WO2017192691A1 - Culture cellulaire contenant des inhibiteurs de bromodomaine - Google Patents

Culture cellulaire contenant des inhibiteurs de bromodomaine Download PDF

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WO2017192691A1
WO2017192691A1 PCT/US2017/030799 US2017030799W WO2017192691A1 WO 2017192691 A1 WO2017192691 A1 WO 2017192691A1 US 2017030799 W US2017030799 W US 2017030799W WO 2017192691 A1 WO2017192691 A1 WO 2017192691A1
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alkyl
optionally substituted
cell culture
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cells
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Mark TIE
Hari KAMADURAI
Scott Estes
Marc Muskavitch
Chapman WRIGHT
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Biogen MA Inc
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Biogen MA Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

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  • the present invention generally pertains to a cell culture medium and methods of using thereof.
  • the cell culture medium described herein generally comprises a compound (e.g., a bromodomain inhibitor) that can enhance product titer, cell viability, cell specific productivity, and/or viable cell density of a cell culture containing the medium.
  • a compound e.g., a bromodomain inhibitor
  • the present invention is directed to a cell culture medium and
  • the present invention provides a cell culture medium comprising a bromodomain inhibitor. In one embodiment, the present invention provides a cell culture medium comprising BI2536 and/or Cl-amidine. In one embodiment, the present invention also provides a cell culture comprising cells (e.g., mammalian cells) and a cell culture medium, e.g., a cell culture medium comprising a bromodomain inhibitor. In one embodiment, the cell culture or cell culture medium can be used in a batch culture, fed-batch culture, a perfusion culture, a shake flask culture, or a bioreactor. In one embodiment, the cell culture medium is a basal medium. In one embodiment, the cell culture medium is a feed medium.
  • the cell culture medium comprises a bromodomain inhibitor selected from the group consisting of RVX208, SGC-CBP30, JQ1, CPI-203, PFI-1, 1-BET-762, OTX-015, 1-BET151, bromosporine, I-CBP112, and a combination thereof.
  • a bromodomain inhibitor selected from the group consisting of RVX208, SGC-CBP30, JQ1, CPI-203, PFI-1, 1-BET-762, OTX-015, 1-BET151, bromosporine, I-CBP112, and a combination thereof.
  • Other suitable bromodomain inhibitors are described herein.
  • the bromodomain inhibitor can be present in the cell culture medium in various concentrations. In one embodiment, the bromodomain inhibitor is present in the cell culture medium in a concentration ranging from about 1 uM to about 100 uM. In one embodiment, the bromodomain inhibitor can also be present in the cell culture medium in a concentration sufficient, when culturing mammalian cells in a fed-batch mode for 14 days (e.g., under the culturing conditions specified in the Examples section herein), (1) to produce a protein or polypeptide of interest at a titer at least about 15% (e.g., about 15% to about 100%)) greater than the titer produced from a control cell culture that does not contain a bromodomain inhibitor; (2) to generate a viable cell density of at least about 20%) (e.g., about 20%> to about 100%>) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor; (3) to generate a cell specific productivity of at least about 10%> (e.
  • the present invention further provides a method of culturing cells, e.g., to produce a protein or polypeptide of interest.
  • the method comprises contacting the cells with a cell culture medium described herein.
  • the culturing is conducted in a batch mode, a fed-batch mode, or a perfusion mode. In a specific embodiment, the culturing is conducted in a fed-batch mode.
  • Various cells can be cultured using the cell culture medium described herein.
  • the cells are mammalian cells.
  • the mammalian cells comprise a polynucleotide encoding a protein or polypeptide of interest.
  • the cell culture medium comprises a bromodomain inhibitor.
  • the mammalian cells are CHO cells (e.g., CHO Kl cells). In one
  • the cell culture is a fed-batch culture.
  • the present invention further provides a method of producing a protein or polypeptide of interest.
  • the method comprises culturing cells (e.g., mammalian cells as described herein) capable of producing the protein or polypeptide of interest in a culture comprising a medium described herein.
  • the method produces the protein or polypeptide of interest in a fed-batch mode.
  • the culture comprises a medium comprising a bromodomain inhibitor
  • the method (1) produces the protein or polypeptide of interest (e.g., at day 14 of the culture) at a titer greater than the titer produced from a control cell culture that does not contain a bromodomain inhibitor; (2) generates a viable cell density (e.g., at day 14 of the culture) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor; (3) generates a cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor; and/or (4) achieves a cell viability (e.g. at day 14 of the culture) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • a viable cell density e.g., at day 14 of the culture
  • a cell specific productivity e.g., a cumulative cell specific productivity at day 14
  • the present invention further provides a method of
  • the method comprises culturing cells (e.g., mammalian cells as described herein) in a culture comprising a medium described herein.
  • the medium comprises a bromodomain inhibitor.
  • FIGs. 1A and IB show structures of certain specific compounds described herein.
  • the chemical compounds shown in FIG. 1A are bromodomain inhibitors, including RVX208, SGC-CBP30, JQ1, CPI-203, PFI-1, 1-BET-762, OTX-015, 1-BET151, bromosporine, and I-CBP112.
  • the chemical structures of BI2536 and Cl-amidine are shown in FIG. IB.
  • FIGs. 2A-2C show the effect on antibody titers by adding SGC-CBP30 to cell cultures at different concentrations.
  • FIG. 2A presents bar graphs showing the antibody titers produced at Day 10, Day 12, and Day 14 by a fed-batch culture with SGC-CBP30 added at different concentrations on Day 8 and Day 11.
  • FIG. 2B presents bar graphs showing the antibody titers produced at Day 10, Day 12, and Day 14 by a fed-batch culture with DMSO added at different volumes on Day 8 and Day 11.
  • 2C presents bar graphs comparing the antibody titers produced at Day 10, Day 12, and Day 14 by fed- batch cultures: (1) cultures with SGC-CBP30 added at different concentrations on Day 8 and Day 11; (2) cultures with DMSO added at 10 uL on Day 8 and Day 11; and (3) control cultures (no SGC-CBP30 and no DMSO).
  • FIGs. 3A-3D show the effect on antibody titer, cumulative cell specific
  • FIG. 3A presents a graph comparing the CD-40 antibody titers produced over 14 days by fed-batch cultures: (1) cultures with SGC-CBP30 added at 3 uM or 30 uM on Day 8 and Day 11, (2) cultures with DMSO added at 1.5 uL or 15 uL on Day 8 and Day 11, and (3) control cultures (no SGC-CBP30 and no DMSO).
  • FIG. 3A presents a graph comparing the CD-40 antibody titers produced over 14 days by fed-batch cultures: (1) cultures with SGC-CBP30 added at 3 uM or 30 uM on Day 8 and Day 11, (2) cultures with DMSO added at 1.5 uL or 15 uL on Day 8 and Day 11, and (3) control cultures (no SGC-CBP30 and no DMSO).
  • FIG. 3A shows that antibody titers were consistently higher in cultures with SGC-CBP30 added at 3 uM or 30 uM than the titers produced by cultures with DMSO added at 1.5 uL or 15 uL or control cultures.
  • FIG. 3B presents a graph comparing the cumulative cell specific productivity on Days 4, 7, 10, 12, and 14 for the different cultures (l)-(3).
  • FIG. 3B shows that the cumulative cell specific productivity was also higher for cultures with SGC-CBP30 added at 3 uM or 30 uM than that observed for cultures with DMSO added at 1.5 uL or 15 uL or control cultures.
  • FIGs. 3C and 3D presents graphs showing the viable cell density (VCD) and viability data of the different cultures (l)-(3). [0014] FIGs.
  • FIG. 4A-4D show the effect on antibody titer, cumulative cell specific productivity, cell viability and variable cell density by adding SGC-CBP30 at 3 uM or 30 uM.
  • CHO-Kl cell line was used to produce OSMR antibody.
  • FIG. 4A presents a graph comparing the OSMR antibody titers produced over 14 days by fed- batch cultures: (1) cultures with SGC-CBP30 added at 3 uM or 30 uM on Day 8 and Day 11, (2) cultures with DMSO added at 1.5 uL or 15 uL on Day 8 and Day 11, and (3) control cultures (no SGC-CBP30 and no DMSO).
  • FIG. 4A shows that antibody titers were consistently higher in cultures with SGC-CBP30 added at 3 uM or 30 uM than the titers produced by cultures with DMSO added at 1.5 uL or 15 uL or control cultures.
  • FIG. 4B presents a graph comparing the cumulative cell specific productivity on Days 4, 7, 10, 12, and 14 for the different cultures (l)-(3).
  • FIG. 4B shows that the cumulative cell specific productivity was also higher for cultures with SGC-CBP30 added at 3 uM or 30 uM than that observed for cultures with DMSO added at 1.5 uL or 15 uL or control cultures.
  • FIGs. 4C and 4D presents graphs showing the viable cell density (VCD) and viability data of the different cultures (l)-(3).
  • FIGs. 5A-5D show the effect on antibody titer, cumulative cell specific
  • FIG. 5A presents a graph comparing the STX-200 antibody titers produced over 14 days by fed-batch cultures: (1) cultures with SGC-CBP30 added at 3 uM or 30 uM on Day 8 and Day 11, (2) cultures with DMSO added at 1.5 uL or 15 uL on Day 8 and Day 11, and (3) control cultures (no SGC-CBP30 and no DMSO).
  • FIG. 5A presents a graph comparing the STX-200 antibody titers produced over 14 days by fed-batch cultures: (1) cultures with SGC-CBP30 added at 3 uM or 30 uM on Day 8 and Day 11, (2) cultures with DMSO added at 1.5 uL or 15 uL on Day 8 and Day 11, and (3) control cultures (no SGC-CBP30 and no DMSO).
  • FIG. 5A shows that antibody titers were consistently higher in cultures with SGC-CBP30 added at 3 uM or 30 uM than the titers produced by cultures with DMSO added at 1.5 uL or 15 uL or control cultures.
  • FIG. 5B presents a graph comparing the cumulative cell specific productivity on Days 4, 7, 10, 12, and 14 for the different cultures (l)-(3).
  • FIG. 5B shows that the cumulative cell specific productivity was also higher for cultures with SGC-CBP30 added at 3 uM or 30 uM than that observed for cultures with DMSO added at 1.5 uL or 15 uL or control cultures.
  • FIGs. 5C and 5D present graphs showing the viable cell density (VCD) and viability data of the different cultures (l)-(3).
  • FIGs. 6A-6D show the effect on antibody titer, cumulative cell specific
  • FIGs. 6A-6D CHO-K1 cell line was used to produce CD-40 antibody.
  • FIGs. 6A-6B present bar graphs comparing CD-40 antibody titers observed on Day 12 and Day 14 in fed-batch cultures: (1) cell cultures with different compounds (RVX-208, SGC-CBP30, 1-BET-762, OTX015, bromosporine, PFI-1) added at 3 uM or 30 uM on Day 8 and Day 11, (2) cultures with DMSO added at 1.5 uL or 15 uL on Day 8 and Day 11, and (3) control cultures (no compound and no DMSO).
  • FIGs. 6A-6B show that adding the tested compounds to the cell cultures improved both antibody titers and cell specific productivity.
  • FIGs. 6C-6D present bar graphs showing the viable cell density (VCD) and viability data of the different cultures (l)-(3).
  • antibody is used to mean an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing etc., through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, monovalent or monospecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments
  • scFv single chain Fv mutants
  • multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, monovalent or monospecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
  • An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • antibody fragment refers to a portion of an intact
  • antibody refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.
  • alkyl refers to a saturated aliphatic
  • the alkyl is a lower alkyl having 1 to 4 carbon atoms, i.e., C 1-4 .
  • a numerical range; e.g., " 1-4” is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, up to and including 4 carbon atoms.
  • the alkyl group is not substituted. However, in one embodiment, the alkyl group can be substituted (e.g., with 1 to 5 substituent groups) when specified.
  • alkylene linker refers to an alkyl linking group, i.e., an alkyl group that links one atom/group to another atom/group in a molecule.
  • alkoxy group as used herein refers to an -O-alkyl group, wherein the alkyl can be any of those as defined herein.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • stereoisomer as used herein includes geometric isomers, such as E or
  • stereoisomeric mixture includes any mixture in any ratio of stereoisomers defined herein.
