.4 mM, or from
about 0.1 mM to about 0.2 mM. In some embodiments, an amount of cysteinesulphinic acid
in cell culture media that results in one or more advantageous property is from about 0.1 mM
to 100 mM, from about 0.2 mM to about 10 mM, from about 0.3 mM to about 1 mM, from
about 0.1 mM to about 1 mM, from about 0.2 mM to about 0.8 mM, or from about 0.3 mM to
about 0.6 mM. In some embodiments, an amount of taurine in cell culture media that results
in one or more advantageous property is from about 0.5 mM to 500 mM, from about 4.0 mM
to about 100 mM, or from about 1.0 mM to about 10 mM. In some embodiments, an amount
of one more media component selected from the group consisting of hypotaurine, s-
carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine in cell culture media
that results in one or more advantageous property is provided in Table 2.
A cell culture medium described herein, in one embodiment, results in one or more
favorable product quality attributes or advantageous property when used in a method of
producing a polypeptide in cell culture as compared to quality attributes of the polypeptide
when produced in a different medium. Reactive oxygen species (ROS) formed through the
use of certain media components may oxidize specific amino acids on the polypeptide and
produce oxidized polypeptide products. The presence of such oxidized protein species may
also alter the product quality attributes of a protein product, such as color intensity, which
may be particularly significant for polypeptide products that are formulated at any
concentration such as, but not limited to, a concentration of greater than any of about 1
mg/mL, about 10 mg/mL, about 25 mg/mL, about 50 mg/mL, or about 75 mg/mL up to 100
mg/mL. In some embodiments, the presence of oxidized protein species may alter the
product quality attributes of a protein product, such as color intensity, which may be
particularly significant for polypeptide products that are formulated at concentrations of
greater than any of about 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175
mg/mL, about 200 mg/mL or about 250 mg/mL. The color intensity of a composition
comprising a polypeptide produced with a media detailed herein (including a composition
comprising at least about 1 mg/mL, about 10 mg/mL, about 50 mg/mL, about 100 mg/mL,
about 150 mg/mL, 200 mg/mL or about 250 mg/mL of the polypeptide, such as an antibody)
can be assessed using a color assay such as one described herein or in, but not limited to, the
United States Pharmacopoeia color standard and the European Pharmacopoeia color standard.
See USP-24 Monograph 631 Color and Achromaticity. United States Pharmacopoeia Inc.,
2000, p. 1926-1927 and Council of Europe. European Pharmacopoeia, 2008, 7 Ed. P.22,
which are incorporated herein by reference in their entirety. In any of the embodiments
herein, a cell culture media described herein can be used for the preparation of compositions
comprising a polypeptide that have a reduced color intensity as compared to a reference
solution as measured by a color assay. For example, the color intensity of a composition
(e.g., pharmaceutical formulation) comprising a polypeptide (e.g., a therapeutic polypeptide)
produced using a cell culture medium as described herein can be reduced by any amount
including, but not limited to, at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% or more as
compared to a composition comprising the polypeptide produced using a cell culture medium
that does not comprise the one or more of components of Table 1 or Table 2.
Commercially available media such as, but not limited to, Ham's F10 (Sigma),
Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), Dulbecco's Modified
Eagle's Medium ([DMEM], Sigma), Luria broth (LB), and Terrific broth (TB) that are
suitable for culturing cells may be supplemented with any of the media components as
detailed herein (e.g., by use of a kit as described). In addition, any of the media described in
Ham and Wallace, Meth. Enz., 58:44 (1979), Barnes and Sato, Anal. Biochem., 102:255
(1980), Vijayasankaran et al., Biomacromolecules., 6:605:611 (2005), Patkar et al., J
Biotechnology, 93:217-229 (2002), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or
4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. No. Re. 30,985; or U.S. Pat. No.
,122,469, the disclosures of all of which are incorporated herein by reference in their
entirety, may be supplemented with any of the media components as detailed herein (e.g., by
use of a kit as described).
In some embodiments, a cell culture medium described herein comprises cystine
and is free of cysteine. In some embodiments, a cell culture medium described herein
comprises ferric citrate and is free of ferrous sulfate. In some embodiments herein, a cell
culture medium described is free from cysteine and ferrous sulfate. In some embodiments, the
medium is free from cysteine and ferrous sulfate and comprises cystine and/or ferric citrate.
In any of the embodiments herein, the cell culture media can be a basal medium or a feed
medium. Amino acids, vitamins, trace elements and other media components at one or two
times the ranges specified in European Patent EP 307,247 or U.S. Pat. No. 6,180,401 may be
used, which documents are herein incorporated by reference in their entireties.
Any media described herein may also be supplemented as necessary with hormones
and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), ions
(such as sodium, chloride, calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleosides (such as adenosine and thymidine), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar range), and glucose or
an equivalent energy source. In some embodiments, a cell culture medium described herein
contains proteins derived from a plant or an animal. In some embodiments, a cell culture
described herein is free of proteins derived from a plant or an animal. Any other necessary
supplements may also be included at appropriate concentrations that would be known to
those skilled in the art.
III. Methods and Uses of the Invention
Described herein are methods of culturing cells in a cell culture media described
herein for the production of polypeptides of interest. In some embodiments, a method is
provided for culturing a cell comprising a nucleic acid encoding a polypeptide of interest,
wherein the method comprises the step of contacting the cell with a cell culture medium,
wherein the cell culture medium comprises one or more of components selected from the
group consisting of hypotaurine, s-carboxymethylcysteine, carnosine, anserine, butylated
hydroxyanisole, lipoic acid, and quercitrin hydrate. In some embodiments, a method is
described for culturing a cell comprising a nucleic acid encoding a polypeptide of interest,
wherein the method comprises the step of contacting the cell with a cell culture medium,
wherein the cell culture medium comprises one or more of components selected from the
group consisting of (a) hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine,
(e) butylated hydroxyanisole, (f) lipoic acid; (g) quercitrin hydrate; and (h) aminoguanidine,
and wherein the cell culture medium comprising one or more of components (a)-(h) reduces
the color intensity of a composition comprising the polypeptide produced by the cell as
compared to a composition comprising the polypeptide produced by the cell cultured in a cell
culture medium that does not comprise the one or more of components (a)-(h). In some
embodiments, the color intensity of the composition comprising the polypeptide is reduced
by at least about 0.1%. In some embodiments, the color intensity of the composition
comprising the polypeptide is reduced by at least about 5%. In some embodiments, the color
intensity of the composition comprising the polypeptide is reduced by about 10% to about
%. In some embodiments, the color intensity of the composition comprising the
polypeptide is reduced by about 5% to about 75%. In some of the embodiments herein, the
cell culture medium comprises one or more components in an amount selected from (a)
hypotaurine at a concentration from about 2.0 mM to about 50.0 mM, (b) s-
carboxymethylcysteine at a concentration from about 8.0 mM to about 12.0 mM, (c)
carnosine at a concentration from about 8.0 mM to about 12.0 mM, (d) anserine at a
concentration from about 3.0 mM to about 5.0 mM, (e) butylated hydroxyanisole at a
concentration from about 0.025 mM to about 0.040 mM, (f) lipoic acid at a concentration
from about 0.040 mM to about 0.060 mM, (g) quercitrin hydrate at a concentration from
about 0.010 mM to about 0.020 mM, and (h) aminoguanidine at a concentration from about
0.0003 mM to about 20 mM. In some of the embodiments herein, the one or more
components selected from the group consisting of (a) hypotaurine, (b) s-
carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylated hydroxyanisole, (f) lipoic
acid; (g) quercitrin hydrate; and (h) aminoguanidine is added to the cell culture medium on
day 0 of a 14 day cell culture cycle.
In some other embodiments, a method is described for culturing a cell comprising a
nucleic acid encoding a polypeptide of interest, wherein the method comprises the step of
contacting the cell with a cell culture medium comprising the hypotaurine or an analog or
precursor thereof. In some embodiments, a method is described for culturing a cell
comprising a nucleic acid encoding a polypeptide of interest, wherein the method comprises
the step of contacting the cell with a cell culture medium comprising the hypotaurine or an
analog or precursor thereof, and wherein the cell culture medium comprising the hypotaurine
or an analog of precursor thereof reduces the color intensity of a composition comprising the
polypeptide produced by the cell as compared to the color intensity of a composition
comprising the polypeptide produced by the cell cultured in a cell culture medium that does
not comprise the hypotaurine or an analog or precursor thereof. In some embodiments, the
color intensity of the composition comprising the polypeptide is reduced by at least about
0.1%. In some embodiments, the color intensity of the composition comprising the
polypeptide is reduced by at least about 5%. In some embodiments, the color intensity of the
composition comprising the polypeptide is reduced by about 10% to about 30%. In some
embodiments herein, the cell culture medium comprises the hypotaurine or an analog or
precursor thereof, at a concentration from at least about 0.0001mM. In some embodiments
herein, the cell culture medium comprises the hypotaurine or an analog or precursor thereof,
at a concentration from about 0.5 mM to about 500 mM. In some embodiments, the cell
culture medium comprises the hypotaurine or an analog or precursor thereof, at a
concentration from about 1.0 mM to about 40 mM. In some embodiments herein, the
hypotaurine or an analog or precursor thereof is selected from the group consisting of
hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine. In
some of the embodiments herein, the hypotaurine or an analog or precursor thereof is added
to the cell culture medium on day 0 of a 14 day cell culture cycle. In some embodiments, the
hypotaurine or an analog or precursor thereof is not added to the cell culture medium
incrementally over the course of a cell culture cycle.
Also described herein are methods of producing a polypeptide of interest
comprising the step of culturing a cell comprising a nucleic acid encoding the polypeptide in
a cell culture medium, wherein the cell culture medium comprises one or more of
components selected from the group consisting of (a) hypotaurine, (b) s-
carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylated hydroxyanisole, (f) lipoic
acid, (g) quercitrin hydrate, and (h) aminoguanidine. In some embodiments, described herein
are methods of producing a polypeptide of interest comprising the step of culturing a cell
comprising a nucleic acid encoding the polypeptide in a cell culture medium, wherein the cell
culture medium comprises one or more of components selected from the group consisting of
(a) hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylated
hydroxyanisole, (f) lipoic acid, (g) quercitrin hydrate, and (h) aminoguanidine, and wherein
the cell culture medium comprising one or more of components (a)-(h) reduces the color
intensity of a composition comprising the polypeptide produced by the cell as compared to a
composition comprising the polypeptide produced by the cell cultured in a cell culture
medium that does not comprise one or more of components (a)-(h). In some embodiments,
the color intensity of the composition comprising the polypeptide is reduced by at least about
0.1%. In some embodiments, the color intensity of the composition comprising the
polypeptide is reduced by at least about 5%. In some embodiments, the color intensity of the
composition comprising the polypeptide is reduced by about 10% to about 30%. In some
embodiments, the color intensity of the composition comprising the polypeptide is reduced
by about 5% to about 75%. In some of the embodiments herein, the cell culture medium
comprises one or more components in an amount selected from (a) hypotaurine at a
concentration from about 2.0 mM to about 50.0 mM, (b) s-carboxymethylcysteine at a
concentration from about 8.0 mM to about 12.0 mM, (c) carnosine at a concentration from
about 8.0 mM to about 12.0 mM, (d) anserine at a concentration from about 3.0 mM to about
.0 mM, (e) butylated hydroxyanisole at a concentration from about 0.025 mM to about 0.040
mM, (f) lipoic acid at a concentration from about 0.040 mM to about 0.060 mM, (g)
quercitrin hydrate at a concentration from about 0.010 mM to about 0.020 mM, and (h)
aminoguanidine at a concentration from about 0.0003 mM to about 20 mM. In some of the
embodiments herein, the one or more components selected from the group consisting of (a)
hypotaurine, (b) s-carboxymethylcysteine, (c) carnosine, (d) anserine, (e) butylated
hydroxyanisole, (f) lipoic acid; (g) quercitrin hydrate; and (h) aminoguanidine is added to the
cell culture medium on day 0 of a 14 day cell culture cycle.
In another embodiment, described herein are methods of producing a polypeptide of
interest comprising the step of culturing a cell comprising a nucleic acid encoding the
polypeptide in a cell culture medium. In some embodiments, described herein are methods of
producing a polypeptide of interest comprising the step of culturing a cell comprising a
nucleic acid encoding the polypeptide in a cell culture medium, wherein the cell culture
medium comprises hypotaurine or an analog or precursor thereof, and wherein the cell culture
medium comprising the hypotaurine or an analog of precursor thereof, reduces the color
intensity of a composition comprising the polypeptide produced by the cell as compared to
the color intensity of a composition comprising the polypeptide produced by the cell cultured
in a cell culture medium that does not comprise the hypotaurine or an analog or precursor
thereof. In some embodiments, the color intensity of the composition comprising the
polypeptide is reduced by at least about 0.1%. In some embodiments, the color intensity of
the composition comprising the polypeptide is reduced by at least about 5%. In some
embodiments, the color intensity of the composition comprising the polypeptide is reduced
by about 10% to about 30%. In some embodiments herein, the cell culture medium
comprises the hypotaurine or an analog or precursor thereof, at a concentration from at least
about 0.0001mM. In some embodiments herein, the cell culture medium comprises the
hypotaurine or an analog or precursor thereof, at a concentration from about 0.5 mM to about
500 mM. In some embodiments, the cell culture medium comprises the hypotaurine or an
analog or precursor thereof, at a concentration from about 1.0 mM to about 40 mM. In some
embodiments herein, the hypotaurine or an analog or precursor thereof is selected from the
group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine, cysteinesulphinic
acid, and taurine. In some of the embodiments herein, the hypotaurine or an analog or
precursor thereof is added to the cell culture medium on day 0 of a 14 day cell culture cycle.
In some embodiments, the hypotaurine or an analog or precursor thereof is not added to the
cell culture medium incrementally over the course of a cell culture cycle.
In any of the embodiments herein, the cell culture medium used in the methods
described herein can be a chemically defined cell culture medium of a chemically undefined
cell culture medium. The cell culture medium described herein can be used a basal cell
culture medium or as a feed cell medium. In some embodiments, a cell culture medium
described herein is used in a method for culturing the cell during the cell’s growth phase. In
some embodiments, a cell culture medium described herein is used in a method for culturing
the cell during the cell’s production phase. In any of the methods herein the cell may be a
mammalian cell such as a CHO cell. In some embodiments, the polypeptide of interest is an
antibody or fragment thereof.
