US20170204362A1 - Cell culture methods and media comprising n-acetylcysteine - Google Patents

Cell culture methods and media comprising n-acetylcysteine Download PDF

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US20170204362A1
US20170204362A1 US15/318,798 US201515318798A US2017204362A1 US 20170204362 A1 US20170204362 A1 US 20170204362A1 US 201515318798 A US201515318798 A US 201515318798A US 2017204362 A1 US2017204362 A1 US 2017204362A1
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cell culture
cells
acetylcysteine
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Brena Holman
Jeong Lee
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MedImmune LLC
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2511/00—Cells for large scale production

Definitions

  • Serum or other animal-protein ingredients are often used to enhance the ability of cells to grow in a laboratory setting.
  • cell culture media and reagents often do not contain serum or other animal-protein ingredients when manufacturing biologic therapeutics. Removal of animal-protein components makes it more difficult for cells to grow in culture and more difficult for cells to thaw and start growing from a frozen stock, thereby reducing product yields and increasing plant occupancy, utilization rates, and costs. Therefore, precisely when production efficiency becomes most important, cell culture medium ingredients are restricted.
  • the art faces challenges in optimizing cell culture ingredients for cell lines used to produce protein-based biologic therapeutics such as heterologous proteins and antibody therapeutics.
  • cell culture media and cell culture methods comprising N-acetylcysteine (NAC), which when added to cell culture media and/or used in cell culture methods involving a cholesterol auxotroph, a myeloma, or a hybridoma surprisingly increases cell viability, cellular growth rate and reduces cell doubling time.
  • NAC N-acetylcysteine
  • N-acetyl cysteine to cell culture media as a generic amino acid source (see, e.g., EP 2351827; at amounts orders of magnitude lower than used herein) or as a generic reducing agent (see, e.g., EP1434856, WO2012095731, US20060258003) to support the growth of T cells, neuronal progenitor/stem cells, or muscle progenitor/stems cells, respectively.
  • cell culture media and cell culture methods comprising N-acetylcysteine that increase cell viability, cellular growth rate and reduce cell doubling time of cholesterol auxotrophs, myeloma, or hybridoma cells.
  • N-acetylcysteine when added to cell culture media already containing amino acids and reducing agents, surprisingly increased cell viability, cellular growth rate, and reduced cell doubling time of NS0 cells.
  • one embodiment provides a cell culture method comprising: (a) providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and (b) culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.
  • a method of increasing cell viability comprises: (a) providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and (b) culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.
  • a method of increasing cell growth rate comprises: (a) providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and (b) culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.
  • a method of reducing cell doubling time comprises: (a) providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine; and (b) culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.
  • the cell is a cholesterol auxotroph.
  • the cell is a myeloma.
  • the cell is a hybridoma.
  • the cells are being thawed from a frozen stock. In another embodiment, the cells are in an expansion phase.
  • the cell culture medium is a serum free and animal-protein free medium. In another embodiment, the cell culture medium is a chemically-defined medium. In another embodiment, the medium comprises lipids.
  • the cells are derived from a mammal.
  • the mammalian cells are murine, hamster, rat, monkey, or human.
  • the cells are cholesterol auxotrophs.
  • a cholesterol auxotroph may comprise NS0, NS1, U937, M19, SRD-12B, SRD-13A, CHO-215, X63 cells, cell lines derived from these cells lines, or any other cell engineered to be a cholesterol auxotroph.
  • the cells are NS0 cells.
  • the cells are a myeloma or a hybridoma.
  • a cell culture medium comprises N-acetylcysteine, a carbohydrate source, an amino acid source, and a cholesterol source.
  • the carbohydrate source and the amino acid source are different.
  • the medium further comprises lipids.
  • the cell culture medium comprises a carbohydrate source, an amino acid source, a cholesterol source, vitamins, inorganic salts, trace metals, surfactants, and a pH buffer.