  • a stereoisomeric mixture includes a racemic mixture.
  • a stereoisomeric mixture includes an enantiomerically enriched mixture.
  • a stereoisomeric mixture includes a mixture of diastereomers in any ratio.
  • enantiomeric excess refers to a measure for how much of one enantiomer is present compared to the other.
  • percent enantiomeric excess is defined as
  • * 100, where R and S are the respective mole or weight fractions of enantiomers in a mixture such that R + S 1.
  • the percent enantiomeric excess is defined as ([a] ObS /[oc]max)* 100, where [a] 0b s is the optical rotation of the mixture of enantiomers and [a] max is the optical rotation of the pure enantiomer.
  • basic media formulation or “basal media” as used herein refers to any cell culture media used to culture cells that has not been modified either by
  • bioreactor refers to any vessel used for the growth of a mammalian cell culture.
  • the bioreactor can be of any size so long as it is useful for the culturing of mammalian cells.
  • the bioreactor will be at least 1 liter and can be 10, 50, 100, 250, 500, 1000, 2000, 2500, 3000, 5000, 8000, 10,000, 12,0000, 15,000, 20,000, 30,000 liters or more, or any volume in between.
  • a bioreactor will be 10 to 5,000 liters, 10 to 10,000 liters, 10 to 15,000 liters, 10 to 20,000 liters, 10 to 30,000 liters, 50 to 5,000 liters, 50 to 10,000 liters, 50 to 15,000 liters, 50 to 20,000 liters, 50 to 30,000 liters, 1,000 to 5,000 liters, or 1,000 to 3,000 liters.
  • the internal conditions of the bioreactor including, but not limited to pH and temperature, are typically controlled during the culturing period.
  • the bioreactor can be composed of any material that is suitable for holding mammalian cell cultures suspended in media under the culture conditions of the present invention, including glass, plastic or metal.
  • production bioreactor refers to the final bioreactor used in the production of the polypeptide or protein of interest.
  • the volume of the large-scale cell culture production bioreactor is typically at least 500 liters and can be 1000, 2000, 2500, 5000, 8000, 10,000, 12,0000, 15,000 liters or more, or any volume in between.
  • the large scale cell culture reactor will be between about 500 liters and about 20,000 liters, about 500 liters and about 10,000 liters, about 500 liters and about 5,000 liters, about 1,000 liters and about 30,000 liters, about 2,000 liters and about 30,000 liters, about 3,000 liters and about 30,000 liters, about 5,000 liters and about 30,000 liters, or about 10,000 liters and about 30,000 liters, or a large scale cell culture reactor will be at least about 500 liters, at least about 1,000 liters, at least about 2,000 liters, at least about 3,000 liters, at least about 5,000 liters, at least about 10,000 liters, at least about 15,000 liters, or at least about 20,000 liters.
  • One of ordinary skill in the art will be aware of and will be able to choose suitable bioreactors for use in practicing the present invention.
  • bromodomain inhibitor includes an inhibitor of any protein that contains a bromodomain. Some exemplary bromodomain-containing proteins are described by Muller et. al. in “Bromodomains as therapeutic targets,” Expert Reviews in Molecular Medicine 13:329 (2011).
  • a "bromodomain inhibitor” includes any compound that has inhibitory effect against BET (Bromodomain and extraterminal domain family), which includes BRD2, BRD3, BRD4 and BRDT.
  • a "bromodomain inhibitor” includes any compound that has inhibitory effect against a protein in human BRD families. Human BRD families are described, for example, in Hay D.A.
  • the bromodomain inhibitor is present in the cell culture medium in a concentration sufficient, when culturing mammalian cells in a fed-batch mode for 14 days (e.g., under the culturing conditions specified in the Examples section herein), to generate a cell specific productivity of about 10% to about 60% (e.g., about 10%), about 20%, about 30%, about 40%, about 50%, about 60%, or any ranges between the specified values) greater than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • a concentration sufficient, when culturing mammalian cells in a fed-batch mode for 14 days (e.g., under the culturing conditions specified in the Examples section herein), to generate a cell specific productivity of about 10% to about 60% (e.g., about 10%), about 20%, about 30%, about 40%, about 50%, about 60%, or any ranges between the specified values) greater than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the medium comprising a bromodomain inhibitor is a feed medium.
  • the bromodomain inhibitor is present in the feed medium in a sufficient concentration such that when added to a culture, the bromodomain inhibitor is present in the culture in a concentration ranging from about 1 uM to about 100 uM, e.g., from about 1 uM to about 75 uM, about 1 uM to about 50 uM, about 1 uM to about 30 uM, about 1 uM to about 10 uM, about 1 uM to about 3 uM, about 3 uM to about 100 uM, about 3 uM to about 75 uM, about 3 uM to about 50 uM, about 3 uM to about 30 uM, about 3 uM to about 10 uM, about 10 uM to about 100 uM, about 10 uM to about 75 uM, about 10 uM to about 50 uM, about 10 uM to about 30 uM, about 3 u
  • the bromodomain inhibitor can be added to reach a concentration in the culture of about 1 uM, about 3 uM, about 10 uM, about 30 uM, about 50 uM, about 75 uM, or about 100 uM. In one embodiment, the bromodomain inhibitor can be added to reach a concentration in the culture of less than about 1 uM (e.g., about 0.5 uM, about 0.1 uM, about 0.01 uM, or any ranges between the specified values).
  • the bromodomain inhibitor is present in the feed medium in a sufficient concentration such that when added to a culture, the bromodomain inhibitor is present in the culture in a concentration sufficient, when culturing mammalian cells in a fed-batch mode for 14 days (e.g., under the culturing conditions specified in the Examples section herein), to produce a protein or polypeptide of interest at a titer at least about 15% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%), or at least about 200%) greater than the titer produced from a control cell culture that does not contain a bromodomain inhibitor.
  • a titer at least about 15% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%
  • the bromodomain inhibitor can be added to reach a concentration in the culture sufficient, when culturing mammalian cells in a fed-batch mode for 14 days (e.g., under the culturing conditions specified in the Examples section herein), to achieve a cell viability of about 10% to about 60% (e.g., about 10%, about 20%), about 30%), about 40%, about 50%, about 60%, or any ranges between the specified values) greater than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • a concentration in the culture sufficient, when culturing mammalian cells in a fed-batch mode for 14 days (e.g., under the culturing conditions specified in the Examples section herein), to achieve a cell viability of about 10% to about 60% (e.g., about 10%, about 20%), about 30%), about 40%, about 50%, about 60%, or any ranges between the specified values) greater than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the bromodomain inhibitor can be added to cell culture medium at day 8 and day 11, at day 8 and day 10, or at day 9 and day 12, when culturing mammalian cells in a fed-batch mode for 14 days.
  • the bromodomain inhibitor can be added at day 8 and day 11 to reach a concentration in the culture sufficient, when culturing mammalian cells in a fed-batch mode for 14 days (e.g., under the culturing conditions specified in the Examples section herein), to achieve a cell viability of about 10% to about 60%> (e.g., about 10%>, about 20%), about 30%), about 40%>, about 50%>, about 60%>, or any ranges between the specified values) greater than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • ring A is optionally substituted with 1 to 4 R 10 , each independently selected from the group consisting of halogen, OH, C 1-4 alkyl, and C 1-4 alkoxyl, wherein the C 1-4 alkyl and Ci- 4 alkoxyl are optionally substituted with 1-3 halogens; and
  • ring B is optionally substituted with 1 to 5 R 11 each independently selected from the group consisting of OH, halogen, C 1-4 alkyl, C 1-4 alkoxyl, wherein the C 1-4 alkyl and C 1-4 alkoxyl are optionally substituted with 1-3 halogens or OR 100 ,
  • the bromodomain inhibitor of Formula I can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula I has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • R 11 at each occurrence can be independently selected from the group consisting of F, CI, OH, Ci -4 alkyl (e.g., Me, Et, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl), and Ci -4 alkoxyl (e.g., MeO, EtO, n-propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, isobutoxyl, or tert-butoxyl), wherein the Ci -4 alkyl and Ci-4 alkoxyl are not substituted with halogens or OR 100 .
  • Ci -4 alkyl e.g., Me, Et, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butoxyl
  • Ci -4 alkyl e.g., Me, Et, n-propyl
  • ring A in Formula la is not substituted with R 10 .
  • ring A in Formula la is substituted with 1 or 2 R 10 , each independently selected from the group consisting of F, CI, Me, Et, OMe, and OEt.
  • ring A in Formula la is substituted with 1 or 2 methoxyl groups.
  • ring B in Formula la is not substituted with R 11 .
  • ring B in Formula la is substituted with 1 or 2 R 11 , each independently selected from the group consisting of F, CI, Me, and Et.
  • ring B in Formula la is substituted with 1 or 2 methyl groups.
  • L 10 is CH 2 CH 2 . In one embodiment, L 10 is CH 2 CH 2 CH 2 . In one embodiment, L 10 is CH 2 CH 2 CH 2 CH 2 .
  • the cell culture medium comprises RVX-208,
  • the only bromodomain inhibitor present in the cell culture medium is RVX-208.
  • the cell culture medium comprises RVX-208 and at least one additional bromodomain inhibitor.
  • the cell culture medium comprises a bromodomain inhibitor of Formula II:
  • ring B is optionally substituted with 1-3 R 21 , each independently selected from the group consisting of halogen and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens;
  • ring D is optionally substituted with 1-5 R 23 , each independently selected from the group consisting of halogen, OH, C 1-4 alkyl, and C 1-4 alkoxyl, wherein the C 1-4 alkyl and C 1-4 alkoxyl are optionally substituted with 1-3 halogens;
  • L 20 is a C 2 -4 alkylene linker, optionally substituted with 1 or 2 C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens;
  • L 21 is a Ci-4 alkylene linker, optionally substituted with 1 or 2 C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens.
  • the bromodomain inhibitor of Formula II can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula II has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • ring A of Formula II is not substituted with R 20 .
  • ring A of Formula II is substituted with 1 or 2 R 20 .
  • ring A of Formula II is substituted with two methyl groups.
  • ring B of Formula II is substituted with 1, 2, or 3 R 21 , each independently selected from the group consisting of F, CI, Me, Et, OMe, and OEt.
  • ring C of Formula II is substituted with 1, 2, or 3 R 22 . In one embodiment, two R 22 can be substituted on the same carbon. [0084] In one embodiment, ring D of Formula II is not substituted with R . In one embodiment, ring D of Formula II is substituted with 1, 2, 3, 4, or 5 R 23 . For example, ring D of Formula II is substituted with 1 or 2 R 23 , each independently selected from the group consisting of halogen, C 1-4 alkyl, and Ci -4 alkoxyl.
  • the cell culture medium comprises a bromodomain inhibitor of Formula Ila:
  • ring B is optionally substituted with 1 or 2 R 21 , each independently selected from the group consisting of halogen and C 1-4 alkyl;
  • ring D is optionally substituted with 1 or 2 R 23 , each independently selected from the group consisting of halogen, C 1-4 alkyl, and Ci -4 alkoxyl,
  • L 20 is a C 2 -4 alkylene linker, optionally substituted with one methyl or ethyl group; and L 21 is a Ci-4 alkylene linker, optionally substituted with one methyl or ethyl group.
  • the bromodomain inhibitor of Formula Ila can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula Ila has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • ring B of Formula Ila is not substituted with R 21 .
  • CH(Me)CH 2 wherein the CH 2 can be directly attached to the nitrogen atom of the morpholine ring or the nitrogen atom of the benzoimidazole ring.
  • L 21 of Formula Ila is an unsubstituted C 1-4 alkylene linker, e.g., CH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , or CH 2 CH 2 CH 2 CH 2 .
  • L 21 of Formula Ila is C 1-4 alkylene linker which is substituted with one methyl or ethyl group, e.g., CH(Me)CH 2 , wherein the CH 2 can be directly attached to the carbon atom on ring D or the carbon atom of the benzoimidazole ring.
  • L is a C 2- 4 alkylene linker, optionally substituted with one methyl group
  • the bromodomain inhibitor of Formula lib can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula lib has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • ring D is substituted with 2 R , each independently selected from the group consisting of F, CI, Me, Et, OMe, and OEt, wherein the two R can be ortho, meta, or para to each other.
  • the two R are ortho to each other, and neither of the two R 23 is ortho to L 21 .