In further embodiments herein the polypeptide of interest is recovered. A
composition comprising the recovered polypeptide can be subjected to at least one
purification step before assessment of color intensity using a quantitative or qualitative color
assay as described herein. In some embodiments, the composition comprising the recovered
polypeptide is a liquid composition or a non-liquid composition. In some embodiments, the
liquid composition or non-liquid composition comprising a recovered polypeptide can be
assessed for color intensity using a color assay as described herein or known in the art. For
example, a non-liquid composition comprising the recovered polypeptide can be a lyophilized
composition that is subsequently reconstituted before measurement of color intensity. In
some embodiments herein, the color intensity of a composition comprising the polypeptide
produced by the cell cultured in a cell culture medium described herein is reduced by at least
0.1% as compared to the color intensity of a composition comprising the polypeptide
produced by the cell cultured in a cell culture medium that does not comprise a media
component as described herein (e.g., hypotaurine or an analog or precursor thereof). In some
embodiments, the color intensity is reduced by at least about 0.1%, by at least about 0.2%, by
at least about 0.3%, by at least about 0.4%, by at least about 0.5%, by at least about 0.6%, by
at least about 0.7%, by at least about 0.8%, by at least about 0.8%, or by at least about 0.9%
to about 1.0%. In some embodiments, the color intensity is reduced by at least about 1%, by
at least about 2%, by at least about 3%, by at least about 4%, by at least about 5%, by at least
about 10%, by at least about 15%, by at least about 20%, by at least about 25%, by at least
about 30%, by at least about 35%, by at least about 40%, by at least about 45%, by at least
about 50%, by at least about 60%, by at least about 70%, by at least about 80%, or by at least
about 90% to about 100%. In some embodiments, the color intensity is reduced by about
0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about
0.8%, about 0.9% to about 1.0%. In some embodiments, the color intensity is reduced by
about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about
9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,
about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about
24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%,
about 32%, about 33%, about 34%, about 35%, about 45%, about 50%, about 60%, about
70%, about 80%, about 90% to about 100%. In some embodiments, the color intensity is
reduced by from about 1% to about 10%, from about 5% to about 15%, from about 5% to
about 20%, from about 5% to about 25%, from about 5% to about 30%, from about 5% to
about 35%, from about 5% to about 40%, from about 5% to about 45%, from about 5% to
about 50%, from about 10% to about 20%, or from about 15% to about 25%. In some
embodiments, a composition comprising a recovered polypeptide appears as a colorless or
slightly colored liquid or composition. A liquid or composition can be determined to be
colorless or slightly colored using a color assay as described herein or a color assay known to
one of skill in the art. In a further embodiment, the composition is a pharmaceutical
composition that optionally further comprises a pharmaceutically acceptable carrier as
described herein.
Methods of administering a polypeptide as detailed herein are also described. For
example, a method is described for administering to an individual a formulation comprising a
polypeptide, wherein the formulation has the polypeptide at a concentration greater than at
least about 100 mg/mL, at least about 125 mg/mL, or at least about 150 mg/mL and has a
color intensity value greater than B3, B4, B5, B6, B7, B8, or B9 as measured by the COC
assay. In some embodiments, the color intensity value as determined by the COC assay can
be any one of, but not limited to, B, BY, Y, GY, or R, wherein higher values indicate a lighter
color intensity. Formulations comprising a polypeptide of interest may be suitable for
injection, such as subcutaneous injection into an individual (e.g., subcutaneous injection into
a human). In some embodiments, a formulation comprising a polypeptide of interest suitable
for injection (e.g., suitable for subcutaneous injection) is at a concentration greater than at
least 100 mg/mL, at least 125 mg/mL, or at least 150 mg/mL and has a color intensity value
greater than B3, B4, B5, B6, B7, B8, or B9 as measured by the COC assay. In some
embodiments, the color intensity value as determined by the COC assay can be any one of,
but not limited to, B, BY, Y, GY, or R, wherein higher values indicate a lighter color
intensity.
Other methods are described throughout, such as in the Brief Summary of the
Invention and elsewhere.
Polypeptide Production
The cell culture media detailed herein can be used in a method of culturing cells to
produce polypeptides, including particular antibodies. The medium may be used in a method
of culturing cells, whether by batch culture, fed batch culture or perfusion culture, and can be
used in a method of producing any polypeptide including any embodiments or embodiments
of the polypeptide as described herein. The polypeptides produced by the compositions (e.g.,
a cell cultured in a cell culturing medium described herein) and methods detailed herein and
present in the compositions (e.g., cell culture media comprising the produced polypeptide)
described herein may be homologous to the host cell, or preferably, may be exogenous,
meaning that they are heterologous, i.e., foreign, to the host cell being utilized, such as a
human protein produced by a Chinese hamster ovary cell, or a yeast polypeptide produced by
a mammalian cell. In one variation, the polypeptide is a mammalian polypeptide (such as an
antibody) directly secreted into the medium by the host cell. In another variation, the
polypeptide is released into the medium by lysis of a cell comprising a nucleic acid encoding
the polypeptide.
Any polypeptide that is expressible in a host cell may be produced in accordance
with the present disclosure and may be present in the compositions described. The
polypeptide may be expressed from a gene that is endogenous to the host cell, or from a gene
that is introduced into the host cell through genetic engineering. The polypeptide may be one
that occurs in nature, or may alternatively have a sequence that was engineered or selected by
the hand of man. An engineered polypeptide may be assembled from other polypeptide
segments that individually occur in nature, or may include one or more segments that are not
naturally occurring.
Polypeptides that may desirably be expressed in accordance with the present
description will often be selected on the basis of an interesting biological or chemical
activity. For example, the present description may be employed to express any
pharmaceutically or commercially relevant enzyme, receptor, antibody, hormone, regulatory
factor, antigen, binding agent, etc.
Methods for producing polypeptides, such as antibodies, in cell culture are well
known in the art. Described herein are non-limiting exemplary methods for producing an
antibody (e.g., full length antibodies, antibody fragments and multispecific antibodies) in cell
culture. The methods herein can be adapted by one of skill in the art for the production of
other proteins, such as protein-based inhibitors. See Molecular Cloning: A Laboratory
Manual (Sambrook et al., 4 ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 2012); Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., 2003); Short
Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Current
Protocols in Protein Science, (Horswill et al., 2006); Antibodies, A Laboratory Manual
(Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and
Specialized Applications (R.I. Freshney, 6 ed., J. Wiley and Sons, 2010) for generally well
understood and commonly employed techniques and procedures for the production of
proteins (e.g., therapeutic proteins), which are all incorporated herein by reference in their
entirety.
(A) Antibody Preparation
The antibody produced in cell culture using a cell culture medium described herein
is directed against an antigen of interest. Preferably, the antigen is a biologically important
polypeptide and administration of compositions comprising the antibody to a mammal
suffering from a disorder can result in a therapeutic benefit in that mammal.
(i) Antigen Preparation
Soluble antigens or fragments thereof, optionally conjugated to other molecules, can
be used as immunogens for generating antibodies. For transmembrane molecules, such as
receptors, fragments of these (e.g. the extracellular domain of a receptor) can be used as the
immunogen. Alternatively, cells expressing the transmembrane molecule can be used as the
immunogen. Such cells can be derived from a natural source (e.g. cancer cell lines) or may be
cells which have been transformed by recombinant techniques to express the transmembrane
molecule. Other antigens and forms thereof useful for preparing antibodies will be apparent
to those in the art.
(ii) Certain Antibody-Based Methods
Monoclonal antibodies of interest can be made using the hybridoma method first
described by Kohler et al., Nature, 256:495 (1975), and further described, e.g., in Hongo et
al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), and Ni, Xiandai
Mianyixue, 26(4):265-268 (2006) regarding human-human hybridomas. Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826 regarding production of
monoclonal human natural IgM antibodies from hybridoma cell lines. Human hybridoma
technology (Trioma technology) is described in Vollmers and Brandlein, Histology and
Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in
Experimental and Clinical Pharmacology, 27(3):185-91 (2005).
For various other hybridoma techniques, see, e.g., US 2006/258841; US
2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US
2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507. An exemplary
protocol for producing monoclonal antibodies using the hybridoma method is described as
follows. In one embodiment, a mouse or other appropriate host animal, such as a hamster, is
immunized to elicit lymphocytes that produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization. Antibodies are raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of interest or a
fragment thereof, and an adjuvant, such as monophosphoryl lipid A (MPL)/trehalose
dicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.). Serum from
immunized animals is assayed for anti-antigen antibodies, and booster immunizations are
optionally administered. Lymphocytes from animals producing anti-antigen antibodies are
isolated. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such
as polyethylene glycol, to form a hybridoma cell. See, e.g., Goding, Monoclonal Antibodies:
Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma cells may be used that
fuse efficiently, support stable high-level production of antibody by the selected antibody-
producing cells, and are sensitive to a medium such as HAT medium. Exemplary myeloma
cells include, but are not limited to, murine myeloma lines, such as those derived from
MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution
Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American
Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human
heteromyeloma cell lines also have been described for the production of human monoclonal
antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture
medium, e.g., a medium that contains one or more substances that inhibit the growth or
survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the
culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and
thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Preferably, serum-free hybridoma cell culture methods are used to reduce use of animal-
derived serum such as fetal bovine serum, as described, for example, in Even et al., Trends in
Biotechnology, 24(3), 105-108 (2006).
Oligopeptides as tools for improving productivity of hybridoma cell cultures are
described in Franek, Trends in Monoclonal Antibody Research, 111-122 (2005). Specifically,
standard culture media are enriched with certain amino acids (alanine, serine, asparagine,
proline), or with protein hydrolyzate fractions, and apoptosis may be significantly suppressed
by synthetic oligopeptides, constituted of three to six amino acid residues. The peptides are
present at millimolar or higher concentrations.
Culture medium in which hybridoma cells are growing may be assayed for
production of monoclonal antibodies. The binding specificity of monoclonal antibodies
produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro
binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay
(ELISA). The binding affinity of the monoclonal antibody can be determined, for example,
by Scatchard analysis. See, e.g., Munson et al., Anal. Biochem., 107:220 (1980).
After hybridoma cells are identified that produce antibodies of the desired
specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution
procedures and grown by standard methods. See, e.g., Goding, supra. Suitable culture media
for this purpose include, for example, D-MEM or RPMI-1640 medium. In some
embodiments, the hybridoma cells are cultured in a cell culture medium described herein. In
some embodiments, the hybridoma cells are cultured in a cell culture medium comprising one
or more media components selected from the group consisting of hypotaurine, s-
carboxymethylcysteine, anserine, butylated hydroxyanisole, carnosine, lipoic acid, and
quercitrin hydrate. In some embodiments, the one or more media component is hypotaurine
or an analog or precursor thereof. In some embodiments, the hypotaurine or an analog or
precursor thereof is selected from the group consisting of hypotaurine, s-
carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine.
Antibodies may be produced using recombinant methods. For recombinant
production of an anti-antigen antibody, nucleic acid encoding the antibody is isolated and
inserted into a replicable vector for further cloning (amplification of the DNA) or for
expression. DNA encoding the antibody may be readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the antibody). Many vectors are
available. The vector components generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, one or more marker genes, an enhancer
element, a promoter, and a transcription termination sequence.
(iii) Certain Library Screening Methods
Antibodies can be made by using combinatorial libraries to screen for antibodies
with the desired activity or activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries for antibodies possessing
the desired binding characteristics. Such methods are described generally in Hoogenboom et
al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa,
N.J., 2001). For example, one method of generating antibodies of interest is through the use
of a phage antibody library as described in Lee et al., J. Mol. Biol. (2004), 340(5):1073-93.
In principle, synthetic antibody clones are selected by screening phage libraries
containing phage that display various fragments of antibody variable region (Fv) fused to
phage coat protein. Such phage libraries are panned by affinity chromatography against the
desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are
adsorbed to the antigen and thus separated from the non-binding clones in the library. The
binding clones are then eluted from the antigen, and can be further enriched by additional
cycles of antigen adsorption/elution. Any of the antibodies of interest can be obtained by
designing a suitable antigen screening procedure to select for the phage clone of interest
followed by construction of a full length antibody clone using the Fv sequences from the
phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al.,
Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242,
Bethesda Md. (1991), vols. 1-3.
In certain embodiments, the antigen-binding domain of an antibody is formed from
two variable (V) regions of about 110 amino acids, one each from the light (VL) and heavy
(VH) chains, that both present three hypervariable loops (HVRs) or complementarity-
determining regions (CDRs). Variable domains can be displayed functionally on phage,
either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked
through a short, flexible peptide, or as Fab fragments, in which they are each fused to a
constant domain and interact non-covalently, as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). As used herein, scFv encoding phage clones and Fab
encoding phage clones are collectively referred to as “Fv phage clones” or “Fv clones.”
Repertoires of VH and VL genes can be separately cloned by polymerase chain
reaction (PCR) and recombined randomly in phage libraries, which can then be searched for
antigen-binding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
Libraries from immunized sources provide high-affinity antibodies to the immunogen without
the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned
to provide a single source of human antibodies to a wide range of non-self and also self
antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V-
gene segments from stem cells, and using PCR primers containing random sequence to
encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as
described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
In certain embodiments, filamentous phage is used to display antibody fragments by
fusion to the minor coat protein pIII. The antibody fragments can be displayed as single chain
Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by
a flexible polypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol., 222: 581-597
(1991), or as Fab fragments, in which one chain is fused to pIII and the other is secreted into
the bacterial host cell periplasm where assembly of a Fab-coat protein structure which
becomes displayed on the phage surface by displacing some of the wild type coat proteins,
e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).
In general, nucleic acids encoding antibody gene fragments are obtained from
immune cells harvested from humans or animals. If a library biased in favor of anti-antigen
clones is desired, the subject is immunized with antigen to generate an antibody response, and
spleen cells and/or circulating B cells other peripheral blood lymphocytes (PBLs) are
recovered for library construction. In one embodiment, a human antibody gene fragment
library biased in favor of anti-antigen clones is obtained by generating an anti-antigen
antibody response in transgenic mice carrying a functional human immunoglobulin gene
array (and lacking a functional endogenous antibody production system) such that antigen
immunization gives rise to B cells producing human antibodies against antigen. The
generation of human antibody-producing transgenic mice is described below.
Additional enrichment for anti-antigen reactive cell populations can be obtained by
using a suitable screening procedure to isolate B cells expressing antigen-specific membrane
bound antibody, e.g., by cell separation using antigen affinity chromatography or adsorption
of cells to fluorochrome-labeled antigen followed by flow-activated cell sorting (FACS).
Alternatively, the use of spleen cells and/or B cells or other PBLs from an
unimmunized donor provides a better representation of the possible antibody repertoire, and
also permits the construction of an antibody library using any animal (human or non-human)
species in which antigen is not antigenic. For libraries incorporating in vitro antibody gene
construction, stem cells are harvested from the subject to provide nucleic acids encoding
unrearranged antibody gene segments. The immune cells of interest can be obtained from a
variety of animal species, such as human, mouse, rat, lagomorpha, luprine, canine, feline,
porcine, bovine, equine, and avian species, etc.
Nucleic acid encoding antibody variable gene segments (including VH and VL
segments) are recovered from the cells of interest and amplified. In the case of rearranged VH
and VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or
mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers
matching the 5’ and 3’ ends of rearranged VH and VL genes as described in Orlandi et al.,
Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse V gene
repertoires for expression. The V genes can be amplified from cDNA and genomic DNA,
with back primers at the 5’ end of the exon encoding the mature V-domain and forward
primers based within the J-segment as described in Orlandi et al. (1989) and in Ward et al.,
Nature, 341: 544-546 (1989). However, for amplifying from cDNA, back primers can also be
based in the leader exon as described in Jones et al., Biotechnol., 9: 88-89 (1991), and
forward primers within the constant region as described in Sastry et al., Proc. Natl. Acad. Sci.