  • the cell culture medium comprises N-acetylcysteine at a concentration of from about 0.25 mM to about 3 mM. In another embodiment, the cell culture medium comprises N-acetylcysteine at a concentration of from about 0.5 to about 2.5 mM. In another embodiment, the cell culture medium comprises N-acetylcysteine at a concentration of from about 1.0 to about 1.5 mM. In another embodiment, the cell culture medium comprises N-acetylcysteine at a concentration of about 1 mM. In another embodiment, the cell culture medium comprises N-acetylcysteine at a concentration of about 1.5 mM.
  • the cell culture medium comprises N-acetylcysteine at a concentration of at least about 0.5 mM, at least about 1.0 mM, at least about 1.5 mM or at least about 2.0 mM.
  • the cell culture medium comprises yeastolate.
  • the cell culture medium comprises 1 g/L of yeastolate.
  • the average doubling time is shorter than in a cell culture with a control medium excluding N-acetylcysteine. In another embodiment, the average doubling time is reduced by at least 10% compared to a cell culture medium without N-acetylcysteine. In another embodiment, the average doubling time is reduced by at least 15% compared to a cell culture medium without N-acetylcysteine. In another embodiment, the average doubling time is reduced by at least 20% compared to a cell culture medium without N-acetylcysteine. In another embodiment, the average doubling time is reduced by at least 25% compared to a cell culture medium without N-acetylcysteine.
  • the average doubling time is reduced by at least 50% compared to a cell culture medium without N-acetylcysteine. In another embodiment, the average doubling time is 60 hours or less in a cell culture medium containing N-acetylcysteine. In another embodiment, the average doubling time is 42 hours or less in a cell culture medium containing N-acetylcysteine. In another embodiment, the average doubling time is 34 hours or less in a cell culture medium containing N-acetylcysteine. In another embodiment, the average doubling time is 30 hours or less in a cell culture medium containing N-acetylcysteine. In another embodiment, the average doubling time is 29 hours or less in a cell culture medium containing N-acetylcysteine.
  • the cell viability is increased over a cell culture with a control medium excluding N-acetylcysteine. In another embodiment, the cell viability is increased by at least 5% compared to a cell culture medium without N-acetylcysteine. In another embodiment, the cell viability is increased by at least 7% compared to a cell culture medium without N-acetylcysteine. In another embodiment, the cell viability is increased by at least 10% compared to a cell culture medium without N-acetylcysteine. In another embodiment, the cell viability is at least 90%. In another embodiment, the cell viability is at least 92%. In another embodiment, the cell viability is at least 93%.
  • the cells do not express a heterologous protein.
  • the cells express a heterologous protein.
  • the cells are transformed with a heterologous nucleic acid.
  • the heterologous nucleic acid is cDNA, a vector, a plasmid, a nucleic acid operably linked to a promoter, and/or a nucleic acid that incorporates into the genome.
  • the heterologous protein is transiently expressed.
  • the heterologous protein is stably expressed.
  • the heterologous protein is an antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is an IL-13 antibody.
  • the antibody is BAK502G9 (as represented by the VH and VL domains of SEQ ID NOs 1-2 and/or the heavy and light chain CDRs of SEQ ID NOs 3-8), BAK278D6 (as represented by the VH and VL domains of SEQ ID NOs 9-10 and/or the heavy and light chain CDRs of SEQ ID NOs 11-16), BAK1183H4 (as represented by the VH and VL domains of SEQ ID NOs 17-18 and/or the heavy and light chain CDRs of SEQ ID NOs 19-24), or BAK1167F2 (as represented by the VH and VL domains of SEQ ID NOs 25-26 and/or the heavy and light chain CDRs of SEQ ID NOs 27-32).
  • FIG. 1 shows population doubling time of NS0 Cell Line 1 expressing a monoclonal antibody (“mAb”) against IL-9 in animal-protein free (APF) medium 1 at vial thaw.