  • L 20 of Formula lib is an unsubstituted C 2-4 alkylene linker, e.g., CH 2 CH 2 , CH 2 CH 2 CH 2 , or CH 2 CH 2 CH 2 CH 2 .
  • L 20 of Formula lib is CH(Me)CH 2 , wherein the CH 2 can be directly attached to the nitrogen atom of the morpholine ring or the nitrogen atom of the benzoimidazole ring.
  • L 21 of Formula lib is an unsubstituted C 1-4 alkylene linker, e.g., CH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , or CH 2 CH 2 CH 2 CH 2 .
  • L 21 of Formula lib is CH(Me)CH 2 , wherein the CH 2 can be directly attached to the carbon atom of ring D or the carbon atom of the benzoimidazole ring.
  • L 21 of Formula lib is CH 2 CH 2 .
  • CBP30 has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • the only bromodomain inhibitor present in the cell culture medium is SGC-CBP30.
  • the cell culture medium comprises SGC- CBP30 and at least one additional bromodomain inhibitor.
  • the cell culture medium comprises a bromodomain inhibitor of Formula III:
  • G 31 is O or R 300 ,
  • ring A is optionally substituted with 1-4 R 30 , each independently selected from the group consisting of halogen, C 1-4 alkyl, and Ci -4 alkoxyl, wherein the C 1-4 alkyl and Ci -4 alkoxyl are optionally substituted with 1-3 halogens;
  • R 31 is H or a Ci-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens; ring B is optionally substituted with 1-5 R 32 , each independently selected from the group consisting of halogen and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens; and
  • R 33 is H or a Ci-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens, wherein R 300 is H, a C 1-4 alkyl optionally substituted with 1-3 halogens, or a phenyl optionally substituted with 1-5 substituents selected from the group consisting of halogen, OH, Ci-4 alkyl, and C 1-4 alkoxyl, wherein the C 1-4 alkyl and C 1-4 alkoxyl are optionally substituted with 1-3 halogens.
  • the bromodomain inhibitor of Formula III can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula III has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • halogen e.g., F, CI
  • R 31 is Me.
  • ring B of Formula III is not substituted with R 32 .
  • ring B of Formula III is substituted with 1 or 2 R 32 , each independently selected from the group consisting of halogen (e.g., F, CI), C 1-4 alkyl, and C M alkoxyl.
  • R 32 at each occurrence can be independently F, CI, Me, or Et.
  • ring B of Formula III is substituted with one halogen (e.g., F, CI).
  • ring B of Formula III is substituted with one CI, e.g., at the para position to the diazepine ring.
  • the cell culture medium comprises JQ1, CPI-203, 1-BET-762, and/or OTX-015: (OTX-015).
  • BET-762, or OTX-015 can have an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • the only bromodomain inhibitor present in the cell culture medium is JQl, CPI-203, I-BET-762, and/or OTX-015.
  • the cell culture medium comprises JQl, CPI-203, I-BET-762, and/or OTX-015 and at least one additional bromodomain inhibitor.
  • the cell culture medium comprises a bromodomain inhibitor Formula IV: (Formula IV),
  • ring A is optionally substituted with 1-5 R 40 , each independently selected from the group consisting of halogen, OH, C 1-4 alkyl, and Ci -4 alkoxyl, wherein the C 1-4 alkyl and Ci -4 alkoxyl are optionally substituted with 1-3 halogens;
  • ring B is optionally substituted with 1-3 R 41 , each independently selected from the group consisting of halogen and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens;
  • each of R 42 , R 43 , and R 44 is independently selected from the group consisting of hydrogen and Ci -4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens.
  • the bromodomain inhibitor of Formula IV can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula IV has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • ring A of Formula IV is not substituted with R 40 .
  • ring A of Formula IV is substituted with 1 or 2 R 40 , each independently selected from the group consisting of halogen (e.g., F, CI), Ci -4 alkyl (e.g., Me, Et), and Ci -4 alkoxyl (e.g., MeO).
  • halogen e.g., F, CI
  • Ci -4 alkyl e.g., Me, Et
  • Ci -4 alkoxyl e.g., MeO
  • ring A of Formula IV is substituted with one methoxyl group, e.g., at either of the ortho positions to the sulfonamide.
  • ring B of Formula IV is not substituted with R 41 .
  • R 42 is Me or Et. Preferably, R 42 is Me.
  • R 43 and R 44 are each independently H or a C 1-4 alkyl (e.g.,
  • the cell culture medium comprises PFI-1 :
  • the only bromodomain inhibitor present in the cell culture medium is PFI-1.
  • the cell culture medium comprises PFI-1 and at least one additional bromodomain inhibitor.
  • the cell culture medium comprises a bromodomain inhibitor of Formula V:
  • ring A is optionally substituted with 1 or 2 R , each independently selected from the group consisting of halogen and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens;
  • ring B is optionally substituted with 1-3 R 51 , each independently selected from the group consisting of halogen, OH, C 1-4 alkyl, and C 1-4 alkoxyl, wherein the C 1-4 alkyl and C 1-4 alkoxyl are optionally substituted with 1-3 halogens;
  • R 52 is H or a Ci-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens; ring C is optionally substituted with 1-4 R 53 , each independently selected from the group consisting of halogen and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens; and
  • L 50 is a Ci-4 alkylene linker, optionally substituted with a C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens.
  • the bromodomain inhibitor of Formula V can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula V has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • ring A of Formula V is not substituted with R 50 .
  • ring A of Formula V is substituted with 1 or 2 R 50 , each independently selected from the group consisting of halogen (e.g., F, CI) and C 1-4 alkyl (e.g., Me, Et). In one embodiment, ring A of Formula V is substituted with two methyl groups.
  • halogen e.g., F, CI
  • C 1-4 alkyl e.g., Me, Et
  • the cell culture medium comprises a bromodomain inhibitor of Formula Va:
  • ring B is optionally substituted with 1 or 2 R 51 , each independently selected from the group consisting of halogen, C 1-4 alkyl, and C 1-4 alkoxyl;
  • ring C is optionally substituted with 1 or 2 R 53 , each independently selected from the group consisting of halogen and C 1-4 alkyl; and
  • L 50 is a Ci-4 alkylene linker, optionally substituted with a Me or Et.
  • the bromodomain inhibitor of Formula Va can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula Va has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • ring B of Formula Va is not substituted with R 51 .
  • ring B of Formula Va is substituted with one C 1-4 alkoxyl group (e.g., MeO, EtO).
  • ring B of Formula Va is substituted with one methoxyl group.
  • ring C of Formula Va is not substituted with R .
  • ring C of Formula Va is substituted with one or two R 53 , each independently selected from the group consisting of halogen (e.g., F, CI) and C 1-4 alkyl (Me, Et).
  • L 50 of Formula Va is an unsubstituted C 1-4 alkylene linker, e.g., CH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 , or CH 2 CH 2 CH 2 CH 2 .
  • L 50 of Formula Va is a C 1-4 alkylene linker substituted with one methyl or ethyl group.
  • L 50 of Formula Va is CH(Me).
  • the cell culture medium comprises I-BET-151 :
  • I-BET-151 can have an enantiomeric purity of about 80% ee or more, e.g., about 80%> ee, about 85%> ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96%) ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • the only bromodomain inhibitor present in the cell culture medium is I-BET-151.
  • the cell culture medium comprises I-BET-151 and at least one additional bromodomain inhibitor.
  • the cell culture medium comprises a bromodomain inhibitor of Formula VI:
  • ring A is optionally substituted with 1-4 R 61 , each independently selected from the group consisting of halogen and C M alkyl, wherein the C M alkyl is optionally substituted with 1-3 halogens;
  • R 62 is selected from the group consisting of H, halogen and C M alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens;
  • R 64 is H or a C M alkyl, wherein the C M alkyl is optionally substituted with 1-3 halogens.
  • the bromodomain inhibitor of Formula VI can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula IV has an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • R 60 is a C M alkyl (e.g., Me, Et). Preferably, R 60 is Me.
  • R 62 is H or a C alkyl (e.g., Me, Et). Preferably, R 62 is H.
  • R 64 is H or a C M alkyl (e.g., Me, Et). Preferably, R 64 is Me.
  • ring A of Formula VI is not substituted with R 61 .
  • ring A of Formula VI is substituted with 1 or 2 R 61 , each independently selected from the group consisting of halogen (e.g., F, CI) and C M alkyl (e.g., Me, Et).
  • ring A is substituted with one methyl group, e.g., at the ortho position to the sulfonamide but para to the pyridazine ring.
  • R 63 is a C 1-4 alkyl.
  • R 63 is Me or Et.
  • the cell culture medium comprises bromosporine:
  • bromodomain inhibitor present in the cell culture medium is bromosporine.
  • the cell culture medium comprises bromosporine and at least one additional bromodomain inhibitor.
  • the cell culture medium comprises a bromodomain inhibitor of Formula VII:
  • ring A is optionally substituted with 1-5 R , each independently selected from group consisting of halogen, OH, C 1-4 alkyl, and C 1-4 alkoxyl, wherein the C 1-4 alkyl and alkoxyl are optionally substituted with 1-3 halogens;
  • ring B is optionally substituted with 1 or 2 R 71 , each independently selected from the group consisting of halogen and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens;
  • R 72 is a Ci-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens; ring C is optionally substituted with 1-3 R 73 , each independently selected from the group consisting of halogen and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens;
  • ring D is optionally substituted with 1-3 R 74 , each independently selected from the group consisting of halogen and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens; and
  • R 75 is H or a Ci-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1-3 halogens.
  • the bromodomain inhibitor of Formula VII can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula VII can have an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • ring A of Formula VII is not substituted with R .
  • ring A of Formula VII is substituted with 1 or 2 R 70 , each independently selected from the group consisting of halogen (e.g., F, CI), C 1-4 alkyl (e.g., Me, Et), and Ci-4 alkoxyl (e.g., MeO, EtO).
  • halogen e.g., F, CI
  • C 1-4 alkyl e.g., Me, Et
  • Ci-4 alkoxyl e.g., MeO, EtO
  • ring B of Formula VII is not substituted with R 71 .
  • ring B of Formula VII is substituted with 1 or 2 R 71 , each independently selected from the group consisting of halogen (e.g., F, CI) and C 1-4 alkyl (e.g., Me, Et).
  • halogen e.g., F, CI
  • C 1-4 alkyl e.g., Me, Et.
  • ring B is not substituted with R 71 .
  • ring C of Formula VII is not substituted with R 73 .
  • ring C of Formula VII is substituted with 1, 2, or 3 R 73 , each independently a Ci-4 alkyl (e.g., Me, Et).
  • two R 73 can be substituted on the same carbon on ring C.
  • ring C of Formula VII is not substituted with R 73 .
  • ring D of Formula VII is not substituted with R 74 .
  • ring D of Formula VII is substituted with 1, 2, or 3 R 74 , each independently a halogen (e.g., F, CI) or a C 1-4 alkyl (e.g., Me, Et).
  • two R 74 can be substituted on the same carbon on ring D.
  • ring D of Formula VII is not substituted with R 74 .
  • R 75 is H. In another embodiment, R 75 is a C 1-4 alkyl,
  • the cell culture medium comprises a bromodomain inhibitor of Formula Vila:
  • ring A is optionally substituted with 1 or 2 R , each independently selected from the group consisting of halogen, C 1-4 alkyl, and C 1-4 alkoxyl;
  • ring C is optionally substituted with 1 or 2 R 73 , each independently a C 1-4 alkyl; and R 72 is a Ci-4 alkyl; and R 75 is H or a C 1-4 alkyl.
  • the bromodomain inhibitor of Formula Vila can be in the form of a tautomer, a stereoisomeric mixture, or a salt.
  • the bromodomain inhibitor of Formula Vila can have an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • ring A of Formula Vila is substituted with 1 or 2 R 70 , each independently selected from the group consisting of halogen, C 1-4 alkyl, and C 1-4 alkoxyl,
  • ring C of Formula Vila is not substituted with R , R is a C 1-4 alkyl, and R is H or methyl.
  • ring A of Formula Vila is substituted with one or two
  • the cell culture medium comprises I-CBPl 12:
  • I-CBPl 12 can have an enantiomeric purity of about 80% ee or more, e.g., about 80%> ee, about 85%> ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96%) ee, about 97% ee, about 98%> ee, about 99% ee, about 99.5% ee or more.