(USA), 86: 5728-5732 (1989). To maximize complementarity, degeneracy can be
incorporated in the primers as described in Orlandi et al. (1989) or Sastry et al. (1989). In
certain embodiments, library diversity is maximized by using PCR primers targeted to each
V-gene family in order to amplify all available VH and VL arrangements present in the
immune cell nucleic acid sample, e.g. as described in the method of Marks et al., J. Mol.
Biol., 222: 581-597 (1991) or as described in the method of Orum et al., Nucleic Acids Res.,
21: 4491-4498 (1993). For cloning of the amplified DNA into expression vectors, rare
restriction sites can be introduced within the PCR primer as a tag at one end as described in
Orlandi et al. (1989), or by further PCR amplification with a tagged primer as described in
Clackson et al., Nature, 352: 624-628 (1991).
Repertoires of synthetically rearranged V genes can be derived in vitro from V gene
segments. Most of the human VH-gene segments have been cloned and sequenced (reported
in Tomlinson et al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in Matsuda et
al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all the major
conformations of the H1 and H2 loop) can be used to generate diverse VH gene repertoires
with PCR primers encoding H3 loops of diverse sequence and length as described in
Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). VH repertoires can also be
made with all the sequence diversity focused in a long H3 loop of a single length as described
in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Human V κ and V λ
segments have been cloned and sequenced (reported in Williams and Winter, Eur. J.
Immunol., 23: 1456-1461 (1993)) and can be used to make synthetic light chain repertoires.
Synthetic V gene repertoires, based on a range of VH and VL folds, and L3 and H3 lengths,
will encode antibodies of considerable structural diversity. Following amplification of V-
gene encoding DNAs, germline V-gene segments can be rearranged in vitro according to the
methods of Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Repertoires of antibody fragments can be constructed by combining VH and VL
gene repertoires together in several ways. Each repertoire can be created in different vectors,
and the vectors recombined in vitro, e.g., as described in Hogrefe et al., Gene, 128: 119-126
(1993), or in vivo by combinatorial infection, e.g., the loxP system described in Waterhouse
et al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivo recombination approach exploits
the two-chain nature of Fab fragments to overcome the limit on library size imposed by E.
coli transformation efficiency. Naive VH and VL repertoires are cloned separately, one into a
phagemid and the other into a phage vector. The two libraries are then combined by phage
infection of phagemid-containing bacteria so that each cell contains a different combination
and the library size is limited only by the number of cells present (about 10 clones). Both
vectors contain in vivo recombination signals so that the VH and VL genes are recombined
onto a single replicon and are co-packaged into phage virions. These huge libraries provide
-1 -8
large numbers of diverse antibodies of good affinity (K of about 10 M).
Alternatively, the repertoires may be cloned sequentially into the same vector, e.g.
as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled
together by PCR and then cloned, e.g. as described in Clackson et al., Nature, 352: 624-628
(1991). PCR assembly can also be used to join VH and VL DNAs with DNA encoding a
flexible peptide spacer to form single chain Fv (scFv) repertoires. In yet another technique,
“in cell PCR assembly” is used to combine VH and VL genes within lymphocytes by PCR
and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res.,
: 3831-3837 (1992).
The antibodies produced by naive libraries (either natural or synthetic) can be of
-1 6 7 -1
moderate affinity (K of about 10 to 10 M ), but affinity maturation can also be mimicked
in vitro by constructing and reselecting from secondary libraries as described in Winter et al.
(1994), supra. For example, mutation can be introduced at random in vitro by using error-
prone polymerase (reported in Leung et al., Technique 1: 11-15 (1989)) in the method of
Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc.
Natl. Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturation can be
performed by randomly mutating one or more CDRs, e.g. using PCR with primers carrying
random sequence spanning the CDR of interest, in selected individual Fv clones and
screening for higher affinity clones. WO 9607754 (published 14 Mar. 1996) described a
method for inducing mutagenesis in a complementarity determining region of an
immunoglobulin light chain to create a library of light chain genes. Another effective
approach is to recombine the VH or VL domains selected by phage display with repertoires
of naturally occurring V domain variants obtained from unimmunized donors and screen for
higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol.,
: 779-783 (1992). This technique allows the production of antibodies and antibody
fragments with affinities of about 10 M or less.
Screening of the libraries can be accomplished by various techniques known in the
art. For example, antigen can be used to coat the wells of adsorption plates, expressed on host
cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture
with streptavidin-coated beads, or used in any other method for panning phage display
libraries.
The phage library samples are contacted with immobilized antigen under conditions
suitable for binding at least a portion of the phage particles with the adsorbent. Normally, the
conditions, including pH, ionic strength, temperature and the like are selected to mimic
physiological conditions. The phages bound to the solid phase are washed and then eluted by
acid, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-7982 (1991), or
by alkali, e.g. as described in Marks et al., J. Mol. Biol., 222: 581-597 (1991), or by antigen
competition, e.g. in a procedure similar to the antigen competition method of Clackson et al.,
Nature, 352: 624-628 (1991). Phages can be enriched 20-1,000-fold in a single round of
selection. Moreover, the enriched phages can be grown in bacterial culture and subjected to
further rounds of selection.
The efficiency of selection depends on many factors, including the kinetics of
dissociation during washing, and whether multiple antibody fragments on a single phage can
simultaneously engage with antigen. Antibodies with fast dissociation kinetics (and weak
binding affinities) can be retained by use of short washes, multivalent phage display and high
coating density of antigen in solid phase. The high density not only stabilizes the phage
through multivalent interactions, but favors rebinding of phage that has dissociated. The
selection of antibodies with slow dissociation kinetics (and good binding affinities) can be
promoted by use of long washes and monovalent phage display as described in Bass et al.,
Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating density of antigen as
described in Marks et al., Biotechnol., 10: 779-783 (1992).
It is possible to select between phage antibodies of different affinities, even with
affinities that differ slightly, for antigen. However, random mutation of a selected antibody
(e.g. as performed in some affinity maturation techniques) is likely to give rise to many
mutants, most binding to antigen, and a few with higher affinity. With limiting antigen, rare
high affinity phage could be competed out. To retain all higher affinity mutants, phages can
be incubated with excess biotinylated antigen, but with the biotinylated antigen at a
concentration of lower molarity than the target molar affinity constant for antigen. The high
affinity-binding phages can then be captured by streptavidin-coated paramagnetic beads.
Such “equilibrium capture” allows the antibodies to be selected according to their affinities of
binding, with sensitivity that permits isolation of mutant clones with as little as two-fold
higher affinity from a great excess of phages with lower affinity. Conditions used in washing
phages bound to a solid phase can also be manipulated to discriminate on the basis of
dissociation kinetics.
Anti-antigen clones may be selected based on activity. In certain embodiments,
described herein are anti-antigen antibodies that bind to living cells that naturally express
antigen or bind to free floating antigen or antigen attached to other cellular structures. Fv
clones corresponding to such anti-antigen antibodies can be selected by (1) isolating anti-
antigen clones from a phage library as described above, and optionally amplifying the
isolated population of phage clones by growing up the population in a suitable bacterial host;
(2) selecting antigen and a second protein against which blocking and non-blocking activity,
respectively, is desired; (3) adsorbing the anti-antigen phage clones to immobilized antigen;
(4) using an excess of the second protein to elute any undesired clones that recognize antigen-
binding determinants which overlap or are shared with the binding determinants of the
second protein; and (5) eluting the clones which remain adsorbed following step (4).
Optionally, clones with the desired blocking/non-blocking properties can be further enriched
by repeating the selection procedures described herein one or more times.
DNA encoding hybridoma-derived monoclonal antibodies or phage display Fv
clones of interest is readily isolated and sequenced using conventional procedures (e.g. by
using oligonucleotide primers designed to specifically amplify the heavy and light chain
coding regions of interest from hybridoma or phage DNA template). Once isolated, the DNA
can be placed into expression vectors, which are then transfected into host cells such as E.
coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired
monoclonal antibodies in the recombinant host cells. Review articles on recombinant
expression in bacteria of antibody-encoding DNA include Skerra et al., Curr. Opinion in
Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151 (1992).
DNA encoding the Fv clones can be combined with known DNA sequences
encoding heavy chain and/or light chain constant regions (e.g. the appropriate DNA
sequences can be obtained from Kabat et al., supra) to form clones encoding full or partial
length heavy and/or light chains. It will be appreciated that constant regions of any isotype
can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and
that such constant regions can be obtained from any human or animal species. An Fv clone
derived from the variable domain DNA of one animal (such as human) species and then fused
to constant region DNA of another animal species to form coding sequence(s) for “hybrid,”
full length heavy chain and/or light chain is included in the definition of “chimeric” and
“hybrid” antibody as used herein. In certain embodiments, an Fv clone derived from human
variable DNA is fused to human constant region DNA to form coding sequence(s) for full- or
partial-length human heavy and/or light chains.
DNA encoding anti-antigen antibody derived from a hybridoma can also be
modified, for example, by substituting the coding sequence for human heavy- and light-chain
constant domains in place of homologous murine sequences derived from the hybridoma
clone (e.g. as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855
(1984)). DNA encoding a hybridoma- or Fv clone-derived antibody or fragment can be
further modified by covalently joining to the immunoglobulin coding sequence all or part of
the coding sequence for a non-immunoglobulin polypeptide. In this manner, “chimeric” or
“hybrid” antibodies are prepared that have the binding specificity of the Fv clone or
hybridoma clone-derived antibodies of interest.
(iv) Humanized and Human Antibodies
Various methods for humanizing non-human antibodies are known in the art. For
example, a humanized antibody has one or more amino acid residues introduced into it from a
source which is non-human. These non-human amino acid residues are often referred to as
“import” residues, which are typically taken from an “import” variable domain.
Humanization can be essentially performed following the method of Winter and co-workers
(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody. Accordingly, such
“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein
substantially less than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice, humanized antibodies are
typically human antibodies in which some CDR residues and possibly some FR residues are
substituted by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in making
the humanized antibodies is very important to reduce antigenicity. According to the so-called
“best-fit” method, the sequence of the variable domain of a rodent antibody is screened
against the entire library of known human variable-domain sequences. The human sequence
which is closest to that of the rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol.,
196:901 (1987)). Another method uses a particular framework derived from the consensus
sequence of all human antibodies of a particular subgroup of light or heavy chains. The same
framework may be used for several different humanized antibodies (Carter et al., Proc. Natl.
Acad Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).
It is further important that antibodies be humanized with retention of high affinity
for the antigen and other favorable biological properties. To achieve this goal, according to
one embodiment of the method, humanized antibodies are prepared by a process of analysis
of the parental sequences and various conceptual humanized products using three-
dimensional models of the parental and humanized sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin sequences. Inspection of
these displays permits analysis of the likely role of the residues in the functioning of the
candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of
the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected
and combined from the recipient and import sequences so that the desired antibody
characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially involved in influencing
antigen binding.
Human antibodies of interest can be constructed by combining Fv clone variable
domain sequence(s) selected from human-derived phage display libraries with known human
constant domain sequence(s) as described above. Alternatively, human monoclonal
antibodies of interest can be made by the hybridoma method. Human myeloma and mouse-
human heteromyeloma cell lines for the production of human monoclonal antibodies have
been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).
It is possible to produce transgenic animals (e.g., mice) that are capable, upon
immunization, of producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been described that the
homozygous deletion of the antibody heavy-chain joining region (J ) gene in chimeric and
germ-line mutant mice results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice
will result in the production of human antibodies upon antigen challenge. See, e.g.,
Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,
362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and Duchosal et al.
Nature 355:258 (1992).
Gene shuffling can also be used to derive human antibodies from non-human, e.g.
rodent, antibodies, where the human antibody has similar affinities and specificities to the
starting non-human antibody. According to this method, which is also called “epitope
imprinting”, either the heavy or light chain variable region of a non-human antibody fragment
obtained by phage display techniques as described herein is replaced with a repertoire of
human V domain genes, creating a population of non-human chain/human chain scFv or Fab
chimeras. Selection with antigen results in isolation of a non-human chain/human chain
chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed
upon removal of the corresponding non-human chain in the primary phage display clone, i.e.
the epitope governs (imprints) the choice of the human chain partner. When the process is
repeated in order to replace the remaining non-human chain, a human antibody is obtained
(see PCT WO 93/06213 published Apr. 1, 1993). Unlike traditional humanization of non-
human antibodies by CDR grafting, this technique provides completely human antibodies,
which have no FR or CDR residues of non-human origin.
(v) Antibody Fragments
Antibody fragments may be generated by traditional means, such as enzymatic
digestion, or by recombinant techniques. In certain circumstances there are advantages of
using antibody fragments, rather than whole antibodies. The smaller size of the fragments
allows for rapid clearance, and may lead to improved access to solid tumors. For a review of
certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.
Various techniques have been developed for the production of antibody fragments.
Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see,
e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992);
and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced
directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed
in and secreted from E. coli, thus allowing the facile production of large amounts of these
fragments. Antibody fragments can be isolated from the antibody phage libraries discussed
above. Alternatively, Fab’-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab’) fragments (Carter et al., Bio/Technology 10:163-167
(1992)). According to another approach, F(ab’) fragments can be isolated directly from
recombinant host cell culture. Fab and F(ab’) fragment with increased in vivo half-life
comprising salvage receptor binding epitope residues are described in U.S. Pat. No.
,869,046. Other techniques for the production of antibody fragments will be apparent to the
skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv).
See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species
with intact combining sites that are devoid of constant regions; thus, they may be suitable for
reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to
yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See
Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear
antibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibodies
may be monospecific or bispecific.
(vi) Multispecific Antibodies
Multispecific antibodies have binding specificities for at least two different
epitopes, where the epitopes are usually from different antigens. While such molecules
normally will only bind two different epitopes (i.e. bispecific antibodies, BsAbs), antibodies
with additional specificities such as trispecific antibodies are encompassed by this expression
when used herein. Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab’) bispecific antibodies).
Methods for making bispecific antibodies are known in the art. Traditional
production of full length bispecific antibodies is based on the coexpression of two
immunoglobulin heavy chain-light chain pairs, where the two chains have different
specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment
of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a
potential mixture of 10 different antibody molecules, of which only one has the correct
bispecific structure. Purification of the correct molecule, which is usually done by affinity
chromatography steps, is rather cumbersome, and the product yields are low. Similar
procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
According to a different approach, antibody variable domains with the desired
binding specificities (antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain
constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is typical to
have the first heavy-chain constant region (CH1) containing the site necessary for light chain
binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy
chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate
expression vectors, and are co-transfected into a suitable host organism. This provides for
great flexibility in adjusting the mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the coding sequences for two or
all three polypeptide chains in one expression vector when the expression of at least two
polypeptide chains in equal ratios results in high yields or when the ratios are of no particular
significance.