  • mAb monoclonal antibody
  • APC N-acetylcysteine
  • FIG. 1 shows population doubling time of NS0 Cell Line 1 expressing a monoclonal antibody (“mAb”) against IL-9 in animal-protein free (APF) medium 1 at vial thaw.
  • Frozen NS0 Cell Line 1 cells thawed in animal-protein free (APF) medium 1 were supplemented with N-acetylcysteine (NAC) (1.5 mM, 2.0 mM, or 2.5 mM) and viable cell density during exponential growth phase was used to calculate average doubling time.
  • Addition of 1.5 mM to 2.5 mM NAC improved cell viability, increased cell growth and reduced average cell doubling time from vial thaw of NS0 Cell Line 1.
  • FIG. 2 shows population doubling time of NS0 Cell Line 1 expressing a monoclonal antibody (“mAb”) against IL-9 in APF medium 2 at vial thaw.
  • mAb monoclonal antibody
  • APF animal-protein free
  • NAC N-acetylcysteine
  • FIG. 2 shows population doubling time of NS0 Cell Line 1 expressing a monoclonal antibody (“mAb”) against IL-9 in APF medium 2 at vial thaw.
  • Frozen NS0 Cell Line 1 cells thawed in animal-protein free (APF) medium 2 were supplemented with N-acetylcysteine (NAC) (1.5 mM, 2.0 mM, or 2.5 mM) and viable cell density during exponential growth phase was used to calculate average doubling time.
  • FIG. 3 shows population doubling time of NS0 Cell Line 2 expressing a monoclonal antibody (“mAb”) against IL-13 in APF medium 1 at vial thaw.
  • mAb monoclonal antibody
  • APF animal-protein free
  • NAC N-acetylcysteine
  • FIG. 2 shows population doubling time of NS0 Cell Line 2 expressing a monoclonal antibody (“mAb”) against IL-13 in APF medium 2 at vial thaw.
  • mAb monoclonal antibody
  • APF animal-protein free
  • NAC N-acetylcysteine
  • FIG. 5 shows population doubling time of NS0 Cell Line 2 expressing a monoclonal antibody against IL-13 in commercially available NS0 cell culture media (CD Hybridoma, Gibco) supplemented with cholesterol (1 ⁇ Invitrogen Cholesterol Lipid Concentrate) at vial thaw.
  • Frozen NS0 Cell Line 2 cells thawed in CD Hybridoma medium with 1 ⁇ Cholesterol Lipid Concentrate were supplemented with N-acetylcysteine (NAC) (0.5 mM, 1.0 mM or 2.0 mM) and viable cell density during exponential growth phase was used to calculate average doubling time.
  • NAC N-acetylcysteine
  • FIG. 6 shows population doubling time of an NS0 null cell line (an untransfected NS0 cell line not expressing a recombinant protein) in APF medium 2 at vial thaw.
  • Frozen NS0 Null Cell Line cells thawed in animal-protein free (APF) medium 2 were supplemented with N-acetylcysteine (NAC) (0.5 mM, 1.0 mM or 1.5 mM) and viable cell density during exponential growth phase was used to calculate average doubling time.
  • NAC N-acetylcysteine
  • FIG. 7 shows population doubling time of NS0 Cell Line 1 expressing a monoclonal antibody (“mAb”) against IL-9 in APF medium 1 during expansion.
  • NS0 Cell Line 1 cells were cultured in animal-protein free (APF) medium 1 supplemented with N-acetylcysteine (NAC) (1.5 mM, 2.0 mM or 2.5 mM) and viable cell density during exponential growth phase was used to calculate average doubling time.
  • APF animal-protein free
  • NAC N-acetylcysteine
  • FIG. 8 shows population doubling time of NS0 Cell Line 1 expressing a monoclonal antibody (“mAb”) against IL-9 in APF medium 2 during expansion.
  • NS0 Cell Line 1 cells were cultured in animal-protein free (APF) medium 2 supplemented with various levels of N-acetylcysteine (NAC) (1.5 mM, 2.0 mM or 2.5 mM) and viable cell density during exponential growth phase was used to calculate average doubling time.