  • the only bromodomain inhibitor present in the cell culture medium is I- CBP112.
  • the cell culture medium comprises I-CBPl 12 and at least one additional bromodomain inhibitor.
  • the cell culture medium comprises a bromodomain inhibitor selected from the group consisting of:
  • cell culture medium comprises two or more bromodomain inhibitor selected from the group consisting of:
  • the bromodomain inhibitors described herein can be prepared by those skilled in the art.
  • the specific compounds including RVX208, SGC-CBP30, JQ1, CPI-203, PFI-1, I-BET-762, OTX-015, 1-BET151, bromosponne, I-CBP112 are known and commercially available. Analogs of these specific compounds can be prepared following similar synthetic routes.
  • compounds of Formula II can be generally synthesized by following the methods described in Hay D.A. et al, "Discovery and Optimization of Small-molecule Ligands for the CBP/p300 Bromodomains," J. Am. Chem. Soc. 73(5:9308 (2014).
  • the cell culture medium of the present invention may not comprise a bromodomain inhibitor.
  • the cell culture medium com rises BI2536 and/or Cl-amidine:
  • BI2536 and/or Cl-amidine is present in the form of a tautomer, a stereoisomeric mixture, or a salt thereof.
  • Cl-amidine can be present in the form of a trifluoroacetic acid (TFA) salt.
  • BI2536 or Cl-amidine can have an enantiomeric purity of about 80% ee or more, e.g., about 80% ee, about 85% ee, about 90% ee, about 91% ee, about 92% ee, about 93% ee, about 94% ee, about 95% ee, about 96% ee, about 97% ee, about 98% ee, about 99% ee, about 99.5% ee or more.
  • the cell culture does not comprise a bromodomain inhibitor.
  • the medium comprises BI2536 and/or Cl-amidine and at least one bromodomain inhibitor (e.g., as described herein).
  • BI2536 and/or Cl-amidine can be present in the cell culture medium ranging from about 1 uM to about 100 uM, e.g., from about 1 uM to about 75 uM, about 1 uM to about 50 uM, about 1 uM to about 30 uM, about 1 uM to about 10 uM, about 1 uM to about 3 uM, about 3 uM to about 100 uM, about 3 uM to about 75 uM, about 3 uM to about 50 uM, about 3 uM to about 30 uM, about 3 uM to about 10 uM, about 10 uM to about 100 uM, about 10 uM to about 75 uM, about 10 uM to about 50 uM, about 10 uM to about 30 uM, about 30 uM to about 100 uM, about 30 uM to about 75 uM, about 30 uM to about 50 uM, about 50 uM to about 30
  • BI2536 and/or Cl-amidine is present in the cell culture medium in a concentration of about 1 uM, about 3 uM, about 10 uM, about 30 uM, about 50 uM, about 75 uM, or about 100 uM. In one embodiment, the BI2536 and/or Cl-amidine can be present can also be present in the cell culture medium in a
  • BI2536 and/or Cl-amidine can also be present in the cell culture medium in a concentration greater than about 100 uM (e.g., about 120 uM, about 200 uM, about 400 uM, about 600 uM, about 1000 uM, or any ranges between the specified values).
  • the medium comprising BI2536 and/or Cl-amidine is a feed medium.
  • the BI2536 and/or Cl-amidine is present in the feed medium in a sufficient concentration such that when added to a culture, the BI2536 and/or Cl-amidine is present in the culture in a concentration ranging from about 1 uM to about 100 uM, e.g., from about 1 uM to about 75 uM, about 1 uM to about 50 uM, about 1 uM to about 30 uM, about 1 uM to about 10 uM, about 1 uM to about 3 uM, about 3 uM to about 100 uM, about 3 uM to about 75 uM, about 3 uM to about 50 uM, about 3 uM to about 30 uM, about 3 uM to about 10 uM, about 10 uM to about 100 uM, about 10 uM to about 75 uM, about 10 uM to about 75 uM,
  • Certain embodiments of the present invention are directed to a cell culture
  • a cell culture composition comprising the cell culture medium described herein and cells (e.g., mammalian cells).
  • a cell culture composition according to the invention can be a batch culture, fed-batch culture or a perfusion culture.
  • a cell culture composition of the invention is a fed batch culture.
  • a cell culture composition described herein comprises
  • a cell culture composition described herein comprises CHO cells (e.g., CHO-K1 cells).
  • a cell culture composition described herein comprises HEK-293 cells.
  • a cell culture composition described herein comprises HeLa cells.
  • a cell culture composition described herein comprises hybridoma cells.
  • a cell culture composition described herein comprises non-mammalian Eukaryotic cells.
  • a cell culture composition described herein can comprise cells that have been adapted to grow in serum free medium, animal protein free medium or chemically defined medium. Or it can comprise cells that have been genetically modified to increase their life-span in culture. In one embodiment, the cells have been modified to express an anti- apoptotic gene. In a specific embodiment, the cells have been modified to express the bcl-xL antiapoptotic gene. Additional anti-apoptotic genes that can be used in accordance with the present invention include, but are not limited to, E1B-9K, Aven, Mcl.
  • the cell culture comprises i) mammalian cells (e.g.,
  • mammalian cells comprising a polynucleotide encoding a protein or polypeptide of interest); and ii) a cell culture medium comprising a bromodomain inhibitor.
  • a cell culture medium comprising a bromodomain inhibitor.
  • bromodomain inhibitors include any of those described herein.
  • the present invention provides a method of culturing cells, comprising contacting the cells with a medium disclosed herein.
  • Cell cultures can be cultured in a batch culture, fed batch culture, shake flask, a perfusion culture, or a bioreactor.
  • a cell culture according to a method of the present invention is a batch culture.
  • a cell culture according to a method of the present invention is a fed batch culture.
  • a cell culture according to a method of the present invention is a shake flask culture.
  • a cell culture according to a method of the present invention is a bioreactor culture.
  • a cell culture according to a method of the present invention is a perfusion culture.
  • a cell culture according to a method of the present invention is a serum-free culture.
  • a cell culture according to a method of the present invention is a chemically defined culture.
  • a cell culture according to a method of the present invention is an animal protein free culture.
  • a cell culture is contacted with a medium described herein during the growth phase of the culture. In another embodiment, a cell culture is contacted with a medium described herein during the production phase of the culture.
  • a cell culture according to the invention is contacted with a feed medium described herein during the production phase of the culture.
  • the culture is supplemented with the feed medium between about 1 and about 25 times during production phase of the culture.
  • a culture is supplemented with the feed medium between about 1 and about 20 times, between about 1 and about 15 times, or between about 1 and about 10 times during the first time period.
  • a culture is supplemented with the feed medium at least once, at least twice, at least three times, at least four times, at least five times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 1 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 20 times, at least 25 times.
  • the culture is a fed batch culture.
  • the culture is a perfusion culture.
  • a culture according to the invention can be contacted with a feed medium
  • the regular interval is about once a day, about once every two days, about once every three days, about once every 4 days, or about once every 5 days.
  • the culture is a fed batch culture. In another specific embodiment, the culture is a perfusion culture.
  • a medium described herein is a feed medium for a fed batch cell culture.
  • a fed batch cell culture can be contacted with a feed medium more than once.
  • a fed batch cell culture is contacted with a medium described herein only once.
  • a fed batch cell culture is contacted with a medium described herein more than once, for example, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, or at least ten times.
  • the total volume of feed medium added to a cell culture should optimally be kept to a minimal amount.
  • the total volume of the feed medium added to the cell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume of the cell culture prior to adding the feed medium.
  • a culture according to the invention can be contacted with a feed medium
  • the regular interval is about once a day, about once every two days, about once every three days, about once every 4 days, or about once every 5 days.
  • the culture is a fed batch culture. In another specific embodiment, the culture is a perfusion culture.
  • a medium described herein is a feed medium for a fed batch cell culture.
  • a fed batch cell culture can be contacted with a feed medium more than once.
  • a fed batch cell culture is contacted with a medium described herein only once.
  • a fed batch cell culture is contacted with a medium described herein more than once, for example, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, or at least ten times.
  • Cell cultures can be grown to achieve a particular cell density, depending on the needs of the practitioner and the requirement of the cells themselves, prior to being contacted with a medium described herein.
  • the cell culture is contacted with a medium described herein at 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.
  • the medium is a feed medium.
  • Cell cultures can be allowed to grow for a defined period of time before they are contacted with a medium described herein.
  • the cell culture is contacted with a medium described herein at day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the cell culture.
  • the cell culture is contacted with a medium described herein at week 1, 2, 3, 4, 5, 6, 7, or 8 of the cell culture.
  • the medium is a feed medium.
  • Cell cultures can be cultured in the production phase for a defined period of time.
  • the cell culture is contacted with a feed medium described herein at day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the production phase.
  • a culture according to the invention can be maintained in production phase for between about 1 day and about 30 days.
  • a culture is maintained in production phase for between about 1 day and about 30 days , between about 1 day and about 25 days , between about 1 day and about 20 days, about 1 day and about 15 days, about 1 day and about 14 days, about 1 day and about 13 days, about 1 day and about 12 days, about 1 day and about 11 days, about 1 day and about 10 days, about 1 day and about 9 days, about 1 day and about 8 days, about 1 day and about 7 days, about 1 day and about 6 days, about 1 day and about 5 days, about 1 day and about 4 days, about 1 day and about 3 days, about 2 days and about 25 days, about 3 days and about 25 days, about 4 days and about 25 days, about 5 days and about 25 days, about 6 days and about 25 days, about 7 days and about 25 days, about 8 days and about 25 days, about 9 days and about 25 days, about 10 days and about 25 days, about 15 days and about 25 days, about 20 days and about 25 days, about 2 days and about 30 days, about 3 days and about 30 days, about 4 days and about about 40 days
  • a culture is maintained in production phase for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days , at least about 12 days, at least about 15 days, at least about 20 days, at least about 25 days, or at least about 30 days.
  • a culture is maintained in production phase for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 15 days, about 20 days, about 25 days, or about 30 days.
  • a cell culture comprising a medium described herein can be maintained in production phase longer than a cell culture that does not comprise a bromodomain inhibitor (e.g., as described herein).
  • a method of producing a protein or polypeptide of interest according to the present invention produces more polypeptide than the amount produced by a method that does not comprise maintaining cells capable of producing the polypeptide in a culture comprising a bromodomain inhibitor (e.g., as described herein).
  • a method according to the present invention produces between about 5% and about 500%, about 5% and about 250%, about 5% and about 100%, about 5% and about 80%, about 5% and about 50%), about 5% and about 30%>, about 10%> and about 500%>, about 20% and about 500%, about 30% and about 500%, about 50% and about 500%, or about 100% and about 500%) more protein or polypeptide.
  • a method according to the present invention produces at least about 5%, at least about 10%>, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%), at least about 70%, at least about 90%, or at least about 100% more protein or polypeptide.
  • a method according to the present invention produces at least about 2 times, three times, four times, five times or ten times more protein or polypeptide.
  • the protein or polypeptide is an antibody.
  • the present invention further provides a method of producing a protein or
  • polypeptide of interest comprising culturing cells (e.g., mammalian cells as described herein) capable of producing the protein or polypeptide of interest in a culture comprising a medium described herein; and optionally isolating the protein or polypeptide from the culture.
  • the protein or polypeptide of interest is a recombinant protein or polypeptide.
  • the protein or polypeptide of interest is an enzyme, receptor, antibody, hormone, regulatory factor, antigen, or binding agent.
  • the protein is an antibody.
  • the method comprises a) providing a cell culture comprising i) mammalian cells comprising a polynucleotide encoding the protein or polypeptide of interest; and ii) a cell culture medium described herein (e.g., a cell culture medium comprising a bromodomain inhibitor (e.g., as described herein)); and b) culturing the mammalian cells under conditions that allow expression of the protein or polypeptide of interest.
  • the culturing is conducted in a batch mode, fed-batch mode or a perfusion mode.
  • the culturing is conducted in a fed-batch mode.
  • the mammalian cells are selected from the group consisting of: CHO cells, HEK-293 cells, VERO cells, NSO cells, PER.C6 cells. Sp2/0 cells, BHK cells, MDCK cells, MDBK cells, COS cells and HeLa cells. Other suitable mammalian cells are known in the art and can be used in embodiments described herein.