In one embodiment of this approach, the bispecific antibodies are composed of a
hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid
immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the
other arm. It was found that this asymmetric structure facilitates the separation of the desired
bispecific compound from unwanted immunoglobulin chain combinations, as the presence of
an immunoglobulin light chain in only one half of the bispecific molecule provides for a
facile way of separation. This approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
According to another approach described in WO96/27011, the interface between a
pair of antibody molecules can be engineered to maximize the percentage of heterodimers
which are recovered from recombinant cell culture. One interface comprises at least a part of
the C 3 domain of an antibody constant domain. In this method, one or more small amino
acid side chains from the interface of the first antibody molecule are replaced with larger side
chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to
the large side chain(s) are created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This
provides a mechanism for increasing the yield of the heterodimer over other unwanted end-
products such as homodimers.
Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For
example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to
biotin. Such antibodies have, for example, been proposed to target immune system cells to
unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360,
WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any
convenient cross-linking methods. Suitable cross-linking agents are well known in the art,
and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking
techniques.
Techniques for generating bispecific antibodies from antibody fragments have also
been described in the literature. For example, bispecific antibodies can be prepared using
chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact
antibodies are proteolytically cleaved to generate F(ab’) fragments. These fragments are
reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal
dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are
then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’-TNB derivatives is
then reconverted to the Fab’-thiol by reduction with mercaptoethylamine and is mixed with
an equimolar amount of the other Fab’-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the selective immobilization of
enzymes.
Recent progress has facilitated the direct recovery of Fab’-SH fragments from E.
coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp.
Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody
F(ab’) molecule. Each Fab’ fragment was separately secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific antibody.
Various techniques for making and isolating bispecific antibody fragments directly
from recombinant cell culture have also been described. For example, bispecific antibodies
have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553
(1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’
portions of two different antibodies by gene fusion. The antibody homodimers were reduced
at the hinge region to form monomers and then re-oxidized to form the antibody
heterodimers. This method can also be utilized for the production of antibody homodimers.
The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-
6448 (1993) has provided an alternative mechanism for making bispecific antibody
fragments. The fragments comprise a heavy-chain variable domain (V ) connected to a light-
chain variable domain (V ) by a linker which is too short to allow pairing between the two
domains on the same chain. Accordingly, the V and V domains of one fragment are forced
to pair with the complementary V and V domains of another fragment, thereby forming
two antigen-binding sites. Another strategy for making bispecific antibody fragments by the
use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol,
152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific
antibodies can be prepared. Tuft et al. J. Immunol. 147: 60 (1991).
(vii) Single-Domain Antibodies
In some embodiments, an antibody of interest is a single-domain antibody. A single-
domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain
variable domain or all or a portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human single-domain antibody
(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In one embodiment,
a single-domain antibody consists of all or a portion of the heavy chain variable domain of an
antibody.
(viii) Antibody Variants
In some embodiments, amino acid sequence modification(s) of the antibodies
described herein are contemplated. For example, it may be desirable to improve the binding
affinity and/or other biological properties of the antibody. Amino acid sequence variants of
the antibody may be prepared by introducing appropriate changes into the nucleotide
sequence encoding the antibody, or by peptide synthesis. Such modifications include, for
example, deletions from, and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antibody. Any combination of deletion, insertion, and
substitution can be made to arrive at the final construct, provided that the final construct
possesses the desired characteristics. The amino acid alterations may be introduced in the
subject antibody amino acid sequence at the time that sequence is made.
(B) Vectors, Host Cells, and Recombinant Methods
Antibodies produced by a cell cultured in a cell culture medium described herein
may also be produced using recombinant methods. For recombinant production of an anti-
antigen antibody, nucleic acid encoding the antibody is isolated and inserted into a replicable
vector for further cloning (amplification of the DNA) or for expression. DNA encoding the
antibody may be readily isolated and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to genes encoding the heavy
and light chains of the antibody). Many vectors are available. The vector components
generally include, but are not limited to, one or more of the following: a signal sequence, an
origin of replication, one or more marker genes, an enhancer element, a promoter, and a
transcription termination sequence.
(i) Signal Sequence Component
An antibody may be produced recombinantly not only directly, but also as a fusion
polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other
polypeptide having a specific cleavage site at the N-terminus of the mature protein or
polypeptide. The heterologous signal sequence selected preferably is one that is recognized
and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells
that do not recognize and process a native antibody signal sequence, the signal sequence is
substituted by a prokaryotic signal sequence selected, for example, from the group of the
alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast
secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, a
factor leader (including Saccharomyces and Kluyveromyces α-factor leaders), or acid
phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO
90/13646. In mammalian cell expression, mammalian signal sequences as well as viral
secretory leaders, for example, the herpes simplex gD signal, are available.
(ii) Origin of Replication
Both expression and cloning vectors contain a nucleic acid sequence that enables
the vector to replicate in one or more selected host cells. Generally, in cloning vectors this
sequence is one that enables the vector to replicate independently of the host chromosomal
DNA, and includes origins of replication or autonomously replicating sequences. Such
sequences are well known for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2µ,
plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus,
VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of
replication component is not needed for mammalian expression vectors (the SV40 origin may
typically be used only because it contains the early promoter.
(iii) Selection Gene Component
Expression and cloning vectors may contain a selection gene, also termed a
selectable marker. Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
One example of a selection scheme utilizes a drug to arrest growth of a host cell.
Those cells that are successfully transformed with a heterologous gene produce a protein
conferring drug resistance and thus survive the selection regimen. Examples of such
dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
Another example of suitable selectable markers for mammalian cells are those that
enable the identification of cells competent to take up antibody-encoding nucleic acid, such
as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably
primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
For example, cells transformed with the DHFR gene are identified by culturing the
transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist
of DHFR. Under these conditions, the DHFR gene is amplified along with any other co-
transformed nucleic acid. A Chinese hamster ovary (CHO) cell line deficient in endogenous
DHFR activity (e.g., ATCC CRL-9096) may be used.
Alternatively, cells transformed with the GS gene are identified by culturing the
transformants in a culture medium containing L-methionine sulfoximine (Msx), an inhibitor
of GS. Under these conditions, the GS gene is amplified along with any other co-transformed
nucleic acid. The GS selection/amplification system may be used in combination with the
DHFR selection/amplification system described above.
Alternatively, host cells (particularly wild-type hosts that contain endogenous
DHFR) transformed or co-transformed with DNA sequences encoding an antibody of
interest, wild-type DHFR gene, and another selectable marker such as aminoglycoside 3’-
phosphotransferase (APH) can be selected by cell growth in medium containing a selection
agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin,
neomycin, or G418. See U.S. Pat. No. 4,965,199.
A suitable selection gene for use in yeast is the trp1 gene present in the yeast
plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trp1 gene provides a selection
marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example,
ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1 lesion
in the yeast host cell genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains
(ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
In addition, vectors derived from the 1.6 µm circular plasmid pKD1 can be used for
transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale
production of recombinant calf chymosin was reported for K. lactis. Van den Berg,
Bio/Technology, 8:135 (1990). Stable multi-copy expression vectors for secretion of mature
recombinant human serum albumin by industrial strains of Kluyveromyces have also been
disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
(iv) Promoter Component
Expression and cloning vectors generally contain a promoter that is recognized by
the host organism and is operably linked to nucleic acid encoding an antibody. Promoters
suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose
promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and
hybrid promoters such as the tac promoter. However, other known bacterial promoters are
suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA encoding an antibody.
Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have
an AT-rich region located approximately 25 to 30 bases upstream from the site where
transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3’
end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of
the poly A tail to the 3’ end of the coding sequence. All of these sequences are suitably
inserted into eukaryotic expression vectors.
Examples of suitable promoter sequences for use with yeast hosts include the
promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase,
glyceraldehydephosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucosephosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional
advantage of transcription controlled by growth conditions, are the promoter regions for
alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes
associated with nitrogen metabolism, metallothionein, glyceraldehydephosphate
dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable
vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast
enhancers also are advantageously used with yeast promoters.
Antibody transcription from vectors in mammalian host cells can be controlled, for
example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox
virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), or from
heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter,
from heat-shock promoters, provided such promoters are compatible with the host cell
systems.
The early and late promoters of the SV40 virus are conveniently obtained as an
SV40 restriction fragment that also contains the SV40 viral origin of replication. The
immediate early promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using the
bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of
this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-
601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of a
thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus
long terminal repeat can be used as the promoter.
(v) Enhancer Element Component
Transcription of a DNA encoding an antibody of this description by higher
eukaryotes is often increased by inserting an enhancer sequence into the vector. Many
enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-
fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell
virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-
270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of
the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on
enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into
the vector at a position 5’ or 3’ to the antibody-encoding sequence, but is preferably located
at a site 5’ from the promoter.
(vi) Transcription Termination Component
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal,
human, or nucleated cells from other multicellular organisms) will also contain sequences
necessary for the termination of transcription and for stabilizing the mRNA. Such sequences
are commonly available from the 5’ and, occasionally 3’, untranslated regions of eukaryotic
or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA encoding antibody. One
useful transcription termination component is the bovine growth hormone polyadenylation
region. See WO94/11026 and the expression vector disclosed therein.
(vii) Selection and Transformation of Host Cells
Suitable host cells for cloning or expressing the DNA in the vectors herein are the
prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this
purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example,
Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,
Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P
disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although
other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC
27,325) are suitable. These examples are illustrative rather than limiting.
Full length antibody, antibody fusion proteins, and antibody fragments can be
produced in bacteria, in particular when glycosylation and Fc effector function are not
needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a
toxin) that by itself shows effectiveness in tumor cell destruction. Full length antibodies have
greater half-life in circulation. Production in E. coli is faster and more cost efficient. For
expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.
,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523
(Simmons et al.), which describes translation initiation region (TIR) and signal sequences for
optimizing expression and secretion. See also Charlton, Methods in Molecular Biology, Vol.
248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing
expression of antibody fragments in E. coli. After expression, the antibody may be isolated
from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A
or G column depending on the isotype. Final purification can be carried out similar to the
process for purifying antibody expressed e.g., in CHO cells.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast
are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces
cerevisiae, or common baker’s yeast, is the most commonly used among lower eukaryotic
host microorganisms. However, a number of other genera, species, and strains are commonly
available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such
as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii
(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K.
thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);
Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. For a review
discussing the use of yeasts and filamentous fungi for the production of therapeutic proteins,
see, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004).
Certain fungi and yeast strains may be selected in which glycosylation pathways
have been “humanized,” resulting in the production of an antibody with a partially or fully
human glycosylation pattern. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describing
humanization of the glycosylation pathway in Pichia pastoris); and Gerngross et al., supra.
Suitable host cells for the expression of glycosylated antibody are also derived from
multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes
aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori have been identified. A variety of viral strains for transfection are publicly
available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of
Bombyx mori NPV, and such viruses may be used as the virus herein according to the
description, particularly for transfection of Spodoptera frugiperda cells.
Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed
(Leninaceae), alfalfa (M. truncatula), and tobacco can also be utilized as hosts. See, e.g., U.S.
Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing
PLANTIBODIES technology for producing antibodies in transgenic plants).
Vertebrate cells may be used as hosts, and propagation of vertebrate cells in culture
(tissue culture) has become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham
et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1
ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human
cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL
34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC
CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562,
ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5
cells; FS4 cells; and a human hepatoma line (Hep G2). Other useful mammalian host cell
lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al.,
Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and Sp2/0.
For a review of certain mammalian host cell lines suitable for antibody production, see, e.g.,
Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,
Totowa, N.J., 2003), pp. 255-268.
Host cells are transformed with the above-described expression or cloning vectors
for antibody production and cultured in a cell culture medium described herein modified as
appropriate for inducing promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
Cell Growth and Polypeptide Production
Generally the cells are combined (contacted) with any of the cell culture media
described herein under one or more conditions that promote any of cell growth, maintenance
and/or polypeptide production. Methods of culturing a cell and producing a polypeptide
employ a culturing vessel (bioreactor) to contain the cell and cell culture medium. The
culturing vessel can be composed of any material that is suitable for culturing cells, including
glass, plastic or metal. Typically, the culturing vessel will be at least 1 liter and may be 10,
100, 250, 500, 1000, 2500, 5000, 8000, 10,000 liters or more. The culture conditions, such as
temperature, pH, and the like, are those previously used with the host cell selected for
expression, and will be apparent to the ordinarily skilled artisan. Culturing conditions that
may be adjusted during the culturing process include but are not limited to pH and
temperature.
A cell culture is generally maintained in the initial growth phase under conditions
conducive to the survival, growth and viability (maintenance) of the cell culture. The precise
conditions will vary depending on the cell type, the organism from which the cell was
derived, and the nature and character of the expressed polypeptide.
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 °C. In general, most mammalian
cells grow well within a range of about 25 °C. to 42 °C. Preferably, mammalian cells grow
well within the range of about 35 °C. to 40 °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.
In one embodiment of the present description, 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.
The cells may be cultured during the initial growth phase for a greater or lesser
amount of time. In one variation, the cells are cultured for a period of time sufficient to
achieve a viable cell density that is a given percentage of the maximal viable cell density that
the cells would eventually reach if allowed to grow undisturbed. For example, the cells may
be cultured for a period of time sufficient to achieve a desired viable cell density of 1, 5, 10,
, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximal
viable cell density.
In another embodiment the cells are allowed to grow for a defined period of time.
For example, depending on the starting concentration of the cell culture, the temperature at
which the cells are cultured, and the intrinsic growth rate of the cells, the cells may be
cultured for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days.
In some cases, the cells may be allowed to grow for a month or more.
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 description, 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.
An initial culturing step is a growth phase, wherein batch cell culture conditions are
modified to enhance growth of recombinant cells, to produce a seed train. The growth phase
generally refers to the period of exponential growth where cells are generally rapidly
dividing, e.g. growing. During this phase, cells are cultured for a period of time, usually, but
not limited to, 1 to 4 days, e.g. 1, 2, 3, or 4 days, and under such conditions that cell growth is
optimal. The determination of the growth cycle for the host cell can be determined for the
particular host cell by methods known to those skilled in the art.
In the growth phase, a basal culture medium described herein and cells may be
supplied to the culturing vessel in batch. The culture medium in one embodiment contains
less than about 5% or less than 1% or less than 0.1% serum and other animal-derived
proteins. However, serum and animal-derived proteins can be used if desired. At a particular
point in their growth, the cells may form an inoculum to inoculate a culture medium at the
start of culturing in the production phase. Alternatively, the production phase may be
continuous with the growth phase. The cell growth phase is generally followed by a
polypeptide production phase.