  • APC N-acetylcysteine
  • FIG. 9 shows population doubling time of NS0 Cell Line 2 expressing a monoclonal antibody (“mAb”) against IL-13 in APF medium 1 during expansion.
  • NS0 Cell Line 2 cells were cultured in animal-protein free (APF) medium 1 supplemented with N-acetylcysteine (NAC) (0.5 mM, 1.0 mM or 2.5 mM) and viable cell density during exponential growth phase was used to calculate average doubling time.
  • APC N-acetylcysteine
  • Addition of 0.5 mM to 2.5 mM NAC increased cell growth and reduced average cell doubling time of NS0 Cell Line 2.
  • FIG. 10 shows population doubling time of NS0 Cell Line 2 expressing a monoclonal antibody (“mAb”) against IL-13 in APF medium 2 during expansion.
  • NS0 Cell Line 2 cells were cultured in animal-protein free (APF) medium 2 supplemented with N-acetylcysteine (NAC) (0.5 mM, 1.0 mM or 2.0 mM) and viable cell density during exponential growth phase was used to calculate average doubling time.
  • APF animal-protein free
  • NAC N-acetylcysteine
  • Addition of 0.5 mM to 2.0 mM NAC increased cell growth and reduced average cell doubling time of NS0 Cell Line 2.
  • FIG. 11 illustrates population doubling time of NS0 Cell Line 2 expressing a monoclonal antibody against IL-13 in commercially available NS0 cell culture media (CD Hybridoma, Gibco) supplemented with cholesterol (1 ⁇ Invitrogen Cholesterol Lipid Concentrate) during expansion.
  • NS0 Cell Line 2 cells were cultured in CD Hybridoma medium with 1 ⁇ Cholesterol Lipid Concentrate supplemented with N-acetylcysteine (NAC) (0.5 mM, 1.0 mM or 2.0 mM) and viable cell density during exponential growth phase was used to calculate average doubling time.
  • NAC N-acetylcysteine
  • FIG. 12 shows population doubling time of an NS0 null cell line (an untransfected NS0 cell line not expressing a recombinant protein) in APF medium 2 during expansion.
  • NS0 Null Cell Line cells were cultured in animal-protein free (APF) medium 2 supplemented with N-acetylcysteine (NAC) (0.5 mM, 1.0 mM or 1.5 mM) and viable cell density during exponential growth phase was used to calculate the average doubling time.
  • APF animal-protein free
  • NAC N-acetylcysteine
  • Table 1 provides a listing of certain sequences referenced in present embodiments.
  • a cell may refer to a single cell or a population of cells.
  • N-acetylcysteine refers to a compound derived from cysteine having an acetyl group attached to the nitrogen atom.
  • N-acetylcysteine is also referred to as (2R)-2-acetamido-3-sulfanylpropanoic acid (IUPAC) and has a Chemical Abstracts Service (CAS) Registry Number of 616-91-1.
  • IUPAC 2R-2-acetamido-3-sulfanylpropanoic acid
  • CAS Chemical Abstracts Service
  • cholesterol auxotroph refers to a cell or cell line that requires cholesterol for growth but is unable to synthesize it.
  • Exemplary cholesterol auxotroph include, but are not limited to, NS0, NS1, U937, M19, SRD-12B, SRD-13A, CHO-215, X63 cells, cell lines derived from these cells lines, or any other cell engineered to be a cholesterol auxotroph.
  • Methods of identifying and/or culturing cholesterol auxotrophs are well known in the art. See, e.g., Keen et al., Cytotechnology. 17(3):203-11 (1995); Gorfien et al., Biotechnol Prog.
  • myeloma and “myeloma cells” refer to an immortalized cell line derived from bone marrow cells such as myelocytes, plasma cells or B cells.