  • the mammalian cells are CHO cells.
  • the mammalian cells are CHO-K1 cells.
  • the method produces the protein or polypeptide of interest in a fed-batch mode.
  • the culture comprises a medium comprising a bromodomain inhibitor, and the method produces the protein or polypeptide of interest (e.g., at day 14 of the culture) at a titer greater than the titer produced from a control cell culture that does not contain a bromodomain inhibitor.
  • the method produces between about 15% and about 100%>, about 15%> and about 80%>, about 15%> and about 50%), about 15%> and about 30%>, about 30%> and about 100%>, about 30%> and about 80%, about 30% and about 50%, about 50% and about 80%, or about 50% and about 100%) higher titer (e.g., at day 14 of the culture) than the titer produced from a control cell culture that does not contain a bromodomain inhibitor.
  • titer e.g., at day 14 of the culture
  • the method produces a titer (e.g., at day 14 of the culture) at least about 15%> (e.g., at least about 20%), at least about 30%>, at least about 40%, at least about 50%, at least about 60%>, at least about 70%, at least about 80%>, at least about 90%, at least about 100%, or at least about 200%o) greater than the titer produced from a control cell culture that does not contain a bromodomain inhibitor.
  • the method produces at least about 2 times, three times, four times, five times or ten times higher titer.
  • the titer produced from a control cell culture that does not contain a bromodomain inhibitor ranges from about lg/L to about 10 g/L (e.g., about lg/L, about 2g/L, about 3g/L, about 4g/L, about 5 g/L, about 6g/L, about 7g/L, about 8g/L, about 9g/L, about lOg/L, or any ranges between the specified values).
  • the culture comprises a medium comprising a bromodomain inhibitor
  • the method generates a viable cell density (e.g., at day 14 of the culture) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the method generates a viable cell density (e.g., at day 14 of the culture) between about 20% and about 100%>, about 20% and about 80%>, about 20% and about 50%, about 20% and about 30%, about 30% and about 100%, about 30% and about 80%, about 30%> and about 50%, or about 50% and about 80%> higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the method produces at least about 20% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or at least about 200%) higher viable cell density (e.g., at day 14 of the culture) than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the viable cell density (e.g., at day 14 of the culture) that observed for a control cell culture that does not contain a bromodomain inhibitor ranges from about 5 x 10 6 cells/mL to about 25 x 10 6 cells/mL (e.g., about 5 x 10 6 cells/mL, about 10 x 10 6 cells/mL, about 15 x 10 6 cells/mL, about 20 x 10 6 cells/mL, about 15 x 10 6 cells/mL, or any ranges between the specified values).
  • the culture comprises a medium comprising a bromodomain inhibitor
  • the method generates a cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the method generates a cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture) between about 10% and about 100%, about 10% and about 80%, about 10% and about 60%, about 10% and about 40%, about 20% and about 100%, about 20% and about 80%, about 20% and about 60%, or about 20% and about 40%, higher cell specific productivity.
  • the method generates at least about 10% (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%), or at least about 100%) higher cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture).
  • the method generates a cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture) between about 10% and about 60% higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture) that observed for a control cell culture that does not contain a bromodomain inhibitor ranges from about 2 pg/cell/day to about 20 pg/cell/day (e.g., about 2 pg/cell/day, about 4 pg/cell/day, about 6 pg/cell/day, about 8 pg/cell/day, about 10 pg/cell/day, about 12 pg/cell/day, about 14 pg/cell/day, about 16 pg/cell/day, about 18 pg/cell/day, about 20 pg/cell/day, or any ranges between the specified values).
  • the culture comprises a medium comprising a bromodomain inhibitor
  • the method achieves a cell viability (e.g., at day 14 of the culture) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the method achieves between about 10% and about 100%>, about 10% and about 80%, about 10% and about 60%, about 10% and about 40%, about 20% and about 100%, about 20% and about 80%, about 20% and about 60%, or about 20%) and about 40%, higher cell viability(e.g., at day 14 of the culture).
  • the method achieves at least about 10% (e.g., at least about 10%, at least about 20%), at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%) higher cell viability (e.g., at day 14 of the culture).
  • the method achieves a cell viability (e.g., at day 14 of the culture) between about 10% and about 60% higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the cell specific productivity (e.g., at day 14 of the culture) that observed for a control cell culture that does not contain a bromodomain inhibitor ranges from about 40% to about 90% (e.g., about 40%, about 50%, about 60%, about 70%, about 80%), about 90%), or any ranges between the specified values).
  • a method of producing a protein or polypeptide of interest according to the present invention produces a maximum protein or polypeptide titer of at least about 2 g/liter, at least about 2.5 g/liter, at least about 3 g/liter, at least about 3.5 g/liter, at least about 4 g/liter, at least about 4.5 g/liter, at least about 5 g/liter, at least about 6 g/liter, at least about 7 g/liter, at least about 8 g/liter, at least about 9 g/liter, or at least about 10 g/liter.
  • the method according to the present invention produces a maximum protein or polypeptide titer of between about 1 g/liter and about 10 g/liter, about 1.5 g/liter and about 10 g/liter, about 2 g/liter and about 10 g/liter, about 2.5 g/liter and about 10 g/liter, about 3 g/liter and about 10 g/liter, about 4 g/liter and about 10 g/liter, about 5 g/liter and about 10 g/liter, about 1 g/liter and about 5 g/liter, about 1 g/liter and about 4.5 g/liter, or about 1 g/liter and about 4 g/liter.
  • Method of modulating viability and/or product titer of between about 1 g/liter and about 10 g/liter, about 1.5 g/liter and about 10 g/liter, about 2 g/liter and about 10 g/liter, about 2.5 g/liter and about 10 g/liter, about 3 g/liter and about 10 g/liter, about 4
  • the present invention further provides a method of modulating the viability the cells of a cell culture and/or the product titer of a cell culture, comprising culturing cells (e.g., mammalian cells as described herein) a culture comprising a medium described herein.
  • culturing cells e.g., mammalian cells as described herein
  • the culturing is conducted in a batch mode, fed-batch mode or a perfusion mode.
  • the culturing is conducted in a fed-batch mode.
  • the cells are mammalian cells selected from the group consisting of
  • CHO cells consisting of: CHO cells, HEK-293 cells, VERO cells, NSO cells, PER.C6 cells. Sp2/0 cells, BHK cells, MDCK cells, MDBK cells, COS cells and HeLa cells.
  • suitable mammalian cells are known in the art and can be used in embodiments described herein.
  • the mammalian cells are CHO cells.
  • the mammalian cells are CHO-K1 cells.
  • the culture comprises a medium comprising a bromodomain inhibitor
  • the method generates a viable cell density (e.g., at day 14 of the culture) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the method generates a viable cell density (e.g., at day 14 of the culture) between about 20% and about 100%>, about 20% and about 80%>, about 20% and about 50%, about 20% and about 30%, about 30% and about 100%, about 30% and about 80%>, about 30%> and about 50%, or about 50% and about 80% higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the method produces at least about 20% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or at least about 200%) higher viable cell density (e.g., at day 14 of the culture) than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the viable cell density (e.g., at day 14 of the culture) that observed for a control cell culture that does not contain a bromodomain inhibitor ranges from about 5 x 10 6 cells/mL to about 25 x 10 6 cells/mL (e.g., about 5 x 10 6 cells/mL, about 10 x 10 6 cells/mL, about 15 x 10 6 cells/mL, about 20 x 10 6 cells/mL, about 15 x 10 6 cells/mL, or any ranges between the specified values).
  • the culture comprises a medium comprising a bromodomain inhibitor
  • the method achieves a cell viability (e.g., at day 14 of the culture) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the method achieves between about 10% and about 100%>, about 10% and about 80%, about 10% and about 60%, about 10% and about 40%, about 20% and about 100%, about 20% and about 80%, about 20% and about 60%, or about 20%) and about 40%, higher cell viability(e.g., at day 14 of the culture).
  • the method achieves at least about 10%> (e.g., at least about 10%>, at least about 20%), at least about 30%>, at least about 40%, at least about 50%, at least about 60%>, at least about 70%, at least about 80%, at least about 90%, or at least about 100%) higher cell viability (e.g., at day 14 of the culture).
  • the method achieves a cell viability (e.g., at day 14 of the culture) between about 10% and about 60% higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the cell specific productivity (e.g., at day 14 of the culture) that observed for a control cell culture that does not contain a bromodomain inhibitor ranges from about 40% to about 90% (e.g., about 40%, about 50%, about 60%, about 70%, about 80%), about 90%), or any ranges between the specified values).
  • the culture comprises a medium comprising a bromodomain inhibitor
  • the method produces a product (e.g., a protein or polypeptide of interest) at a titer (e.g., at day 14 of the culture) greater than the titer produced from a control cell culture that does not contain a bromodomain inhibitor.
  • the method produces between about 15% and about 100%, about 15% and about 80%, about 15% and about 50%), about 15% and about 30%, about 30% and about 100%, about 30% and about 80%, about 30% and about 50%, about 50% and about 80%, or about 50% and about 100%) higher titer (e.g., at day 14 of the culture) than the titer produced from a control cell culture that does not contain a bromodomain inhibitor.
  • the method produces a titer (e.g., at day 14 of the culture) at least about 15% (e.g., at least about 20%), at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or at least about 200%o) greater than the titer produced from a control cell culture that does not contain a bromodomain inhibitor.
  • the method produces at least about 2 times, three times, four times, five times or ten times higher titer.
  • the titer produced from a control cell culture that does not contain a bromodomain inhibitor ranges from about lg/L to about 10 g/L (e.g., about lg/L, about 2g/L, about 3g/L, about 4g/L, about 5g/L, about 6g/L, about 7g/L, about 8g/L, about 9g/L, about lOg/L, or any ranges between the specified values).
  • the culture comprises a medium comprising a bromodomain inhibitor
  • the method generates a cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture) higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the method generates a cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture) between about 10% and about 100%>, about 10%> and about 80%>, about 10% and about 60%, about 10% and about 40%, about 20% and about 100%, about 20% and about 80%, about 20% and about 60%, or about 20% and about 40%, higher cell specific productivity.
  • the method generates at least about 10% (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%), or at least about 100%) higher cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture).
  • the method generates a cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture) between about 10% and about 60% higher than that observed for a control cell culture that does not contain a bromodomain inhibitor.
  • the cell specific productivity (e.g., a cumulative cell specific productivity at day 14 of the culture) that observed for a control cell culture that does not contain a bromodomain inhibitor ranges from about 2 pg/cell/day to about 20 pg/cell/day (e.g., about 2 pg/cell/day, about 4 pg/cell/day, about 6 pg/cell/day, about 8 pg/cell/day, about 10 pg/cell/day, about 12 pg/cell/day, about 14 pg/cell/day, about 16 pg/cell/day, about 18 pg/cell/day, about 20 pg/cell/day, or any ranges between the specified values).
  • the cells are mammalian cells comprising a polynucleotide encoding a protein or polypeptide of interest as described herein.
  • the protein or polypeptide of interest is an enzyme, receptor, antibody, hormone, regulatory factor, antigen, or binding agent.
  • the protein is an antibody.
  • the invention further provides a conditioned cell culture medium produced by a method described herein.
  • a conditioned cell culture medium according to the invention comprises a recombinant protein or polypeptide.
  • a conditioned cell culture medium according to the invention comprises a recombinant protein or polypeptide at a titer of at least about 2 g/liter, at least about 2.5 g/liter, at least about 3 g/liter, at least about 3.5 g/liter, at least about 4 g/liter, at least about 4.5 g/liter, at least about 5 g/liter, at least about 6 g/liter, at least about 7 g/liter, at least about 8 g/liter, at least about 9 g/liter, or at least about 10 g/liter, or a titer of between about 1 g/liter and about 10 g/liter, about 1.5 g/liter and about 10 g/liter, about 2 g/liter and about 10 g/liter, about 2.5 g/liter and about 10 g/liter, about 3 g/liter and about
  • a conditioned cell culture medium according to the invention comprises a recombinant protein or polypeptide at a higher titer than the titer obtained without the use of a medium described herein.
  • the protein or polypeptide is an antibody.