During the polypeptide production phase, the cell culture may be maintained under
a second set of culture conditions (as compared to the growth phase) conducive to the
survival and viability of the cell culture and appropriate for expression of the desired
polypeptide. For example, during the subsequent production phase, CHO cells express
recombinant polypeptides and proteins well within a range of 25°C to 38°C. Multiple discrete
temperature shifts may be employed to increase cell density or viability or to increase
expression of the recombinant polypeptide or protein. In one embodiment, a medium as
described herein reduces the presence of metabolic by-products when used in a method of
increasing polypeptide production as compared to contaminants obtained when the
polypeptide is produced in a different medium. In one variation, the contaminants are
reactive oxygen species. In one embodiment, a medium as described herein reduces color
intensity of a polypeptide product when used in a method of increasing production of the
polypeptide as compared to color intensity obtained when the polypeptide product is
produced in a different media. In one variation, a method of increasing polypeptide
production comprises a temperature shift step during the polypeptide production phase. In a
further variation, a temperature shift step comprises a shift of the temperature from 31°C to
38°C, from 32°C to 38°C, from 33°C to 38°C, from 34°C to 38°C, from 35°C to 38°C, from
36°C to 38°C , from 31°C to 32°C, from 31°C to 33°C, from 31°C to 34°C, from 31°C to
°C, or from 31°C to 36°C.
The cells may be maintained in the subsequent production phase until a desired cell
density or production titer is reached. In one embodiment, the cells are maintained in the
subsequent production phase until the titer to the recombinant polypeptide reaches a
maximum. In other embodiments, the culture may be harvested prior to this point. For
example, the cells may be maintained for a period of time sufficient to achieve a viable cell
density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99
percent of maximal viable cell density. In some cases, it may be desirable to allow the viable
cell density to reach a maximum, and then allow the viable cell density to decline to some
level before harvesting the culture.
In certain cases, it may be beneficial or necessary to supplement the cell culture
during the subsequent production phase with nutrients or other medium components that have
been depleted or metabolized by the cells. For example, it might be advantageous to
supplement the cell culture with nutrients or other medium components observed to have
been depleted during monitoring of the cell culture. Alternatively or additionally, it may be
beneficial or necessary to supplement the cell culture prior to the subsequent production
phase. As non-limiting examples, it may be beneficial or necessary to supplement the cell
culture with hormones and/or other growth factors, particular ions (such as sodium, chloride,
calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace
elements (inorganic compounds usually present at very low final concentrations), amino
acids, lipids, or glucose or other energy source.
A component described herein (e.g., hypotaurine an analog or precursor thereof) can
be added to the cell culture medium at any time during the cell culture cycle. For example,
hypotaurine may be added at any one or more of days 0-14 for a 14 day cell culture cycle
(e.g., at any one or more of days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) at any amount
to provide a cell culture medium comprising hypotaurine at a concentration described herein
(e.g., at least 0.0001 mM). It is therefore appreciated that for a 14 day cell culture cycle,
hypotaurine may be added at any one or more of days 0-14 (e.g., at any one or more of days
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) in any amount. As used herein “day 0” can
refer to a cell culture medium that has been supplemented with a component described herein
(e.g., hypotaurine) before the cell culture medium has been applied to the cell culture. It is
understood that a cell culture cycle can be any amount of days as long as the cells remain
viable and/or sufficient levels of polypeptide are produced as can be determined by one of
skill in the art. For example, a cell culture cycle can be at least 3 days, 4 days, 5 days, 6 days,
at 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17
days, 18 days, 19 days, or 20 days in duration. In some embodiments, a component described
herein (e.g., hypotaurine or an analog or precursor thereof) is added to the cell culture
medium on at least on day of a cell culture cycle.
Polypeptide Purification
The polypeptide of interest preferably is recovered from the culture medium as a
secreted polypeptide, although it also may be recovered from host cell lysates when directly
expressed without a secretory signal. In one embodiment, the polypeptide produced is an
antibody, such as a monoclonal antibody.
The culture medium or lysate may be centrifuged to remove particulate cell debris.
The polypeptide thereafter may be purified from contaminant soluble proteins and
polypeptides, with the following procedures being exemplary of suitable purification
procedures: by fractionation on immunoaffinity or ion-exchange columns; ethanol
precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin
such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel
filtration using, for example, Sephadex G-75; and protein A Sepharose columns to remove
contaminants such as IgG. A protease inhibitor such as phenyl methyl sulfonyl fluoride
(PMSF) also may be useful to inhibit proteolytic degradation during purification. One skilled
in the art will appreciate that purification methods suitable for the polypeptide of interest may
require modification to account for changes in the character of the polypeptide upon
expression in recombinant cell culture. Polypeptides can be generally purified using
chromatographic techniques (e.g., protein A, affinity chromatography with a low pH elution
step and ion exchange chromatography to remove process impurities). For antibodies, the
suitability of protein A as an affinity ligand depends on the species and isotype of any
immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify
antibodies that are based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol.
Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3
(Guss et al., EMBO J. 5:15671575 (1986)). Purified proteins may concentrated to provide a
concentrated protein drug product as described herein, e.g., one with a protein concentration
of at least 1 mg/mL or 10 mg/mL or 50 mg/mL or 75 mg/mL or 100 mg/mL or 125 mg/mL or
150 mg/mL or a concentration of about 1 mg/mL or 10 mg/mL or 50 mg/mL or 75 mg/mL or
100 mg/mL or 125 mg/mL or 150 mg/mL. It is understood that concentrated polypeptide
products may be concentrated up to levels that are permissible under the concentration
conditions, e.g., up to a concentration at which the polypeptide is no longer soluble in
solution. For example, a polypeptide purification process can comprise the steps of
harvesting cell culture fluid from polypeptide-producing cells and purifying the polypeptide
through protein A affinity chromatography with further purification through anion and cation
exchange chromatography, filtration for removal of virus, and a final ultrafiltration and
diafiltration step for final formulation and concentration of the polypeptide. Non-limiting
examples of methods for producing and purifying polypeptides for drug formulations are
described in Kelley, B. MAbs., 2009, 1(5):443-452, which is incorporated herein in its
entirety by reference.
Polypeptide Color Assessment
The polypeptides produced by the methods detailed herein and present in the
compositions described may be assessed for color at any step of the protein purification
process. A method for assessing color may involve harvesting the cell culture fluid from
cells cultured in the media detailed herein, purifying the polypeptide from cell culture fluid to
obtain a composition (e.g., a solution) comprising the polypeptide and assessing the solution
comprising the polypeptide for color. In one variation, a composition comprising the
polypeptide is assessed for color after purification with Protein A affinity chromatography. In
a further variation, a composition comprising the polypeptide is assessed for color after
purification by ion exchange chromatography. In another variation, a composition
comprising the polypeptide is assessed for color after purification by high performance liquid
chromatography. In yet another variation, a composition comprising the polypeptide is
assessed for color after purification by hydrophobic interaction chromatography. In still
another variation, a composition comprising the polypeptide is assessed for color after
purification by size exclusion chromatography. In one variation, a composition comprising
the polypeptide is assessed for color after purification by filtration including microfiltration
or ultrafiltration. In one variation, the composition comprising the polypeptide is
concentrated prior to assessing for color (e.g., the composition may comprise at least 1
mg/mL, 10 mg/mL, 50 mg/mL, 75 mg/mL, 100 mg/mL, 125 mg/mL or 150 mg/mL
polypeptide, such as an antibody). The composition comprising the polypeptide can be
concentrated by centrifugation, filter devices, semi-permeable membranes, dialysis,
precipitation, ion exchange chromatography, affinity chromatography, high performance
liquid chromatography, or hydrophobic interaction chromatography. In one variation, the
polypeptide can be concentrated by lyophilization and resuspended prior to assessment for
color. The composition comprising the polypeptide may be assessed for color after
purification with one or more of the techniques detailed herein. Color assessment of the
composition comprising the polypeptide after the composition has undergone one or more
freeze thaw cycle(s) is contemplated herein. Methods for color assessment of cell culture
fluid containing the polypeptide prior to purification or concentration of the polypeptide is
further contemplated herein.
The polypeptides produced by the methods detailed herein with the media described
herein (or present in the compositions described) may be assessed for color by use of one or
more visual color standards. Methods for color assessment of composition comprising the
polypeptide include use of an international or national color standard such as, but not limited
to, the United States Pharmacopoeia color standard and the European Pharmacopoeia color
standard. See USP-24 Monograph 631 Color and Achromaticity. United States
Pharmacopoeia Inc., 2000, p. 1926-1927 and Council of Europe. European Pharmacopoeia,
2008, 7 Ed. P.22, which are incorporated herein by reference in their entirety. For example,
the Color, Opalescence and Coloration (COC) assay may be used to assess color of a solution
containing the polypeptide. In one variation, identical tube of colorless, transparent, neutral
glass of 12 mm external diameter are used to compare 2.0 mL of the composition comprising
the polypeptide with 2.0 mL of water or of the solvent or of the reference solution prescribed
in the monograph. The colors are compared in diffused daylight and viewed horizontally
against a white background for color determination, measurement, or assessment. In another
variation, identical tubes of colorless, transparent, neutral glass with a flat base and an
internal diameter of 15 mm to 25 mm are used to compare the composition comprising the
polypeptide with water or the solvent or the reference solution prescribed in the monograph,
the depth of the layer being 40 mm. The colors are compared in diffused daylight and viewed
vertically against a white background for color determination, measurement, or assessment.
In one variation, color determination, measurement or assessment can be done by human
visual inspection. In another variation, color determination, measurement, or assessment can
be done by using an automated process. For example, the tubes can be loaded in a machine
that images the tubes for processing of the images with an algorithm to determine, measure,
or assess the color. It is understood that the reference standards for the COC assay can be
any one of, but not limited to, brown (B), brownish-yellow (BY), yellow (Y), greenish-
yellow (GY), or red (R). Compositions comprising the polypeptide that are compared to the
brown reference standard can be given a brown reference standard value of B1 (darkest), B2,
B3, B4, B5, B6, B7, B8, or B9 (lightest). Compositions comprising the polypeptide that are
compared to the brownish-yellow reference standard can be given a brownish-yellow
reference standard value of BY1 (darkest), BY2, BY3, BY4, BY5, BY6, or BY7 (lightest).
Compositions comprising the polypeptide that are compared to the yellow reference standard
can be given a yellow reference standard value of Y1 (darkest), Y2, Y3, Y4, Y5, Y6, or Y7
(lightest). Compositions comprising the polypeptide that are compared to the greenish-
yellow reference standard can be given a greenish-yellow reference standard value of GY1
(darkest), GY2, GY3, GY4, GY5, GY6, or GY7 (lightest). Compositions comprising the
polypeptide that are compared to the red reference standard can be given a red reference
standard value of R1 (darkest), R2, R3, R4, R5, R6, or R7 (lightest). In one embodiment, an
acceptable color is any color except that which measures darkest on a scale provided herein
(e.g., except R1 for a red reference standard value). In one variation, the color of the
composition comprising the polypeptide produced by cells cultured in the media detailed
herein has a reference standard value as described in Table 3. As is described herein, it is
understood that in one embodiment the media that may be used in the methods and
compositions herein result in a polypeptide composition (which in one variation is a
composition comprising at least 100 mg/mL or 125 mg/mL or 150 mg/ml polypeptide)
having a reference standard color value selected from the group consisting of B3, B4, B5, B6,
B7, B8, B9, BY3, BY4, BY5, BY6, BY7, Y3, Y4, Y5, Y6, Y7, GY3, GY4, GY5, GY6, GY7,
R3, R4, R5, R6 and R7. In one embodiment, the media that may be used in the methods and
compositions herein result in a polypeptide composition (which in one variation is a
composition comprising at least 100 mg/mL or 125 mg/mL or 150 mg/ml polypeptide)
having a reference standard color value of greater than any one of B4, B5, B6, B7, B8, BY4,
BY5, BY6, Y4, Y5, Y6, GY4, GY5, GY6, GY7, R3, R4, R5 and R6. As would be
understood to the skilled artisan, descriptions of reference standard color values are
applicable to, and may further modify descriptions of, any of the media, methods or
compositions detailed herein.
In some embodiments, color intensity is determined using the Total Color assay.
See, e.g., Vijayasankaran et al., Biotechol. Prog. 29:1270-1277, 2013, which is incorporated
herein by reference. For the Total Color assay, a quantitative value of the relative color of
samples is derived by using the CIE System of color measurement as described in Berns et
al., Billmeyer and Saltzman’s Principles of Color Technology, 3 Edition. New York, NY,
John Wiley & Sons, Inc., (2000). Briefly, after blanking with water, the absorption spectrum
of a neat test sample is measured in the visible region (380-780nm) using a HP8453A
spectrophotometer (1cm pathlength cuvette). The absorption spectrum is then converted to
the CIE L*a*b* color scale as previously described in Standard Practice for Calculation of
Color Tolerances and Color Differences from Instrumentally Measured Color Coordinates,
Annual Book of ASTM Standards, Vol. 06.01, (2011). L*a*b* is a three dimensional color
space with an approximately uniform spacing in visual perception. The L*a*b* color space
is able to quantify differences in visual judgment of colors. For example, two solutions that
are visually judged to have very different colors will be further apart in the L*a*b* color
space when compared with two solutions that have similar color which will be closer together
within the L*a*b* color speace. Within the three dimensional L*a*b* space the distance
between points is calculatated as the Euclidian between the points (delta E). This allows for
measuring the delta E between points in the L*a*b* color space and correlating this distance
to visual perception judgment of color differences, Large delta E represents two solutions of
very different colors, and small delta E represents two solutions of similar color. The
transformation of absorption spectrum to L*a*b* color space requires a defined illuminant.
For example, an artificial flat spectrum in the visible region can be used as the illuminant. In
some embodiments, the “Total Color” may represent the Delta E which corresponds to the
Euclidian distance between the test sample and water in the three dimensional CIE L*a*b*
color space. In addition, the “Total Color” may represent the overall color of the test
monoclonal antibody sample without differentiating between differing hues. Total color
measurement can be normalized to the value measured for a reference standard. For
example, the color intensity value is subsequently determined by calculating the ratio of the
“Total Color” measurement of the test monoclonal antibody sample to that of a reference
monoclonal antibody sample containing a COC reading of ≤ B5.
The color intensity can also be determined using NIFTY (Normalized Intrinsic
Fluorescence Tool for Yellow/brown proteins) assay. In this assay, the fluorescence of the
antibody molecule is used as proxy for color as it has been shown that the color intensity and
fluorescence intensity correlate well in the protein A pool (R2=0.84). See Vijayasankaran et
al., Biotechnol Prog 27:1270-1277 (2013). The higher numerical NIFTY value indicates
higher color intensity and lower numerical NIFTY value indicates lower color intensity.
About 50 to 125 µg of monoclonal antibody samples are analyzed by size exclusion
chromatography (SEC) using a G3000SWXL column (TOSOH), with an isocratic flow rate
of 0.5mL/min. Mobile phase for SEC is 0.2M potassium phosphate, 0.25M potassium
chloride, pH 6.2. Column temperature is controlled at 15 °C. For example, the SEC eluent
can be monitored for UV absorption at 280 nm and for fluorescence with excitation
wavelength at 350 nm and emission wavelength at 425 nm. These wavelengths are chosen
based on the strong correlation as well as the maximal fluorescence response observed with
these wavelengths. The SEC peaks of monoclonal antibody species are integrated using
Agilent Chemstation software on the UV absorbance and the fluorescence emission
chromatograms. For each monoclonal antibody sample, the normalized fluorescence is
determined by dividing the fluorescence peak area of the main peak by the UV absorbance
peak area of the main peak, which corrects the fluorescence response by the antibody mass
contribution. The color intensity value is subsequently determined by calculating the ratio of
the normalized fluorescence of the test monoclonal antibody sample to that of a reference
monoclonal antibody sample (e.g., a sample containing a COC reading of ≤ B5). As the
sample requirement for NIFTY is small, it is useful as a surrogate for color when culture
volume is limited.