  • exemplary myeloma cells include, but are not limited to, X63Ag8, Sp2/0, NS1, NS0, J558L, U266, U937, P3U1, XG-1, XG-2, XG-3, XG-4, XG-5, XG-6, XG-7, XG-8, XG-9, U266, RPM1-8226, LP1, L363, OPM1, OPM2, and NCLH929 cells or cell lines derived from these cells lines.
  • hybridomas refer to an immortalized cell line formed by fusing a B cell with an immortalized cell (e.g. a myeloma cell).
  • Methods of generating and/or culturing hybridomas are well known in the art. See, e.g., Kohler and Milstein, Nature 256:495 (1975); Galfrè and Milstein. Methods Enzymol. 73(Pt B):3-46 (1981); and Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), each herein incorporated by reference in its entirety.
  • cell culture medium refers to a liquid or substrate designed to support the growth of cells derived from multi-cellular eukaryotes, especially animal cells.
  • Exemplary cell culture media and methods of culturing cells are described in Doyle et al., “Mammalian cell culture: essential techniques” Wiley, (1997); Freshney, “Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications” John Wiley & Sons, (2011); and Meenakshi, “Cell Culture Media: A Review” Mater Methods. 3:175 (2013) each herein incorporated by reference in its entirety.
  • the term “serum free medium” refers to a cell culture medium that does not contain animal serum such as fetal bovine serum, bovine serum albumin or human serum albumin.
  • animal-protein free medium refers to a cell culture medium that does not contain proteins and/or protein components from higher multicellular non-plant eukaryotes such as albumin, transferrin, insulin or growth factors. Animal proteins and protein components are to be distinguished from non-animal proteins, small peptides and oligopeptides obtainable from plants (usually 10-30 amino acids in length) or lower eukaryotes, such as yeast, which may be included into the animal-protein free cell culture medium according to the invention.
  • Serum free and animal-protein free medium may be based on any basal medium such as DMEM, Ham's F12, Medium 199, McCoy or RPMI generally known to the skilled worker.
  • the basal medium may comprise a number of ingredients, including amino acids, vitamins, organic and inorganic salts, and sources of carbohydrate, each ingredient being present in an amount which supports the cultivation of a cell which is generally known to the person skilled in the art.
  • the medium may contain auxiliary substances, such as buffer substances like sodium bicarbonate, antioxidants, stabilizers to counteract mechanical stress, or protease inhibitors.
  • a non-ionic surfactant such as mixtures of polyethylene glycols and polypropylene glycols (e.g.
  • Pluronic F68®, SERVA can be added as a defoaming agent.
  • serum free and animal-protein free medium are well known in the art as described in Mariani et al., “Commercial serum-free media: hybridoma growth and monoclonal antibody production.” J Immunol Methods. 145:175-83 (1991); Barnes et al., “Methods for growth of cultured cells in serum-free medium.” Anal Biochem. 102:255-70 (1980); Waymouth, “Preparation and use of serum-free culture media.” In: Barnes D W, Sirbasku D A, Sato G H, editors.
  • chemically-defined medium is a cell growth medium suitable for the cell culture of human or animal cells in which all of the chemical components are known.
  • a “cell culture medium sufficient to support cell growth” refers to a cell culture medium capable of supporting the growth, survival and/or proliferation of a cell.
  • a “cell culture medium sufficient to support cell growth” comprises an appropriate energy source and a complement of amino acids, vitamins, salts, and/or nutrients generally known to the skilled person.
  • Exemplary cell culture media sufficient to support cell growth include commercially available media, chemically-defined media, serum-free medium, and animal-protein free media, as generally known to the skilled worker.
  • cell viability refers to the ability of a cell to live or develop. In general, determining “cell viability’ requires measuring the ability of a cell or cell population to live or develop (including, e.g., making a determination of the number of living or dead cells, based on a total cell sample), as generally known to the skilled worker.
  • Cell viability assays are well known in the art and include: cytolysis or membrane leakage assays (e.g. using Propidium iodide, Trypan blue and/or 7-Aminoactinomycin D), Mitochondrial activity or caspase assays (e.g. using Resazurin and/or Formazan), cell functional assays (e.g.