  • Any polypeptide that is expressible in a host cell can be produced in accordance with the present invention.
  • the polypeptide can 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 can be one that occurs in nature, or can alternatively have a sequence that was engineered or selected by the hand of man.
  • An engineered polypeptide can be assembled from other polypeptide segments that individually occur in nature, or can include one or more segments that are not naturally occurring.
  • Antibodies are proteins that have the ability to specifically bind a particular antigen. Any antibody that can be expressed in a host cell can be used in accordance with the present invention. In one embodiment, the antibody to be expressed is a monoclonal antibody.
  • Particular antibodies can be made, for example, by preparing and expressing synthetic genes that encode the recited amino acid sequences or by mutating human germline genes to provide a gene that encodes the recited amino acid sequences.
  • these antibodies can be produced, e.g., using one or more of the following methods.
  • One exemplary method includes screening protein expression libraries, e.g., phage or ribosome display libraries.
  • Phage display is described, for example, U.S. Pat. No. 5,223,409; Smith (1985) Science 228: 1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.
  • the display of Fab's on phage is described, e.g., in U.S. Pat. Nos. 5,658,727; 5,667,988; and 5,885,793.
  • a protein or a peptide thereof can be used as an antigen in a non- human animal, e.g., a rodent, e.g., a mouse, hamster, or rat.
  • the non-human animal includes at least a part of a human immunoglobulin gene.
  • a human immunoglobulin gene For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci.
  • antigen-specific monoclonal antibodies derived from the genes with the desired specificity can be produced and selected. See, e.g., XENOMOUSETM, Green et al. (1994) Nature Genetics 7: 13-21, U.S. 2003-0070185, WO 96/34096, and WO 96/33735.
  • a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized or deimmunized.
  • Winter describes an exemplary CDR-grafting method that can be used to prepare humanized antibodies described herein (U.S. Pat. No. 5,225,539). All or some of the CDRs of a particular human antibody can be replaced with at least a portion of a non-human antibody. In one embodiment, it is only necessary to replace the CDRs required for binding or binding determinants of such CDRs to arrive at a useful humanized antibody that binds to an antigen.
  • Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions.
  • General methods for generating humanized antibodies are provided by Morrison, S. L. (1985) Science 229: 1202-1207, by Oi et al. (1986)
  • Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, can be obtained from a hybridoma producing an antibody against a predetermined target, as described above, from germline immunoglobulin genes, or from synthetic constructs.
  • the expression vector comprises a polynucleotide encoding a glutamine synthetase polypeptide. ⁇ See, e.g., Porter et al, Biotechnol Prog 26(5): 1446-54 (2010).)
  • the antibody can include a human Fc region, e.g., a wild-type Fc region or an Fc region that includes one or more alterations.
  • the constant region is altered, e.g., mutated, to modify the properties of the antibody ⁇ e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).
  • the human IgGl constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237.
  • Antibodies can have mutations in the CH2 region of the heavy chain that reduce or alter effector function, e.g., Fc receptor binding and complement activation.
  • antibodies can have mutations such as those described in U.S. Pat. Nos. 5,624,821 and 5,648,260.
  • Antibodies can also have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of IgG4, as disclosed in the art ⁇ e.g., Angal et al. (1993) o/. Immunol. 30: 105-08). See also, e.g., U.S. 2005-0037000.
  • the antibody can be modified to have an altered
  • glycosylation pattern i.e., altered from the original or native glycosylation pattern.
  • altered means having one or more carbohydrate moieties deleted, and/or having one or more glycosylation sites added to the original antibody.
  • Addition of glycosylation sites to the presently disclosed antibodies can be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences; such techniques are well known in the art.
  • Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. These methods are described in, e.g., WO 87/05330, and Aplin and Wriston (1981) CRC Crit. Rev. Biochem. 22:259-306. Removal of any carbohydrate moieties present on the antibodies can be accomplished chemically or enzymatically as described in the art (Hakimuddin et al. (1987) Arch.
  • the antibodies can be in the form of full length antibodies, or in the form of
  • fragments of antibodies e.g., Fab, F(ab') 2 , Fd, dAb, and scFv fragments.
  • Additional forms include a protein that includes a single variable domain, e.g., a camel or camelized domain. See, e.g., U.S. 2005-0079574 and Davies et al. (1996) Protein Eng. 9(6):531-7.
  • the antibody is an antigen-binding fragment of a full length antibody, e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment.
  • a full length antibody e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment.
  • the antibody is a full length antibody.
  • the antibody can be a monoclonal antibody or a mono-specific antibody.
  • the antibody can be a human, humanized, CDR-grafted, chimeric, mutated, affinity matured, deimmunized, synthetic or otherwise in vitro- generated antibody, and combinations thereof.
  • the heavy and light chains of the antibody can be substantially full-length.
  • the protein can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or can include an antigen-binding fragment (e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment).
  • an antigen-binding fragment e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment.
  • the antibody has a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; particularly, chosen from, e.g., IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl (e.g., human IgGl).
  • the heavy chain constant region is human or a modified form of a human constant region.
  • the antibody has a light chain constant region chosen from, e.g., kappa or lambda, particularly, kappa (e.g., human kappa).
  • a protein or polypeptide of interest may be, but is not limited to, anti-LINGO, anti-LINGO-l(see, e.g., U.S. Patent No. 8,425,910).
  • Anti-LINGO-1 for example, is a fully human monoclonal antibody that targets LINGO-1, a protein expressed selectively in the central nervous system (CNS) that is known to negatively regulate axonal myelination and axonal regeneration (Mi S, et al. Nat Neurosci.
  • interferon e.g., interferon beta la - AVONEX
  • Abciximab REOPRO®
  • Adalimumab HUMIRA®
  • Alemtuzumab CAMPATH®
  • Basiliximab SIMULECT®
  • Bevacizumab AVASTIN®
  • Cetuximab ERBITUX®
  • Certolizumab pegol e.g., interferon beta la - AVONEX
  • interferon beta la - AVONEX Abciximab
  • Adalimumab HUMIRA®
  • Alemtuzumab CAMPATH®
  • Basiliximab SIMULECT®
  • Bevacizumab AVASTIN®
  • Cetuximab ERBITUX®
  • RPTIVA® Gemtuzumab
  • MYLOTARG® Ibritumomab tiuxetan
  • REMICADE® Muromonab-CD3 (ORTHOCLONE OKT3®), Natalizumab (TYSABRI®), Omalizumab (XOLAIR®), Palivizumab (SYNAGIS®), Panitumumab (VECTIBIX®), Ranibizumab (LUCENTIS®), Rituximab (RITUXAN®), Tositumomab (BEXXAR®), and/or Trastuzumab (HERCEPTIN®).
  • the protein or polypeptide of interest is Natalizumab (TYSABRI®).
  • the protein or polypeptide of interest is a blood cascade protein.
  • Blood cascade proteins are known in the art and include, but are not limited to, Factor VII, tissue factor, Factor IX, Factor X, Factor XI, Factor XII, Tissue factor pathway inhibitor, Factor V, prothrombin, thrombin, von WillebrandF actor, kininigen, prekallikrien, kallikrein, fribronogen, fibrin, protein C, thrombomodulin, and antithrombin.
  • the blood cascade protein is Factor IX or Factor VIII.
  • the blood cascade protein is Factor IX- Fc (FIXFc) or Factor VIII - Fc (FVIIIFc).
  • one or more proteins of interest are hormones, regulatory proteins and/or neurotrophic factors.
  • Neurotrophic factors are known in the art and include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT -4), members of the glial cell line-derived neurotrophic factor ligands (GDNF) and ciliary neurotrophic factor (CNTF).
  • the protein or polypeptide of interest is neublastin.
  • the protein or polypeptide of interest is an antibody.
  • elsilimomab enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavi
  • embodiments described herein include without limitation insulin (e.g., HUMULIN®, NOVOLIN®), insulin human inhalation (e.g., EXUBERA®), insulin aspart (e.g., NOVOLOG®), insulin glulisine (e.g., APIDRA®), insulin lispro (e.g., HUMALOG®), isophane insulin (e.g., PH), insulin detemir (e.g., LEVEMIR®), insulin glargine (e.g., LANTUS®), insulin zinc extended (e.g., LENTE®, ULTRALENT®), pramlintide acetate (e.g., SYMLIN®), growth hormone (GH), somatotropin (e.g., GENOTROPIN®
  • insulin e.g., HUMULIN®, NOVOLIN® insulin human inhalation
  • insulin aspart e.g., NOVOLOG®
  • insulin glulisine e.g
  • HUMATROPE® HUMATROPE®, NORDITROPIN®, NUTROPIN®, OMNITROPE®, PROTROPIN®, SIAZEN®, SEROSTIM®, VALTROPIN®), Mecasermin (e.g., INCRELEX®),
  • Mecasermin rinfabate e.g., IPlex
  • Factor Vlll e.g., BIOCLATE®, HELIXATE®, KOGENATE®, REC OMB IN ATE® , REFACTO®
  • Factor IX e.g., BENEFIX®
  • Antithrombin III e.g., THROMBATE III®
  • protein C concentrate e.g., CEPROTIN®
  • ⁇ -Gluco- cerebrosidase e.g., CEREZYME®
  • ⁇ -Gluco-cerebrosidase e.g., CEREDASE® (purified from pooled human placenta), alglucosidase- ⁇ , aronidase/ ⁇ -1-iduronidase (e.g., ALDURAZYME®), Idursulphase/Iduronate-2-sulphatase (e.g., ELAPRASE®),
  • Galsulphase e.g., NAGLAZYME®
  • Agalsidase- ⁇ /human ⁇ -galactosidase A e.g., FABRAZYME®
  • ⁇ -1 -Proteinase inhibitor e.g., ARAL AST®, PROLASTIN®
  • Lactase e.g., LACTAID®
  • pancreatic enzymes e.g., ARCO-LASE®, COTAZYM®, CREON®, DONNAZYME®, PANCREASE®, VIOKAS®E, ZYMASE®
  • Adenosine deaminase e.g., ADAGEN®
  • pooled immunoglobulins e.g., OCTAGAM®
  • Human albumin e.g., ALBUMARC®, ALBUMIN®, ALBUMIN AR®, ALBURX®, ALBUTEIN®
  • EPOGEN® PROCRIT
  • darbepoetin- ⁇ e.g., ARANESP®
  • filrastim e.g.,
  • NEUPOGEN® pegfilgrastim (e.g., NEULASTA®), sargramostim (e.g., LEUKINE®), oprelvekin (e.g., NEUMEGA®), human follicle stimulating hormone (FSH) (e.g.,
  • botulinum toxin type A e.g., BOTOX®
  • botulinum toxin type B e.g., MYOBLOCK®
  • collagenase e.g., Collagenase, SANTYL®
  • human deoxynbonuclease I e.g., dornase - ⁇
  • dornase - ⁇ e.g., PULMOZYME®
  • hyaluronidase e.g., AMPHADASE®
  • hyaluronidase e.g., HYLENEX®
  • papin e.g., ACCUZYME®, PANAFI®L
  • L- Asparaginase e.g., ELSPAR®
  • peg-asparaginase e.g., ONCASPAR®
  • rasbuncase e.g., ELITEK®
  • lepirudin e.g., REFLUDAN®
  • bivalirudin e.g., ANGIOMAX®
  • streptokinase e.g., STREPTASE®
  • Anistreplase e.g., EMINASE®
  • bevacizumab e.g., AVASTIN®
  • cetuximab e.g., ERBITUX®
  • panitumumab e.g., VECTIBIX®
  • alemtuzumab e.g., CAMPATH®
  • alefacept e.g., AMEVIVE®
  • efalizumab e.g., RAPTIVA®
  • natalizumab e.g., TYSABRI®
  • eculizumab e.g., SOLIRIS®
  • antithymocyte globulin e.g., THYMOGLOBULIN®
  • basiliximab e.g., SIMULECT®
  • daclizumab e.g.,
  • ZENAPAX® muromonab-CD3 (e.g., ORTHOCLONE®, OKT3), omalizumab (e.g., XOLAIR®), palivizumab (e.g., SYNAGIS®), enfuviritide (e.g., FUZEON®), abciximab (e.g., REOPRO®), pegvisomant (e.g., SOMA VERT®), crotalidae polyvalent immune Fab (e.g., CROFAB®), digoxin immune serum (e.g., DIGIFAB®), ranibizumab (e.g.,
  • LUCENTIS® denileukin Diftitox
  • ONTAK® denileukin Diftitox
  • ibritumomab tiuxetan e.g.,
  • ZEVALIN® gemtuzumab ozogamicin
  • MYLOTARG® gemtuzumab ozogamicin
  • tositumomab and I- tositumomab e.g., BEXXAR®, BEXXAR® 1-131.