NIFTY value can be calculated as shown below. F= Peak area on fluorescence
chromatogram; U= Peak area on the UV absorption chromatogram; i=variable; S=Sample;
R=Reference.
Table 3. Exemplary reference standard values
Reference Reference standard value
standard
(a) Brown from about B1 to about B9; from about B1 to about B8; from about B1 to
about B7; from about B1 to about B6; from about B1 to about B5; from
about B1 to about B4; from about B1 to about B3; from about B1 to about
B2; from about B2 to about B9; from about B3 to about B9; from about
B4 to about B9; from about B5 to about B9; from about B6 to about B9;
from about B7 to about B9; from about B8 to about B9; from about B2 to
about B8; from about B3 to about B7; from about B4 to about B6; from
about B5 to about B7; from about B6 to about B8; about any of B1 or B2
or B3 or B4 or B5 or B6 or B7 or B8 or B9; at least about any of B1 or B2
or B3 or B4 or B5 or B6 or B7 or B8 or B9. Preferably B3 to B9. Most
preferably B4 to B9.
(b) Brownish- from about BY1 to about BY7; from about BY1 to about BY6; from
Yellow about BY1 to about BY5; from about BY1 to about BY4; from about BY1
to about BY3; from about BY1 to about BY2; from about BY2 to about
BY7; from about BY3 to about BY7; from about BY4 to about BY7; from
about BY5 to about BY7; from about BY6 to about BY7; from about BY2
to about BY6; from about BY3 to about BY5; from about BY4 to about
BY6; from about BY5 to about BY6; about any of BY1 or BY2 or BY3 or
BY4 or BY5 or BY6 or BY7; at least about any of BY1 or BY2 or BY3 or
BY4 or BY5 or BY6 or BY7. Preferably BY3 to BY7. Most preferably
BY4 to BY7.
(c) Yellow from about Y1 to about Y7; from about Y1 to about Y6; from about Y1 to
about Y5; from about Y1 to about Y4; from about Y1 to about Y3; from
about Y1 to about Y2; from about Y2 to about Y7; from about Y3 to
about Y7; from about Y4 to about Y7; from about Y5 to about Y7; from
about Y6 to about Y7; from about Y2 to about Y6; from about Y3 to
about Y5; from about Y4 to about Y6; from about Y5 to about Y6; about
any of Y1 or Y2 or Y3 or Y4 or Y5 or Y6 or Y7; at least about any of Y1
or Y2 or Y3 or Y4 or Y5 or Y6 or Y7. Preferably Y3 to Y7. Most
preferably Y4 to Y7.
(d) Greenish- from about GY1 to about GY7; from about GY1 to about GY6; from
Yellow about GY1 to about GY5; from about GY1 to about GY4; from about
GY1 to about GY3; from about GY1 to about GY2; from about GY2 to
about GY7; from about GY3 to about GY7; from about GY4 to about
GY7; from about GY5 to about GY7; from about GY6 to about GY7;
from about GY2 to about GY6; from about GY3 to about GY5; from
about GY4 to about GY6; from about GY5 to about GY6; about any of
GY1 or GY2 or GY3 or GY4 or GY5 or GY6 or GY7; at least about any
of GY1 or GY2 or GY3 or GY4 or GY5 or GY6 or GY7. Preferably GY3
to GY7. Most preferably GY4 to GY7.
(e) Red from about R1 to about R7; from about R1 to about R6; from about R1 to
about R5; from about R1 to about R4; from about R1 to about R3; from
about R1 to about R2; from about R2 to about R7; from about R3 to about
R7; from about R4 to about R7; from about R5 to about R7; from about
R6 to about R7; from about R2 to about R6; from about R3 to about R5;
from about R4 to about R6; from about R5 to about R6; about any of R1
or R2 or R3 or R4 or R5 or R6 or R7; at least about any of R1 or R2 or R3
or R4 or R5 or R6 or R7. Preferably R3 to R7. Most preferably R4 to R7.
In another example, the polypeptides produced by the methods detailed herein with
the media described herein (or present in the compositions described) may be assessed for
color with a quantitative assay. In some embodiments, the quantitative assay can be done
using an automated process. In some embodiments, a higher value (e.g., higher numerical
value) provided by the quantitative assay indicates a higher color intensity and a lower value
(e.g., lower numerical value) indicates a lower color intensity.
A color assay detailed herein may find use in assessing color of any solution (e.g., a
polypeptide-containing solution), including, but not limited to, the polypeptide compositions
described herein.
IV.Compositions and Pharmaceutical Formulations
Compositions comprising the cell culture medium and one or more other
component, such as a cell or a desired polypeptide (e.g., an antibody), are also described. A
cell comprising a nucleic acid encoding a polypeptide of interest (e.g., an antibody) can
secrete the polypeptide into a cell culture medium of the description during cell culture.
Accordingly, compositions of the description can comprise a cell that produces the
polypeptide and a cell culture medium described herein that the polypeptide is secreted into.
Compositions comprising the produced polypeptide and a cell culture medium described
herein are also contemplated. In some embodiments of the description, a composition
comprises (a) a cell comprising a nucleic acid encoding a polypeptide; and (b) a cell culture
medium are described herein. In some embodiments, the composition comprises (a) a
polypeptide; and (b) a cell culture medium as described herein, wherein the polypeptide is
secreted into the medium by a cell comprising an isolated nucleic acid encoding the
polypeptide. In other embodiments, the composition comprises: (a) a polypeptide; and (b) a
cell culture medium as described herein, wherein the polypeptide is released into the medium
by lysis of a cell comprising an isolated nucleic acid encoding the polypeptide. The cell of the
composition may be any cell detailed herein (e.g., a CHO cell) and the medium of the
composition may be any medium detailed herein, such as a medium comprising one or more
compounds as detailed in Table 1 or Table 2. Likewise, the polypeptide of the composition
may be any polypeptide detailed herein, such as an antibody. In some embodiments, the
composition may have a color. In some embodiments, the color is determined, measured, or
assessed by use of one or more visual color standards. The visual color standard can be an
international or national color standard such as, but not limited to, the United States
Pharmacopoeia color standard and the European Pharmacopoeia color standard. See USP-24
Monograph 631 Color and Achromaticity. United States Pharmacopoeia Inc., 2000, p. 1926-
1927 and Council of Europe. European Pharmacopoeia, 2008, 7 Ed. P.22. Accordingly, in
some embodiments, a composition comprising (a) a polypeptide; and (b) a cell culture
medium described herein is assessed for color intensity. In a further embodiment, the
polypeptide is isolated and/or purified before assessment of color intensity. In some
embodiments, a color intensity of a composition comprising (a) a polypeptide; and (b) a cell
culture medium described herein is used to predict the color intensity of the final protein
composition. For example, a composition comprising a polypeptide and a cell culture
medium described herein is measured for color intensity using the COC assay as described
herein. If the color intensity value is greater than B3, B4, B5, B6, B7, B8, or B9 then there is
an increased likelihood that the final protein composition will have a color intensity value of
greater than B3, B4, B5, B6, B7, B8, or B9. In some embodiments, the composition
comprising a polypeptide and the cell culture medium is subjected to at least one purification
step before measurement of color intensity. In some embodiments, the final protein
composition is a pharmaceutical formulation. In some embodiments, a composition as
described herein comprises a polypeptide at a concentration of at least about 1 mg/mL, 10
mg/mL or 25 mg/mL or 50 mg/mL or 75 mg/mL to about 100 mg/mL or at a concentration of
about 1 mg/mL, 10 mg/mL or 25 mg/mL or 50 mg/mL or 75 mg/mL to about 100 mg/mL. In
some embodiments, a composition as described herein comprises a polypeptide at a
concentration of at least 100 mg/mL or 125 mg/mL or 150 mg/mL or at a concentration of
about 100 mg/mL or 125 mg/mL or 150 mg/mL or 175 mg/mL or 200 mg/mL.
Compositions (e.g., pharmaceutical formulations) of the polypeptides (e.g. a
therapeutic polypeptide) produced by any of the methods described herein are prepared by
mixing a polypeptide having the desired degree of purity with one or more optional
pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to: buffers, antioxidants,
preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins;
hydrophilic polymers; amino acids; monosaccharides, disaccharides, and other carbohydrates,
chelating agents, sugars, salt-forming counter-ions, metal complexes (e.g. Zn-protein
complexes), and/or non-ionic surfactants. Exemplary lyophilized polypeptide formulations
are described in US Patent No. 6,267,958. Aqueous polypeptide formulations include those
described in US Patent No. 6,171,586 and WO2006/044908, the latter formulations including
a histidine-acetate buffer. In some embodiments, the pharmaceutical formulation is
administered to a mammal such as a human. Pharmaceutical formulations of the polypeptide
(e.g., an antibody) can be administered by any suitable means, including parenteral,
intrapulmonary, and intranasal, and, if desired for local treatment, intralesional
administration. Parenteral infusions include intramuscular, intravenous, intraarterial,
intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by
injections, such as intravenous or subcutaneous injections, depending in part on whether the
administration is brief or chronic. Accordingly, polypeptide-containing formulations as
described herein may be suitable for injection, such as subcutaneous injection into an
individual (e.g., subcutaneous injection into a human). The pharmaceutical formulations to be
used for in vivo administration are generally sterile. Sterility may be readily accomplished,
for example by filtration through sterile filtration membranes.
In some embodiments, a composition (e.g., pharmaceutical formulation) as
described herein comprises a polypeptide (e.g., a therapeutic polypeptide) at a concentration
of at least about 1 mg/mL, 10 mg/mL, 25 mg/mL, 50 mg/mL, or 75 mg/mL, or at a
concentration of about 1 mg/mL, about 10 mg/mL, about 25 mg/mL, about 50 mg/mL, or
about 75 mg/mL up to about 100 mg/mL. In other embodiments, a composition (e.g.,
pharmaceutical formulation) as described herein comprises a polypeptide (e.g., a therapeutic
polypeptide) at a concentration of at least about 100 mg/mL, 125 mg/mL, 150 mg/mL, 200
mg/mL, or 250 mg/mL, or at a concentration of about 100 mg/mL, about 125 mg/mL, about
150 mg/mL, about 175 mg/mL, about 200 mg/mL, or about 250 mg/mL. In some
embodiments, a pharmaceutical formulation as described herein comprises a polypeptide at a
concentration greater than at least about 1 mg/mL, at least about 10 mg/mL, at least about 25
mg/mL, at least about 50 mg/mL, or at least about 75 mg/mL and has a color intensity value
greater than B3, B4, B5, B6, B7, B8, or B9 as measured by the COC assay. In some
embodiments, a pharmaceutical formulation as described herein comprises a polypeptide at a
concentration greater than at least about 100 mg/mL, at least about 125 mg/mL, at least about
150 mg/mL, or at least about 200 mg/mL and has a color intensity value greater than B3, B4,
B5, B6, B7, B8, or B9 as measured by the COC assay. In some embodiments, the color
intensity value as determined by the COC assay can be any one of, but not limited to, B, BY,
Y, GY, or R, wherein higher values indicate a lighter color intensity. In some embodiments,
a pharmaceutical formulation as described herein comprises a polypeptide at a concentration
greater than at least about 1 mg/mL, at least about 10 mg/mL, at least about 25 mg/mL, at
least about 50 mg/mL, or at least about 75 mg/mL and has a color intensity value less than a
color intensity value of a reference solution as measured by a color assay. In some
embodiments, a pharmaceutical formulation as described herein comprises a polypeptide at a
concentration greater than at least about 100 mg/mL, at least about 125 mg/mL, at least about
150 mg/mL, or at least about 200 mg/mL and has a color intensity value less than a color
intensity value of a reference solution as measured by a color assay. For example, the color
intensity of a composition (e.g., pharmaceutical formulation) comprising a polypeptide (e.g.,
a therapeutic polypeptide) can be reduced by at least 0.1% or by about 5% to about 50% as
compared to a composition comprising the polypeptide produced by a cell cultured in a cell
culture medium that does not comprise the one or more of components of Table 1 or Table 2.
V.Articles of Manufacture or Kits
A kit for supplementing a cell culture medium with chemically defined constituents
is described. The kit may contain dried constituents to be reconstituted, and may also contain
instructions for use (e.g., for use in supplementing a medium with the kit constituents). The
kit may contain the constituents described herein in amounts suitable to supplement a cell
culture medium. In some embodiments, the kit contains one or more constituent selected
from the group consisting of hypotaurine, s-carboxymethylcysteine, anserine, butylated
hydroxyanisole, carnosine, lipoic acid, and quercitrin hydrate in amounts to supplement a cell
culture medium with a constituent concentration as provided in Table 1 or Table 2. In some
embodiments, a kit comprises one or more of: (a) hypotaurine in an amount to provide from
about 2.0 mM to about 50.0 mM hypotaurine in the cell culture medium; (b) s-
carboxymethylcysteine in an amount to provide from about 8.0 mM to about 12.0 mM s-
carboxymethylcysteine in the cell culture medium; (c) carnosine in an amount to provide
from about 8.0 mM to about 12.0 mM carnosine in the cell culture medium; (d) anserine in an
amount to provide from about 3.0 mM to about 5.0 mM anserine in the cell culture medium;
(e) butylated hydroxyanisole in an amount to provide from about 0.025 mM to about 0.040
mM butylated hydroxyanisole; (f) lipoic acid in an amount to provide from about 0.040 mM
to about 0.060 mM lipoic acid in the cell culture medium; (g) quercitrin hydrate in an amount
to provide from about 0.010 mM to about 0.020 mM quercitrin hydrate in the cell culture
medium; and (h) aminoguanidine in an amount to provide from about 0.0003 mM to about 10
mM aminoguanidine in the cell culture medium. In some embodiments, the kit contains one
or more constituent, wherein the one or more constituent is hypotaurine or an analog or
precursor thereof. In some embodiments, the hypotaurine or an analog or precursor thereof is
selected from the group consisting of hypotaurine, s-carboxymethylcysteine, cysteamine,
cysteinesulphinic acid, and taurine. In some embodiments, a kit for supplementing a cell
culture medium with chemically defined constituents, the kit comprising hypotaurine or an
analog or precursor thereof at a concentration of at least about 0.0001 mM, and wherein the
hypotaurine or an analog or precursor is selected from the group consisting of hypotaurine, s-
carboxymethylcysteine, cysteamine, cysteinesulphinic acid, and taurine.
In another embodiment of the description, an article of manufacture is described
comprising a container which holds the cell culture medium of the description and optionally
provides instructions for its use. Suitable containers include, for example, bottles and bags.