  • motility assays cell proliferation or growth assays
  • Genomic and/or proteomic assays e.g. measuring the expression of various genes or proteins associated with cell death, damage or stress
  • Cytotoxicity assays Cytotoxicity assays
  • vital staining See Chapter 15, “Assays for Cell Viability, Proliferation and Function” In: “The Molecular Probes Handbook. A Guide to Fluorescent Probes and Labeling Technologies” (I. Johnson and M. Spence (eds.) 11th Edition, Life Technologies (2010) herein incorporated by reference in its entirety.
  • cell doubling time refers to the period of time required for a cell or population of cells to double in number.
  • cell doubling time is measured when the relative growth rate of a cell or cell population is constant (e.g. in the exponential growth or log phase).
  • Cell counting assays are well known in the art and include counting cells; using a counting chamber (e.g.
  • heterologous nucleic acid refers to a nucleic acid molecule (e.g. a polynucleotide, cDNA, DNA, RNA, or fragment thereof) introduced into a cell using standard recombinant DNA and molecular cloning techniques including, but not limited to, those described in Sambrook et al., “Molecular Cloning: A Laboratory Manual” Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Silhavy et al., “Experiments with Gene Fusions” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and Ausubel, F. M.
  • Nucleic acids according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic.
  • transformation refers to the transfer of a nucleic acid molecule or fragment thereof into a host cell, resulting in inheritance of the nucleic acid molecule or fragment thereof to daughter cells of the host cell.
  • Host cells containing the transformed nucleic acids or fragments thereof are referred to herein as “transgenic” or “recombinant” or “transformed” cells.
  • promoter refers to a polynucleotide sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a nucleic acid in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid molecule so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • Plasmid and vector refer to a nucleic acid element usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecules of the invention. Expression may also refer to translation of mRNA into a polypeptide.
  • heterologous protein refers to a protein (e.g. a polypeptide, peptide or fragment thereof) that is encoded by a heterologous nucleic acid and expressed in a host cell.
  • a heterologous protein may be expressed transiently (e.g. where the polynucleotide encoding the heterologous protein does not incorporate into the host cell genome) or stably (e.g. where the polynucleotide encoding the heterologous protein incorporates into the host cell genome).
  • the term “antibody” refers to at least the minimal portion of an antibody which is capable of binding to antigen, e.g., at least the variable domain of a heavy chain (VH) and the variable domain of a light chain (VL) in the context of a typical antibody produced by a B cell.
  • VH variable domain of a heavy chain
  • VL variable domain of a light chain
  • Basic antibody structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), which is incorporated by reference herein in its entirety.
  • Antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library.
  • ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019, which is incorporated by reference herein in its entirety.
  • Immunoglobulin or antibody molecules encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • type e.g., IgG, IgE, IgM, IgD, IgA, and IgY
  • class e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2
  • subclass of immunoglobulin molecule e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2
  • Antibodies or antigen-binding fragments, variants, or derivatives thereof disclosed herein can be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target polypeptide that they recognize or specifically bind.
  • an IL-13 or anti-IL-13 antibody is an antibody that binds to an IL-13 polypeptide or a portion thereof.
  • the anti-IL-13 antibody is BAK502G9 (e.g. an anti-IL-13 antibody comprising SEQ ID NOs: 1 and 2).
  • the antibody is BAK502G9 (as represented by the VH and VL domains of SEQ ID NOs 1-2 and/or the heavy and light chain CDRs of SEQ ID NOs 3-8), BAK278D6 (as represented by the VH and VL domains of SEQ ID NOs 9-10 and/or the heavy and light chain CDRs of SEQ ID NOs 11-16), BAK1183H4 (as represented by the VH and VL domains of SEQ ID NOs 17-18 and/or the heavy and light chain CDRs of SEQ ID NOs 19-24), or BAK1167F2 (as represented by the VH and VL domains of SEQ ID NOs 25-26 and/or the heavy and light chain CDRs of SEQ ID NOs 27-32).