  • the present invention also provides methods for the production of viruses using a cell culture according to methods known to those of skill in the field of virology.
  • the viruses to be produced in accordance with the present invention can be chosen from the range of viruses known to infect the cultured cell type. For instance, when utilizing a mammalian cell culture, viruses can be chosen from the genera of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses,
  • the virus used can be a wild-type virus, an attenuated vims, a reassortant virus, or a recombinant virus.
  • an infectious nucleic acid clone can be utilized according to infectious clone transfection methods known to those of skill in the field of virology.
  • the virus produced is an influenza virus.
  • plant cells for example, plant cells, yeast cells, animal cells, insect cells, avian cells or mammalian cells can be utilized in accordance with the present invention.
  • the eukaryotic cells are capable of expressing a recombinant protein or are capable of producing a recombinant or reassortant virus.
  • Non-limiting examples of mammalian cells that can be used in accordance with the present invention include BALB/c mouse myeloma line (NSO/1, ECACC No:
  • PER.C6 CruCell, Leiden, The Netherlands
  • 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
  • Chinese hamster ovary cells ⁇ DHFR CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)
  • mouse Sertoli cells TM4, Mather, Biol.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO- 76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al, Annals N. Y. Acad.
  • the present invention is used in the culturing of and expression of polypeptides from CHO cell lines.
  • the CHO cell line is the CHO Kl cell line.
  • the CHO cell line comprises a vector comprising a polynucleotide encoding a glutamine synthetase polypeptide.
  • the CHO cell line expresses an exogenous glutamine synthetase gene. (See, e.g., Porter et al, Biotechnol Prog 26(5): 1446-54 (2010).)
  • hybridoma cell lines that express polypeptides or proteins can be utilized in accordance with the present invention.
  • hybridoma cell lines might have different nutrition requirements and/or might require different culture conditions for optimal growth and polypeptide or protein expression, and will be able to modify conditions as needed.
  • the eukaryotic cells according to the present invention can be selected or
  • cells are genetically engineered to produce high levels of protein, for example by introduction of a gene encoding the protein or polypeptide of interest and/or by introduction of control elements that regulate expression of the gene (whether endogenous or introduced) encoding the protein or polypeptide of interest.
  • the eukaryotic cells can also be selected or engineered to survive in culture for extended periods of time.
  • the cells can be genetically engineered to express a polypeptide or polypeptides that confer extended survival on the cells.
  • the eukaryotic cells comprise a transgene encoding the Bcl-2 polypeptide or a variant thereof. See, e.g., US 7,785,880.
  • the cells comprise a polynucleotide encoding the bcl-xL polypeptide. See, e.g., Chiang GG, Sisk WP. 2005. Biotechnology and Bioengineering 91(7): 779-792.
  • the eukaryotic cells can also be selected or engineered to modify its
  • the cells are selected or engineered to modify a protein glycolsylation pathway.
  • the cells are selected or engineered to express an aglycosylated protein, e.g., an aglycosylated recombinant antibody.
  • the cells are selected or engineered to express an afucosylated protein, e.g., an afucosylated recombinant antibody.
  • the eukaryotic cells can also be selected or engineered to allow culturing in serum free medium.
  • the cell culture of the present invention is prepared in any medium suitable for the particular cell being cultured.
  • a media formulation of the present invention generally comprises a compound, for example, a bromodomain inhibitor as described herein, that can have beneficial effects on cell growth and/or viability or on expression of polypeptide or protein.
  • a bromodomain inhibitor as described herein.
  • the media formulations of the present invention encompass both defined and non-defined media.
  • the medium contains e.g., inorganic salts, carbohydrates (e.g., sugars such as glucose, galactose, maltose or fructose), amino acids, vitamins (e.g., B group vitamins (e.g., B12), vitamin A vitamin E, riboflavin, thiamine and biotin), fatty acids and lipids (e.g., cholesterol and steroids), proteins and peptides (e.g., albumin, transferrin, fibronectin and fetuin), serum (e.g., compositions comprising albumins, growth factors and growth inhibitors, such as, fetal bovine serum, newborn calf serum and horse serum), trace elements (e.g., zinc, copper, selenium and tricarboxylic acid intermediates), hydrolysates (hydrolyzed proteins derived from plant or animal sources), and combinations thereof.
  • carbohydrates e.g., sugars such as glucose, galactose, maltose or fructose
  • DMEM/F12 5x-concentrated DMEM/F12 (Invitrogen), CD OptiCHO feed (Invitrogen), CD EfficientFeed (Invitrogen), Cell Boost (HyClone), BalanCD CHO Feed (Irvine Scientific), BD Recharge (Becton Dickinson), Cellvento Feed (EMD Millipore), Ex-cell CHOZN Feed (Sigma- Aldrich), CHO Feed Bioreactor Supplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria), ActiCHO (PAA/GE Healthcare), Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are exemplary nutrient solutions.
  • 5x-concentrated DMEM/F12 Invitrogen
  • CD OptiCHO feed Invitrogen
  • CD EfficientFeed Invitrog
  • any of these media can be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamycin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) lipids (such as linoleic or other fatty acids) and their suitable carriers, and glucose or an equivalent energy source.
  • the nutrient media is serum-free media, a protein-free media, or a chemically defined media.
  • the mammalian host cell is a CHO cell and a suitable medium contains a basal medium component such as a DMEM/HAM F-12 based formulation (for composition of DMEM and HAM F 12 media, see culture media formulations in
  • a nucleic acid sufficient to achieve expression (typically a vector containing the gene encoding the polypeptide or protein of interest and any operably linked genetic control elements) can be introduced into the host cell line by any number of well-known techniques. Typically, cells are screened to determine which of the host cells have actually taken up the vector and express the polypeptide or protein of interest.
  • Traditional methods of detecting a particular polypeptide or protein of interest expressed by mammalian cells include but are not limited to immunohistochemistry, immunoprecipitation, flow cytometry,
  • ELISA immunosorbentassay
  • FRPLC high performance liquid chromatography
  • biological activity assays biological activity assays
  • affinity chromatography affinity chromatography
  • the cell is propagated in culture by any of the variety of methods well-known to one of ordinary skill in the art.
  • the cell expressing the polypeptide of interest is typically propagated by growing it at a temperature and in a medium that is conducive to the survival, growth and viability of the cell.
  • the initial culture volume can be of any size, but is often smaller than the culture volume of the production bioreactor used in the final production of the polypeptide or protein of interest, and frequently cells are passaged several times in bioreactors of increasing volume prior to seeding the production bioreactor.
  • the cell culture can be agitated or shaken to increase oxygenation of the medium and dispersion of nutrients to the cells.
  • special sparging devices that are well known in the art can be used to increase and control oxygenation of the culture.
  • it can be beneficial to control or regulate certain internal conditions of the bioreactor, including but not limited to pH, temperature, oxygenation, etc.
  • the cell density useful in the methods of the present invention can be chosen by one of ordinary skill in the art.
  • the cell density can be as low as a single cell per culture volume.
  • starting cell densities can range from about 2xl0 2 viable cells per mL to about 2xl0 3 , 2xl0 4 , 2xl0 5 , 2xl0 6 , 5xl0 6 or lOxlO 6 viable cells per mL and higher.
  • a cell culture size can be any volume that is appropriate for production of polypeptides.
  • the volume of the cell culture is at least 500 liters.
  • the volume of the production cell culture is 10, 50, 100, 250, 1000, 2000, 2500, 5000, 8000, 10,000, 12,000 liters or more, or any volume in between.
  • a cell culture will be 10 to 5,000 liters, 10 to 10,000 liters, 10 to 15,000 liters, 50 to 5,000 liters, 50 to 10,000 liters, or 50 to 15,000 liters, 100 to 5,000 liters, 100 to 10,000 liters, 100 to 15,000 liters, 500 to 5,000 liters, 500 to 10,000 liters, 500 to 15,000 liters, 1,000 to 5,000 liters, 1,000 to 10,000 liters, or 1,000 to 15,000 liters.
  • a cell culture will be between about 500 liters and about 30,000 liters, about 500 liters and about 20,000 liters, about 500 liters and about 10,000 liters, about 500 liters and about 5,000 liters, about 1,000 liters and about 30,000 liters, about 2,000 liters and about 30,000 liters, about 3,000 liters and about 30,000 liters, about 5,000 liters and about 30,000 liters, or about 10,000 liters and about 30,000 liters, or a cell culture will be at least about 500 liters, at least about 1,000 liters, at least about 2,000 liters, at least about 3,000 liters, at least about 5,000 liters, at least about 10,000 liters, at least about 15,000 liters, or at least about 20,000 liters.
  • the production bioreactor for the culture can be constructed of any material that is conducive to cell growth and viability that does not interfere with expression or stability of the produced polypeptide or protein.
  • the temperature of the cell culture 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.
  • the temperature of the initial growth phase is maintained at a single, constant temperature.
  • the temperature of the initial growth phase is maintained within a range of temperatures. For example, the temperature can be steadily increased or decreased during the initial growth phase. Alternatively, the temperature can 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 can be grown during the initial growth phase for a greater or lesser
  • the cells are grown for a period of time sufficient to achieve a viable cell density that is a given percentage of the maximal viable cell density that the cells would eventually reach if allowed to grow undisturbed.
  • the cells can be grown for a period of time sufficient to achieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximal viable cell density.
  • the cells are allowed to grow for a defined period of time.
  • the cells can be grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. In some cases, the cells can be allowed to grow for a month or more.
  • the growth phase is between about 1 day and about 20 days, about 1 day and about 15 days, about 1 day and about 14 days, about 1 day and about 13 days, about 1 day and about 12 days, about 1 day and about 11 days, about 1 day and about 10 days, about 1 day and about 9 days, about 1 day and about 8 days, about 1 day and about 7 days, about 1 day and about 6 days, about 1 day and about 5 days, about 1 day and about 4 days, about 1 day and about 3 days, about 2 days and about 15 days, about 3 days and about 15 days, about 4 days and about 15 days, about 5 days and about 15 days, about 6 days and about 15 days, about 7 days and about 15 days, about 8 days and about 15 days, about 9 days and about 15 days, about 10 days and about 15 days, about 2 days and about 20 days, about 3 days and about 20 days, about 4 days and about 20 days, about 5 days and about 20 days, about 6 days and about 20 days, about 7 days and about 20 days, about 8 days and about 20 days, about 9 days and about 20 days, about
  • the growth phase is at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days , at least about 12 days, at least about 15 days, or at least about 20 days.
  • the growth phase is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days , about 12 days, about 15 days, or about 20 days.
  • the cells would be grown for 0 days in the production bioreactor if their growth in a seed bioreactor, at the initial growth phase temperature, was sufficient that the viable cell density in the production bioreactor at the time of its inoculation is already at the desired percentage of the maximal viable cell density.
  • the practitioner of the present invention will be able to choose the duration of the initial growth phase depending on polypeptide or protein production requirements and the needs of the cells themselves.
  • the cell culture can be agitated or shaken during the initial culture phase in order to increase oxygenation and dispersion of nutrients to the cells.
  • 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.
  • 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.
  • At the end of the initial growth phase at least one of the
  • culture conditions is shifted so that a second set of culture conditions is applied.
  • the shift in culture conditions can be accomplished by a change in the temperature, pH, osmolality or chemical inductant level of the cell culture.
  • the culture conditions are shifted by shifting the temperature of the culture.
  • the temperature change can be relatively gradual. For example, it can take several hours or days to complete the temperature change. Alternatively, the temperature shift can be relatively abrupt. For example, the temperature change can be complete in less than several hours. Given the appropriate production and control equipment, such as is standard in the commercial large-scale production of polypeptides or proteins, the temperature change can even be complete within less than an hour.
  • the cell culture is maintained for a subsequent production phase under a second set of culture conditions conducive to the survival and viability of the cell culture and appropriate for expression of the desired polypeptide or protein at commercially adequate levels.