The container may be formed from a variety of materials such as glass or plastic. The
container holds the cell culture medium and the label on, or associated with, the container
may indicate directions for use (e.g., for use in culturing cells). The article of manufacture
may further include other materials desirable from a commercial and user standpoint,
including other buffers, diluents and package inserts with instructions for use.
The following Examples are provided to illustrate but not to limit the invention.
EXAMPLES
Media have been identified that produce a protein product (e.g., a protein drug
product) with acceptable quality attributes, such as reduced color intensity, particularly when
the protein product is present as a concentrated solution (e.g., to a concentration of at least
about 1 mg/mL or at least about 100 mg/mL). Methods of culturing cells in the media
described herein are described, as are methods of producing a polypeptide using the media. A
media may in one embodiment comprise hypotaurine. In some of the embodiments described
herein, the media comprises one or more hypotaurine analog or precursor thereof, such as
carboxymethylcysteine. Each of the media constituents may be present in any value described
throughout. The media may be chemically defined or chemically undefined. The media may
reduce the presence of reactive oxygen species when used in a method of polypeptide
production as compared to the polypeptide produced in different media. The media finds use
through all phases of cell culture and polypeptide production and may be used in the basal
and/or feed medium. A polypeptide produced by any of the methods described herein is
described, as is a pharmaceutical composition comprising a polypeptide produced as detailed
herein. In one embodiment, the pharmaceutical compositions comprise the polypeptide at a
concentration of at least or about any of 100 mg/mL, 125 mg/mL, or 150 mg/mL. Methods
of making and compositions comprising antibodies are particularly contemplated. Kits for
supplementing a cell culture medium with chemically defined constituents are also described.
Example 1: Identification of antioxidant compounds capable of reducing color in
antibody compositions.
Compounds that have been reported to react with an oxidant were screened for their
ability to reduce the color of protein containing compositions (Table 4). For antioxidant
screening, a total volume of 40 ml media was prepared by mixing 1 part basal Media 1 and
0.3 part feed Media 2 to mirror a representative ratio of media used in cell culture conditions
(Table 5). The mixture of Media 1 and Media 2, which was previously shown to increase the
color intensity of antibody-containing solutions when used for culturing antibody-producing
cells, was supplemented with one of 30 antioxidant compounds and spiked with 2 g/L IgG1
monoclonal antibody. The samples were incubated at 37°C with shaking at 250 rpm for a
five day incubation period. Two control samples were included in the screening assay: 1) a
40 ml sample of a Media 1 and Media 2 mixture containing 2g/L IgG1 monoclonal antibody
that was incubated for 5 days at 37°C with shaking at 250 rpm without antioxidant (positive
control), and 2) a 40 mL sample of a media mixture prepared by mixing 1 part basal Media 3
and 0.3 part feed Media 4 (Table 5), which was previously shown to reduce the color
intensity of antibody-containing solutions when used for culturing antibody-producing cells,
spiked with 2g/L IgG1 monoclonal antibody and incubated for 5 days at 37°C with shaking at
250 rpm without antioxidant (negative control).
Table 4. Representative compounds screened for reduction of color
1X Test
Antioxidant IUPAC CAS #
Concentration
2,3-tert-butyl
2-tert-butylmethoxyphenol 250135 34.68 µM
hydroxyanisole
2,6-di-tert-butyl
2,6-di-tert-butylmethylphenol 971236 102.11 µM
methylphenol
3-aminopropane
3-aminopropanesulfonic acid 36871 9.16 mM
sulfonic acid
Adenosylhomocysteine S-(5'-Deoxyadenos-5'-yl)-L-homocysteine 9790 10.41 µM
(2S)(3-aminopropanamido)(1-methyl-1H-
Anserine 100301 4.12 mM
imidazolyl)propanoic acid; nitric acid
B-Alanine 3-aminopropanoic acid 1079 9.16 mM
1,3,3-trimethyl
[(1E,3E,5E,7E,9E,11E,13E,15E,17E)-3,7,12,16-
B-carotene tetramethyl(2,6,6-trimethylcyclohexen 72357 9.31 µM
yl)octadeca-1,3,5,7,9,11,13,15,17-nonaen
yl]cyclohexene
Butylated
2-tert-butylmethoxyphenol 250135 31.62 µM
hydroxyanisole
Butylated
2,6-di-tert-butylmethylphenol 1280 124.80 µM
hydroxytoluene
(2S)(3-aminopropanamido)(1H-imidazol
Carnosine 3050 10.00 mM
yl)propanoic acid
[3-(9H-carbazolyloxy)hydroxypropyl][2-(2-
Carvedilol 729563 21.53 µM
methoxyphenoxy)ethyl]amine
(1E,4Z,6E)hydroxy-1,7-bis(4-hydroxy
Curcumin 4587 49.95 µM
methoxyphenyl)hepta-1,4,6-trienone
Cysteamine 2-aminoethanethiol 601 12.00 mM
Cysteamine
hydrogen 2-aminoethanethiol chloride 1560 10.00 mM
hydrochloride
(1R,2S,10S,11S,13R,14R,15S,17S)fluoro-
14,17-dihydroxy(2-hydroxyacetyl)-2,13,15-
Dexamethasone 502 9.56 µM
trimethyltetracyclo[8.7.0.0^{2,7}.0^{11,15}]hepta
deca-3,6-dienone
Diallyldisulfide 3-(propenylsulfanyl)propene 5921 1.00 mM
2-amino[(2-amino
DL-Lanthionine 31832 97.96 µM
carboxyethyl)sulfanyl]propanoic acid
DL-Thiorphan 2-(2-benzylsulfanylpropanamido)acetic acid 767216 0.10 mM
Ethoxyquin 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline 912 49.99 µM
Gallic acid 3,4,5-trihydroxybenzoic acid 1497 14.11 µM
Gentisic acid sodium
sodium 2,5-dihydroxybenzoate 49552 2.84 mM
salt hydrate
2-amino({1-[(carboxymethyl)carbamoyl]
Glutathione 708 2.0 mM
sulfanylethyl}carbamoyl)butanoic acid
2-amino[(2-{[2-(4-amino
carboxybutanamido)
Glutathione disulfide [(carboxymethyl)carbamoyl]ethyl]disulfanyl} 270258 2.0 mM
[(carboxymethyl)carbamoyl]ethyl)carbamoyl]buta
noic acid
(2S)amino{[(1R)
Glutathione reduced
[(carboxymethyl)carbamoyl] 926140 0.93 mM
ethyl ester
sulfanylbutyl]carbamoyl}butanoic acid
Glycine 2-aminoacetic acid 566 13.32 mM
(1S,2R,10S,11S,14R,15S,17S)-14,17-dihydroxy-
14-(2-hydroxyacetyl)-2,15-
Hydrocortisone 507 55.03 mM
dimethyltetracyclo[8.7.0.0^{2,7}.0^{11,15}]hepta
decenone
Hypotaurine 2-aminoethanesulfinate 3005 9.16 mM
Isethionic acid
ammonium 2-hydroxyethanesulfonate 572674 9.16 mM
ammonium salt
(2S)amino{[(1R){[(2R)amino-3,3-
L-Cysteine-glutathione dihydroxypropyl]sulfanyl}
130816 0.73 mM
Disulfide [(carboxymethyl)carbamoyl]
sulfanylideneethyl]carbamoyl}butanoic acid
L-Cysteinesulfinic acid (2R)amino[(R)-sulfino]propanoic acid
2071210 9.15 mM
monohydrate hydrate
Lipoic Acid 5-[(3R)-1,2-dithiolanyl]pentanoic acid 12002 50.40 µM
Lipoic acid reduced 6,8-disulfanyloctanoic acid 4624 48.00 µM
Mercaptopropionyl
2-(2-sulfanylpropanamido)acetic acid 19532 10.00 mM
glycine
Methionine 2-amino(methylsulfanyl)butanoic acid 598 5.00 mM
3-({[(2-
Methylenebis(3-
carboxyethyl)sulfanyl]methyl}sulfanyl)propanoic 42650 0.99 mM
thiopropionic acid)
acid
Oxalic acid oxalic acid 1447 500.94 µM
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy
Quercetrin hydrate {[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy 5223 13.94 µM
methyloxanyl]oxy}-4H-chromenone
-[(E)(4-hydroxyphenyl)ethenyl]benzene-1,3-
Resveratrol 5010 98.58 µM
diol
(2E,4E,6E,8E)-3,7-dimethyl(2,6,6-
Retinoic acid trimethylcyclohexenyl)nona-2,4,6,8- 3024 2.0 µM
tetraenoic acid
S-Carboxymethyl-L- (2R)amino
6383 10.00 mM
cysteine [(carboxymethyl)sulfanyl]propanoic acid
Selenium selanylidene 77822 1.40 µM
Selenomethionine (2S)amino(methylselanyl)butanoic acid 32115 30.09 µM
Silver
silver(1+) ion (diethylcarbamothioyl)sulfanide 14707 0.10 mM
diethyldithiocarbamate
Taurine 2-aminoethanesulfonic acid 1077 5.00 mM
Thiolactic acid 2-sulfanylpropanoic acid amine 795 10.00 mM
2-{[1,3-dihydroxy(hydroxymethyl)propan
Tricine 57041 4.46 mM
yl]amino}acetic acid
2-(1,2-dihydroxyethyl)-4,5-dihydroxy-2,3-
Vitamin C 507 9.82 µM
dihydrofuranone
(2R)-2,5,7,8-tetramethyl[(4R,8R)-4,8,12-
Vitamin E trimethyltridecyl]-3,4-dihydro-2Hbenzopyran- 101910 27.86 µM
6-ol
1X test concentration indicates final concentration in the media
After incubation, the monoclonal antibody was purified using affinity
chromatography. Color intensity of the concentrated antibody composition was measured in
the purified pool using an assay wherein higher numerical values indicate higher color
intensity and lower numerical values indicate lower color intensity. The numerical results
were normalized to the positive control, where the value for the positive control was set at
0% change in color intensity. Of the 30 antioxidant compounds tested several compounds,
such as gentisic acid, cysteamine, hydrocortisone, and mercaptopropionyl glycine, were
found to increase the color of the antibody composition (Fig. 1). In comparison, six of the
compounds such as hypotaurine, anserine, butylated hydroxyanisole, carnosine, lipoic acid,
and quercitrin hydrate, were found to reduce the color of the antibody composition (Fig. 2).
Of the antioxidants that reduced color intensity, hypotaurine demonstrated the greatest effect
by reducing the color intensity of the antibody-containing compositions by approximately
%. Taurine, an analog of hypotaurine, also reduced color intensity by approximately 5%.
Table 5. Representative components in media compositions tested
Media 1 Media 2 Media 3 Media 4
Media Components
(Basal) (Feed) (Basal) (Feed)
Iron (µM) 75 0 18 0
Vitamin B2 (mg/L) 1.41 10 0.25 0
Vitamin B6/ Pyridoxine (mg/L) 15.42 7 5.35 0
Vitamin B6/ Pyridoxal (mg/L) 0 60 0 0
Vitamin B9 (mg/L) 9.93 197 8.61 0
Vitamin B12 (mg/L) 3.05 48 1.76 0
Cysteine (mg/L) 525 1500 0 1500
Cystine (mg/L) 0 0 480 0
Hydrocortisone (nM) 150 0 150 0
Iron source is ferrous sulfate
Iron source is ferric citrate
Example 2: Characterization of antioxidant compounds capable of reducing color
intensity in antibody compositions isolated from antibody-producing cell lines.
The ability of hypotaurine to reduce color intensity in antibody containing
compositions obtained directly from cell cultures was evaluated. For these studies a shaker
flask cell culture model was utilized that was found to be representative of larger scale 2L
cell culture. Briefly, for the shaker flask cell culture model, antibody producing CHO cells
were inoculated at approximately 1.0 x 10 cells/mL in a 250 mL flask containing 100 mL of
basal Media 1 or basal Media 3. For the larger scale 2L cell cultures, antibody producing
CHO cells were inoculated at approximately 1.0 x 10 cells/mL in 2-liter stirred bioreactors
(Applikon, Foster City, CA) containing 1L of basal Media 1 or basal Media 3. For the larger
scale cell growth model, cells were cultured in fed-batch mode with addition of 100 mL of
feed Media 2 if cultured in basal Media 1, or with 100 mL of feed Media 4 if cultured in
basal Media 3, per liter of cell culture fluid at days 3, 6 and 9 for initiation of the production
phase. For the shaker flask cell culture model, the cells were cultured in fed-batch mode with
addition of 10 mL of feed Media 2 if cultured in basal Media 1, or with 10 mL mL of feed
Media 4 if cultured in basal Media 3, per liter of cell culture fluid at days 3, 6 and 9 for
initiation of the production phase. The concentration of glucose was analyzed every day and
if the glucose concentration fell below 3 g/L, it was replenished from a 500 g/L stock solution
of glucose for prevention of glucose depletion. Reactors were equipped with calibrated
dissolved oxygen, pH and temperature probes. Dissolved oxygen was controlled on-line
through sparging with air and/or oxygen. For the larger scale 2L cell culture, pH was
controlled through addition of CO or Na CO and antifoam was added to the cultures as
2 2 3
needed. The cell cultures were maintained at pH 7.0 and a temperature of 37ºC from days 0
through 3, and then at 35ºC after day 3. The cell cultures were agitated at 275 rpm and the
dissolved oxygen level was at 30% of air saturation. For the shaker flask cell cultures,
cultures were placed on a shaker platform and agitated at 150 rpm in a 5% CO incubator
with a temperature of 37°C from day 0 up to day 3 of the cell culture cycle with a
temperature shift to 35°C on day 4 until the end of the cell culture cycle at day 14.
Osmolality was monitored using an osmometer from Advanced Instruments (Norwood, MA).
Offline pH and metabolite concentrations were also determined daily using a Nova Bioprofile
400 (Nova Biomedical, Waltham, MA). Viable cell density (VCC) and cell viability was
measured daily using a ViCell automated cell counter (Beckman Coulter, Fullerton, CA).
The cell culture fluid was collected daily by centrifuging 1 mL of cell culture fluid for
determination of antibody titer using high performance liquid chromatography. At the end of
the cell culture duration on day 14, the cell culture fluid from all samples was harvested by
centrifugation. The monoclonal antibody in the harvested cell culture fluid was purified
using affinity chromatography. Color intensity of the concentrated antibody composition was
measured in the purified pool using an assay wherein higher numerical values indicate higher
color intensity and lower numerical values indicate lower color intensity. Growth as
measured by VCC (Fig. 3A) and cell viability (Fig. 3B) were comparable between the larger
scale (2L) and shaker flask (SF) cell culture models regardless of the media used. Antibody
production was slightly lower in the shaker flask cell culture model with the highest antibody
production observed in the larger scale cell culture model incubated in Media 1 and Media 2
(Fig. 3C). Color intensity of antibody compositions obtained from the shaker flask cell
culture model was lower at a value of 1.07 when cultured in Media 3 and Media 4 as
compared to antibody compositions obtained from shaker flask cell culture compositions
when cultured in Media 1 and Media 2 which had a value of 2.25. These experiments
established that the shaker flask model was comparable to the 2L cell culture model and was
suitable for use in subsequent experiments.