  • anti-IL-13 monoclonal antibodies that can be used include those described in U.S. Pat. Appl. Publ. No. 2012-0052060, published Mar. 1, 2012, herein incorporated by reference in its entirety.
  • Other IL-13 antibodies include, without limitation, anti-human-IL-13 antibodies, for example, Lebrikizumab (MILR1444A/RG3637, Roche/Genentech), ABT-308 (Abbott), GSK679586 (GlaxoSmithKline) or QAX576 (Novartis).
  • antibodies, including anti-IL13 antibodies may be produced in cells using various techniques known in the art.
  • Cell culture is the process of placing cells, tissues, or organs removed from an animal into an artificial environment that promotes their survival, growth, and/or proliferation.
  • Basic environmental requirements for cells to grow optimally include: a suitable vessel, a cell culture/growth medium to supply nutrients (including, but not limited to, at least one of amino acids, carbohydrates, vitamins, minerals, growth factors, hormones, etc.), and a controlled physicochemical environment (to control, e.g., pH, osmotic pressure, temperature, O 2 , CO 2 , etc.).
  • nutrients including, but not limited to, at least one of amino acids, carbohydrates, vitamins, minerals, growth factors, hormones, etc.
  • a controlled physicochemical environment to control, e.g., pH, osmotic pressure, temperature, O 2 , CO 2 , etc.
  • Some cells are anchorage-dependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (s
  • the cell culture medium or cell culture method includes a cell culture medium sufficient to support cell growth comprising N-acetylcysteine.
  • the cell culture medium is a serum free and animal-protein free medium.
  • the cell culture medium is a chemically-defined medium.
  • N-acetylcysteine is added to commercially-available cell culture media.
  • the commercially-available cell culture medium is EX-CELL® NS0 serum-free medium for NS0 cells (available from Sigma-Aldrich, catalog number H4281), EX-CELL® CD hybridoma medium (Sigma-Aldrich, catalog number H4409), Ex-Cell 620-HSF serum-free medium for hybridoma cells (Sigma-Aldrich, catalog number 14621C), Ex-Cell NS-serum-free medium for NS0 (Sigma-Aldrich, catalog number 14650C), DMEM (Sigma-Aldrich, catalog number D567), Iscove's Modified Dulbecco's Medium (IMDM) (available from Sigma-Aldrich, catalog number 13390), RPMI-1640 Medium (Sigma-Aldrich, catalog number R8005), Hybridoma-SFM (Life Technologies, catalog number 12045076), CD Hybridoma AGT medium (Life Technologies, catalog number 12372025), CD Hybridom
  • N-acetylcysteine is added to the cell culture medium or the cell culture methods described herein to increase cell viability, cell growth rate, and/or reduce cell doubling time. While not being limited by any particular theory, it is believed that N-acetylcysteine provides benefits to cells growing in culture by protecting them from free radicals, preventing cell membrane breakdown, and/or preventing oxidation of other cell culture medium ingredients, including, but not limited to, lipids (such as cholesterol).
  • a cell culture medium or a cell culture method as described herein comprises N-acetylcysteine at a concentration of from about 0.25 mM to about 3 mM, from about 0.5 mM to about 2.5 mM, from about 0.5 mM to about 2.0 mM, from about 0.5 mM to about 1.5 mM, from about 0.5 mM to about 1.0 mM, from about 1.0 mM to about 2.5 mM, from about 1.0 mM to about 2.0 mM, from about 1.0 mM to about 1.5 mM, from about 1.5 mM to about 2.5 mM, or from about 1.5 mM to about 2.0 mM.
  • a cell culture medium or a cell culture method as described herein comprises N-acetylcysteine at a concentration of about 1 mM. In another embodiment, a cell culture medium or a cell culture method as described herein comprises N-acetylcysteine at a concentration of about 1.5 mM. In another embodiment, a cell culture medium or a cell culture method as described herein comprises N-acetylcysteine at a concentration of about 2.0 mM. In another embodiment, a cell culture medium or a cell culture method as described herein comprises N-acetylcysteine at a concentration of about 2.5 mM.