  • the culture can be shifted by shifting one or more of a number of culture conditions including, but not limited to, temperature, pH, osmolality, and sodium butyrate levels.
  • the temperature of the culture is shifted.
  • the culture is maintained at a temperature or temperature range that is lower than the temperature or temperature range of the initial growth phase.
  • CHO cells express recombinant polypeptides and proteins well within a range of 25°C to 35°C.
  • the cells can be maintained in the subsequent production phase until a desired cell density or production titer is reached.
  • the cells are maintained in the subsequent production phase until the titer to the recombinant polypeptide or protein reaches a maximum. In other
  • the culture can be harvested prior to this point, depending on the production requirement of the practitioner or the needs of the cells themselves.
  • the cells can 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.
  • the cells are allowed to grow for a defined period of time during the subsequent production phase.
  • the cells can be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days.
  • the cells can be allowed to grow for a month or more.
  • the practitioner of the present invention will be able to choose the duration of the subsequent production phase depending on polypeptide or protein production requirements and the needs of the cells themselves.
  • nutrients or other medium components observed to have been depleted.
  • 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.
  • These supplementary components can all be added to the cell culture at one time, or they can be provided to the cell culture in a series of additions.
  • the supplementary components are provided to the cell culture at multiple times in proportional amounts.
  • the cell culture is fed continually with these supplementary components.
  • the total volume added to the cell culture should optimally be kept to a minimal amount.
  • the total volume of the medium or solution containing the supplementary components added to the cell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume of the cell culture prior to providing the supplementary components.
  • the cell culture can be agitated or shaken during the subsequent production phase in order to increase oxygenation and dispersion of nutrients to the cells.
  • it can be beneficial to control or regulate certain internal conditions of the bioreactor during the subsequent growth phase, including but not limited to pH, temperature, oxygenation, etc.
  • 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.
  • the practitioner can find it
  • Monitoring cell culture conditions allows the practitioner to determine whether the cell culture is producing recombinant polypeptide or protein at suboptimal levels or whether the culture is about to enter into a suboptimal production phase.
  • cell density can be measured using a hemacytometer, a Coulter counter, or Cell density examination (CEDEX).
  • Viable cell density can be determined by staining a culture sample with Trypan blue. Since only dead cells take up the Trypan blue, viable cell density can be determined by counting the total number of cells, dividing the number of cells that take up the dye by the total number of cells, and taking the reciprocal.
  • HPLC can be used to determine the levels of lactate, ammonium or the expressed polypeptide or protein.
  • the level of the expressed polypeptide or protein can be determined by standard molecular biology techniques such as coomassie staining of SDS-PAGE gels, Western blotting, Bradford assays, Lowry assays, Biuret assays, and UV absorbance. It can also be beneficial or necessary to monitor the post- translational modifications of the expressed polypeptide or protein, including
  • the practitioner can also monitor the metabolic status of the cell culture, for example
  • cell culture conditions can be analyzed by using NOVA Bioprofile 100 or 400 (NOVA Biomedical, WA). Additionally, the practitioner can monitor the metabolic state of the cell culture by monitoring the activity of mitochondria. In embodiment, mitochondrial activity can be monitored by monitoring the mitochondrial membrane potential using Rhodamine 123. Johnson LV, Walsh ML, Chen LB. 1980. Proceedings of the National Academy of Sciences 77(2):990-994.
  • polypeptides expressed according to the present invention are expressed according to the present invention.
  • the expressed polypeptide or protein is secreted into the medium and thus cells and other solids can be removed, as by centrifugation or filtering for example, as a first step in the purification process.
  • the expressed polypeptide can be bound to the surface of the host cell.
  • the media is removed and the host cells expressing the polypeptide or protein are lysed as a first step in the purification process. Lysis of mammalian host cells can be achieved by any number of means well known to those of ordinary skill in the art, including physical disruption by glass beads and exposure to high pH conditions.
  • the polypeptide can be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation or by any other available technique for the purification of proteins (See, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer- Verlag, New York, 1987; Higgins, S. J. and Hames, B. D. (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. L, Abelson, J. N.
  • the protein can be isolated by binding it to an affinity column comprising antibodies that were raised against that protein and were affixed to a stationary support.
  • affinity tags such as an influenza coat sequence, poly-histidine, or glutathione-S-transferase can be attached to the protein by standard recombinant techniques to allow for easy purification by passage over the appropriate affinity column.
  • Protease inhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin can be added at any or all stages in order to reduce or eliminate degradation of the polypeptide or protein during the purification process. Protease inhibitors are particularly desired when cells must be lysed in order to isolate and purify the expressed polypeptide or protein.
  • PMSF phenyl methyl sulfonyl fluoride
  • leupeptin leupeptin
  • pepstatin aprotinin
  • aprotinin can be added at any or all stages in order to reduce or eliminate degradation of the polypeptide or protein during the purification process.
  • Protease inhibitors are particularly desired when cells must be lysed in order to isolate and purify the expressed polypeptide or protein.
  • purification technique will vary depending on the character of the polypeptide or protein to be purified, the character of the cells from which the polypeptide or protein is expressed, and the composition of
  • shake flask/96-well plate [0248] Briefly, cells were cultured in a single large shake flask (40 mL working volume) and dispensed into a 96-well plate (1 mL working volume) for treatment on day 7. SGC- CBP30 was dissolved in DMSO (10 mM). This solution was then added on Day 8 and Day 11 in the amount of 0.1 uL, 0.3 uL, 1 uL, 3 uL, or 10 uL to reach the targeted concentrations. As a control, no addition of DMSO and SGC-CBP30 on Day 8 and Day 11 was made for some of the cultures.
  • DMSO without SGC-CBP30 i.e., 0.1 uL, 0.3 uL, 1 uL, 3 uL, or 10 uL
  • Feed medium was added daily from day 3 to day 12, ranging from 2.75% to 8.25%.
  • Samples were taken on days 10, 12, and 14 and protein titer were analyzed. Protein titers were measured by Octet® (ForteBio).
  • FIG. 1 A shows that the protein titers produced by cell cultures with added SGC-CBP30, as measured on Day 12 and Day 14, were higher than the titers produced by the control cultures.
  • the enhancement is more pronounced when the SGC-CBP30 was added at a concentration of 1 uM to 30 uM.
  • FIG. IB shows that adding a solvent DMSO only has limited effect on antibody titers. There are only marginal enhancements from the cell cultures with 3 uL or 10 uL DMSO added.
  • FIG. 1C further shows that adding SGC-CBP30 to cell cultures enhances protein titers when compared to the control cell cultures and the cell cultures with 10 uL DMSO added.
  • Table 1 summarizes the effect of SGC-CBP30 on protein titers (ug/mL) in comparison to DMSO addition and control.
  • Table 1 Comparison of titers from cultures containing SGC-CBP30 or DMSO
  • CHO Kl cell line constructed to produce CD-40, OSMR, or STX200 proteins/antibodies
  • CHO DG44 cell line constructed to produce BART, LINGO, or Alpha-SYN proteins/antibodies.
  • SGC- CBP30 was added to each culture to reach targeted concentrations in the culture, 3 uM or 30 uM, on Day 8 and Day 11 of the culture.
  • the cultures were studied with respect to viable cell density (VCD) and cell viability and antibody titer.
  • VCD viable cell density
  • Feed medium was added daily from day 3 to day 12, ranging from 2.75% to 8.25%. Samples were taken on Day 0, Day 3, Day 4, Day 7, Day 8, Day 10, Day 12, and Day 14 and cell density, viability, and protein titers were analyzed. Cell counts were conducted on Vi-CellTM Cell Counter (Beckman Coulter, Inc.). Protein titers were measured by Octet® (ForteBio).
  • Tables 2A-2D summarize the protein titers and cell specific productivities
  • productivity were improved in cultures with SGC-CBP30 added at 3 uM or 30 uM than those observed for cultures with DMSO added or control cultures.
  • the advantageous effects observed are more pronounced in the CHO Kl cell lines.
  • the cell specific productivity improvement is above 40% compared to control.
  • the concentrations of SGC-CBP30 tested in this example may not be optimal for the CHO DG44 cell lines as tested in this example.
  • adding 3 uM or 30 uM SGC-CBP30 to cell cultures improved titers and cell specific productivities over those observed for the cell cultures with DMSO alone added or the control cultures.
  • viable cell densities and viabilities are also generally improved in cell cultures with 3 uM or 30 uM SGC-CBP30 added over the VCD and viability in the cell cultures with DMSO alone added or the control cultures.
  • SGC-CBP30 is able to increase protein titers and cell specific productivities at 3 uM and/or 30 uM.
  • Amidine (as TFA salt) on viable cell density, cell viability, and protein titer in fed-batch shake flask cultures was performed.
  • the study involved CHO Kl cell line, constructed to produce CD-40 antibodies.
  • the compounds in this set of experiments were added to each culture to reach targeted concentrations in the culture, 3 uM or 30 uM, on Day 8 and Day 11 of the cultures.
  • the cultures were studied with respect to viable cell density (VCD) and cell viability and antibody titer.
  • cells were cultured in a single large shake flask (40 mL working volume) and dispensed into a 96-well plate (1 mL working volume) for treatment on day 7.
  • cell cultures were added 1.5 uL or 15 uL of compound solutions in DMSO (10 mM) to reach the targeted concentrations.
  • the compound solutions used in this study are SGC-CBP30 solution, RVX-208 solution, JQ-1 solution, PFI-1 solution, I- BET-762 solution, OTX015 solution, I-BET-151 solution, bromosporin solution, I- CBP112 solution, BI-2536 solution, or CI-Amidine solution.
  • the protein titers (ug/mL) on D12 and Day 14 and cell viability and density data on Day 14 are summarized in Tables 3A and 3B.
  • the Day 12 average titer was 2225.8 mg/L and the Day 14 average titer was 2309.0 mg/L, and the percentage viable cells on Day 14 is 59.4% with the viable cell density of 11.9 x 10 6 cells/mL.
  • the Day 12 average titer was 2123.4 mg/L and the Day 14 average titer was 2153.4 mg/L, and the percentage viable cells on Day 14 is 60.2% with the viable cell density of 14.5 x 10 6 cells/mL.

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Abstract

L'invention concerne un milieu de culture cellulaire et ses procédés d'utilisation. Le milieu de culture cellulaire comprend généralement un composé (par exemple, un inhibiteur de bromodomaine) qui permet d'améliorer le titre du produit, la viabilité cellulaire, la productivité spécifique des cellules et/ou la densité de cellules viables d'une culture cellulaire contenant le milieu. L'invention concerne également un procédé de production d'une protéine ou d'un polypeptide d'intérêt (par exemple, un anticorps), comprenant la culture de cellules dans une culture comprenant le milieu.
PCT/US2017/030799 2016-05-03 2017-05-03 Culture cellulaire contenant des inhibiteurs de bromodomaine Ceased WO2017192691A1 (fr)

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US10131640B2 (en) 2009-03-18 2018-11-20 Resverlogix Corp. Anti-inflammatory agents
US10532054B2 (en) 2007-02-01 2020-01-14 Resverlogix Corp. Compounds for the prevention and treatment of cardiovascular diseases
WO2024043322A1 (fr) 2022-08-25 2024-02-29 富士フイルム株式会社 Procédé de production de virus de ciblage et composition de culture

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WO2008130437A2 (fr) * 2006-10-20 2008-10-30 Arizona Board Of Regents For And On Behalf Of Arizona State University Cyanobactérie modifiée
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Cited By (5)

* Cited by examiner, † Cited by third party
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US10532054B2 (en) 2007-02-01 2020-01-14 Resverlogix Corp. Compounds for the prevention and treatment of cardiovascular diseases
US10131640B2 (en) 2009-03-18 2018-11-20 Resverlogix Corp. Anti-inflammatory agents
US11407719B2 (en) 2009-03-18 2022-08-09 Resverlogix Corp. Anti-inflammatory agents
WO2024043322A1 (fr) 2022-08-25 2024-02-29 富士フイルム株式会社 Procédé de production de virus de ciblage et composition de culture
EP4578943A4 (fr) * 2022-08-25 2026-02-25 Fujifilm Corp Procédé de production de virus de ciblage et composition de culture

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