For experimentation with cell culture media compositions that were supplemented
with the antioxidant hypotaurine, antibody producing CHO cells were inoculated at
approximately 1.0 x 10 cells/mL in a 250 mL flask containing 100 mL of basal Media 1.
Media 1 was supplemented with 9.16 mM (100%), 4.58 mM (50%), or 2.29 mM (25%)
hypotaurine for use in cell culture on Day 0. The cells were cultured in fed-batch mode with
addition of 10 mL of feed Media 2 per liter of cell culture fluid at Days 3, 6 and 9 for
initiation of the production phase. An additional experimental sample involved the
incremental addition of 9.16 mM hypotaurine over the cell culture period. Specifically, 2.29
mM (25%) hypotaurine was added on Day 0 of cell culture in basal Media 1, and 25% was
added on Day 3, Day 6 and Day 9 in feed Media 2. A positive control was included by
culturing cells in Media 1 and 2 without hypotaurine supplementation. The negative control
was included by culturing cells cultured in Media 3 and Media 4 without hypotaurine
supplementation. As described above, the concentration of glucose was analyzed every day
and if the glucose concentration fell below 3 g/L, it was replenished from a 500 g/L stock
solution of glucose for prevention of glucose depletion. The cell cultures were maintained at
pH 7.0 and a temperature of 37ºC from days 0 through 3, and then at 35ºC after day 3. The
cell cultures were agitated at 275 rpm and the dissolved oxygen level was at 30% of air
saturation. VCC and cell viability was measured daily using a ViCell automated cell
counter (Beckman Coulter, Fullerton, CA). The cell culture fluid was collected daily by
centrifuging 1 mL of cell culture fluid for determination of antibody titer using high
performance liquid chromatography. At the end of the cell culture duration on day 14, the
cell culture fluid from all samples was harvested by centrifugation. The monoclonal antibody
in the harvested cell culture fluid was purified using affinity chromatography. Color intensity
of the concentrated antibody composition was measured in the purified pool using an assay
wherein higher numerical values indicate higher color intensity and lower numerical values
indicate lower color intensity. The numerical results were normalized to the positive control,
where the value for the positive control was set at 0% change in color intensity. Growth as
measured by VCC (Fig. 4A) and cell viability (Fig. 4B) was comparable among all the cell
cultures tested. Furthermore, with the exception of incremental addition of hypotaurine, cell
cultures cultured in media supplemented with hypotaurine produced the same level of
antibody titers as cell cultures cultured in media not containing hypotaurine (Fig.4C). Color
intensity was found to be reduced with higher concentration of hypotaurine with the greatest
reduction observed in media containing 9.16 mM hypotaurine (Fig. 5). This reduction in
color intensity was optimal when hypotaurine was added as a bolus at Day 1 rather than
added incrementally over the course of cell culture incubation. Comparison of color intensity
values obtained from cell culture experiments and incubation experiments (See Example 1)
demonstrated that the results of the incubation screening experiments (Fig. 5, empty circles)
correlated well with the results from cell culture experiments (Fig. 5, filled circles).
Similar experiments were conducted for antibody compositions isolated from cell
cultures harvested in basal Media 3 and feed Media 4 to determine if the color reducing effect
of hypotaurine extended to other cell culture media. Briefly, as above, antibody producing
CHO cells were inoculated at approximately 1.0 x 10 cells/mL in a 250 mL flask containing
100 mL of basal Media 3. Media 3 was supplemented with 12.95 mM (1X), 25.9 mM (2X),
or 38.85 mM (3X) hypotaurine for use in cell culture on Day 0. The cells were cultured in
fed-batch mode with addition of 10 mL of feed Media 4 per liter of cell culture fluid at Days
3, 6 and 9 for initiation of the production phase. A positive control was included by culturing
cells in Media 1 and 2 without hypotaurine supplementation. The cultures were placed on a
shaker platform and agitated at 150 rpm in a 5% CO incubator with a temperature of 37°C
from day 0 up to day 3 of the cell culture cycle with a temperature shift to 35°C on day 4
until the end of the cell culture cycle at day 14. Osmolality, offline pH and metabolite
concentrations were measured as described above. VCC and cell viability was measured
daily using a ViCell automated cell counter (Beckman Coulter, Fullerton, CA). The cell
culture fluid was collected daily by centrifuging 1 mL of cell culture fluid for determination
of antibody titer using high performance liquid chromatography. At the end of the cell
culture duration on day 14, the cell culture fluid from all samples was harvested by
centrifugation. The monoclonal antibody in the harvested cell culture fluid was purified
using affinity chromatography. Color intensity of the concentrated antibody composition was
measured in the purified pool using an assay wherein higher numerical values indicate higher
color intensity and lower numerical values indicate lower color intensity. The numerical
results were normalized to the positive control, where the value for the positive control was
set at 0% change in color intensity. Color intensity was found to be reduced with higher
concentration of hypotaurine with the greatest reduction observed in media containing 38.85
mM hypotaurine (Fig. 6).
Example 3: Characterization of hypotaurine analogs in reduction of color in antibody
compositions isolated from antibody-producing cell lines.
Hypotaurine analogs were tested to assess if they demonstrated a color reducing
effect in antibody containing compositions. Antibody producing CHO cells were inoculated
at approximately 1.0 x 10 cells/mL in 2-liter stirred bioreactors (Applikon, Foster City, CA)
containing 1L of basal Media 1 supplemented with 12.95 mM hypotaurine or 10 mM
carboxymethylcysteine (CAS number 6383). Cells were cultured in fed-batch mode with
addition of 100 mL of feed Media 2 per liter of cell culture fluid at days 3, 6 and 9 for
initiation of the production phase. A positive control was included by culturing cells in Media
1 and 2 without hypotaurine supplementation. The concentration of glucose was analyzed
every day and if the glucose concentration fell below 2 g/L, it was replenished from a 1.5 g/L
stock solution of glucose for prevention of glucose depletion. Reactors were equipped with
calibrated dissolved oxygen, pH and temperature probes. Dissolved oxygen was controlled
on-line through sparging with air and/or oxygen. pH was controlled through addition of CO
or Na CO and antifoam was added to the cultures as needed. The cell cultures were
maintained at pH 7.0 and a temperature of 37ºC from days 0 through 3, and then at 35ºC after
day 3. The cell cultures were agitated at 275 rpm and the dissolved oxygen level was at 30%
of air saturation. Osmolality was monitored using an osmometer from Advanced Instruments
(Norwood, MA). Offline pH and metabolite concentrations were also determined daily using
a Nova Bioprofile 400 (Nova Biomedical, Waltham, MA). VCC and cell viability was
measured daily using a ViCell automated cell counter (Beckman Coulter, Fullerton, CA).
The cell culture fluid was collected daily by centrifuging 1 mL of cell culture fluid for
determination of antibody titer using high performance liquid chromatography. At the end of
the cell culture duration on day 14, when the amount of protein in the culture was
approximately 2-10 g/L, the cell culture fluid from all samples was harvested by
centrifugation. The monoclonal antibody in the harvested cell culture fluid was purified
using protein A affinity chromatography. The protein A pool was concentrated to 150 g/L
using Amicon Centricon centrifugal filter devices (Millipore Corporation, Billerica, MA).
Color intensity of the concentrated antibody composition was measured in the concentrated
protein A pool using two different assays wherein higher numerical values indicated higher
color intensity and lower numerical values indicated lower color intensity. Growth as
measured by VCC (Fig. 7A) and cell viability (Fig. 7B) was comparable among all cell
cultures tested. Cell cultures cultured in media supplemented with hypotaurine or
carboxymethylcysteine produced comparable levels of antibody titers (Fig.8). Using a
specific color assay, color intensity of isolated antibody composition was found to be reduced
by 27 % and 13% when antibody-producing cells were cultured media supplemented with
hypotaurine and carboxymethylcysteine, respectively (Fig. 9A). This color intensity
reduction was confirmed by using a second color assay which detected an approximate 17%
and 13% color intensity reduction in antibody compositions isolated from cell cultured in
media supplemented with hypotaurine and carboxymethylcysteine, respectively (Fig. 9B).
Example 4: Characterization of aminoguanidine in reduction of color in antibody
compositions isolated from antibody-producing cell lines.
In order to identify a compound that reduces in antibody compositions and works
under cell culture conditions, a screen assay in cell free medium was conducted. Taurine,
carnosine and aminoguanidine were chosen for screening. These compounds were dissolved
in 25 mL culture media at the concentration of 1.2 g/L (taurine), 13.6 g/L (carnosine), and
27.2 g/L (aminoguanidine hydrochloride). After pH adjustment to a range from 6.8 to 7.2
and sterile filtration with Steriflip filter units (Millipore, Billerica, MA) the solution was
incubated in 50 mL Falcon tubes (BD Biosciences, San Jose, CA) equipped with TubeSpin
caps (TPP Techno Plastic Products AG, Trasadingen, Switzerland). CHO cells were
incubated for 7 days in a moisture controlled cell culture incubator at 37°C and 250 rpm with
no protection from light to produce the monoclonal antibody.
The monoclonal antibody in the harvested cell culture fluid (HCCF) and the
incubation broth was further purified with affinity chromatography. Color intensity of the
concentrated antibody composition was measured in the purified pool using an assay wherein
higher numerical values indicated higher color intensity and lower numerical values indicated
lower color intensity.
The relative color intensity for antibodies produced in culture medium containing
taurine, carnosine, or aminoguanidine are shown in Fig. 10. The data indicated that
aminoguanidine was able to decrease color by about 71%, and the relative color intensity
value was even lower than the value for the negative control in which the antibody was
incubated without any glucose.
[0196A] Certain statements that appear herein are broader than what appears in the
statements of the invention. These statements are provided in the interests of providing the
reader with a better understanding of the invention and its practice. The reader is directed to
the accompanying claim set which defines the scope of the invention.
[0196B] In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that
such documents, or such sources of information, in any jurisdiction, are prior art, or form part
of the common general knowledge in the art.
Claims (25)
1. A method of producing an antibody or fragment thereof comprising the step of culturing a Chinese Hamster Ovary (CHO) cell comprising a nucleic acid encoding the antibody or fragment thereof in a cell culture medium comprising a media component selected from the group consisting of hypotaurine at a concentration of about 1 mM to about 50 mM, s- carboxymethylcysteine at a concentration of about 0.5 mM to about 120 mM, and taurine at a concentration of about 2 mM to about 50 mM, and wherein the cell culture medium comprising the media component reduces the color intensity of a composition comprising the antibody or fragment thereof produced by the cell as compared to the color intensity of a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the media component.
2. The method of claim 1, wherein the cell culture medium comprising the media component reduces the color intensity of a composition comprising the antibody or fragment thereof produced by the cell by at least about 0.1% as compared to a composition comprising the antibody or fragment thereof produced by the cell cultured in a cell culture medium that does not comprise the media component.
3. The method of claim 1, wherein the cell culture medium comprising the media component reduces the color intensity of a composition comprising the antibody or fragment thereof produced by the cell by about 5% to about 50% as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise the media component.
4. The method of claim 1, wherein the cell culture medium comprises the hypotaurine at a concentration from about 1.0 mM to about 40.0 mM.
5. The method of claim 1, wherein the cell culture medium comprises the hypotaurine at a concentration from about 1.0 mM to about 10.0 mM.
6. The method of any one of claims 1-5, wherein the cell culture medium is a chemically defined cell culture medium.
7. The method of any one of claims 1-6, wherein the cell culture medium is a chemically undefined cell culture medium.
8. The method of any one of claims 1-7, wherein the cell culture medium is a basal cell culture medium.
9. The method of any one of claims 1-7, wherein the cell culture medium is a feed cell culture medium.
10. The method of any one of claims 1-9, wherein the media component is added to the cell culture medium on at least one day of a cell culture cycle.
11. The method of any one of claims 1-9, wherein the media component is added to the cell culture medium on day 0 of a 14 day cell culture cycle.
12. The method of any one of claims 1-11, wherein the antibody or fragment thereof is an IgG1 antibody or fragment thereof.
13. The method of claim 12, wherein the antibody or fragment thereof is secreted into the cell culture medium comprising the media component.
14. The method of any one of claims 1-13, further comprising the step of recovering the antibody or fragment thereof from the cell culture medium comprising the media component.
15. The method of claim 14, wherein a composition comprising the recovered antibody or fragment thereof is a liquid composition or a non-liquid composition.
16. The method of claim 15, wherein the composition comprising the recovered antibody or fragment thereof appears as a colorless or slightly colored liquid.
17. A method of producing an antibody or fragment thereof comprising the step of culturing a Chinese Hamster Ovary (CHO) cell comprising a nucleic acid encoding the antibody or fragment thereof in a cell culture medium, wherein the cell culture medium comprises one or more of components (a)-(e): (a) hypotaurine at a concentration of about 1 mM to about 50 mM; (b) s-carboxymethylcysteine at a concentration of about 0.5 mM to about 120 mM; (c) butylated hydroxyanisole at a concentration of about 0.001 mM to about 0.2 mM; (d) lipoic acid at a concentration of about 0.01 mM to about 1.5 mM; and (e) quercitrin hydrate at a concentration of about 0.005 mM to about 0.04 mM; and wherein the cell culture medium comprising one or more of components (a)-(e) reduces the color intensity of a composition comprising the antibody or fragment thereof produced by the cell as compared to a composition comprising the polypeptide produced by the cell cultured in a cell culture medium that does not comprise one or more of components (a)-(e).
18. The method of claim 17, wherein the cell culture medium comprising one or more of components (a)-(e) reduces the color intensity of a composition comprising the antibody or fragment thereof produced by the cells by at least about 0.1% as compared to a composition comprising the antibody or fragment thereof produced by the cell cultured in a cell culture medium that does not comprise the one or more of components (a)-(e).
19. The method of claim 17, wherein the cell culture medium comprising one or more of components (a)-(e) reduces the color intensity of a composition comprising the antibody or fragment thereof produced by the cells by about 5% to about 50% as compared to a composition comprising the antibody or fragment thereof produced by the cell cultured in a cell culture medium that does not comprise the one or more of components (a)-(e).
20. The method of any one of claims 17-19, wherein the cell culture medium comprises hypotaurine at a concentration from about 2.0 mM to about 50.0 mM.
21. The method of any one of claims 17-20, wherein the cell culture medium comprises s- carboxymethylcysteine at a concentration from about 8.0 mM to about 12.0 mM.
22. The method of any one of claims 17-21, wherein the cell culture medium comprises butylated hydroxyanisole at a concentration from about 0.025 mM to about 0.040 mM.
23. The method of any one of claims 17-22, wherein the cell culture medium comprises lipoic acid at a concentration from about 0.040 mM to about 0.060 mM.
24. The method of any one of claims 17-23, wherein the cell culture medium comprises quercitrin hydrate at a concentration from about 0.010 mM to about 0.020 mM.
25. The method of any one of claims 17-24, wherein the cell culture medium is a chemically defined cell culture medium.
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