  • a cell culture medium or a cell culture method as described herein comprises N-acetylcysteine at a concentration of about 0.5 mM. In another embodiment, the cell culture medium or a cell culture method as described herein comprises N-acetylcysteine at a concentration of at least about 0.5 mM, at least about 1.0 mM, at least about 1.5 mM, at least about 2.0 mM, or at least about 2.5 mM.
  • the cell culture medium or cell culture method described herein further comprises a carbon source, a nitrogen source, and/or a phosphorous source. These can be provided by the same ingredient or different ingredients.
  • the cell culture medium or cell culture methods described herein further comprise a carbon source, a nitrogen source, a phosphorous source, and/or mineral salts.
  • a carbon source suitable for use in the cell culture medium or cell culture methods described herein includes a carbohydrate (such as a sugar) or an amino acid, such as L-glutamine and/or pyruvate or any combination thereof.
  • the cell culture medium or cell culture method described herein further comprises carbohydrates and amino acids.
  • the cell culture medium or cell culture method described herein further comprises at least one of salts, vitamins, metabolic precursors, growth factors, hormones, and trace elements.
  • the cell culture medium or cell culture methods described herein further comprise basal media, containing amino acids, vitamins, inorganic salts, and a carbon source such as glucose.
  • Exemplary carbohydrates suitable for use in the cell culture medium or cell culture methods described herein include glucose, galactose, trehalose, glucosamine, mannose, raffinose, fructose, ribose, glucuronic acid, lactose, maltose, sucrose, turanose, any other carbohydrate suitable for cell culture generally known in the art, or any combinations thereof.
  • the carbohydrate may be glucose or galactose.
  • amino acids suitable for use in the cell culture medium or cell culture methods described herein include one or more essential amino acids (i.e. histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and/or valine), and/or one or more nonessential amino acids (i.e. alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, and asparagine), and/or any combination of essential and non-essential amino acids thereof.
  • essential amino acids i.e. histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and/or valine
  • nonessential amino acids i.e. alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine
  • NS0 cells lack or contain very low levels of the endogenous glutamine synthetase enzyme and, as such, glutamine is an essential amino acid for NS0 cells unless glutamine synthetase is included in the expression system for a heterologous protein.
  • amino acids this disclosure includes any amino acids, including, but not limited to, D- or L-amino acids and non-standard amino acids.
  • amino acid encompasses any organic compound with an amine (—NH 2 ) and a carboxylic acid (—COOH) functional group.
  • the cell culture medium or cell culture methods described herein further comprise a lipid.
  • lipids suitable for use in the cell culture medium or cell culture methods described herein include one or more of cholesterol, arachadonic acid, tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid, a phospholipid (such as phosphatidylcholine) any other lipid suitable for cell culture generally known in the art, or any combinations thereof.
  • Inositol as a component of membrane phospholipids, may also be optionally included.
  • Synthetic or plant-derived lipids may also be optionally used in applications where the medium is desired to be kept free of animal-derived components.
  • Lipids may be added in a cyclodextrin-based lipid supplement. Cyclodextrins may be used to solubilize lipids and/or other ingredients such as fat-soluble vitamins and hormones.
  • Cholesterol may be synthetically produced or it may be animal derived. For instance, cholesterol may be isolated from sheep wool. An animal-protein free medium may contain cholesterol derived from an animal source. In some embodiments, cholesterol is obtained from a commercial source. See, e.g., Chemically Defined Lipid Concentrate from Gibco; Lipid Concentrate from SAFC.
  • Cholesterol may be added to the medium in a cholesterol-cyclodextrin solution, as a synthetic cholesterol (such as but not limited to SyntheCholTM), as cholesterol nanoparticles (see, e.g., Wu et al., Enhanced Productivity of NS0 cells in fed-batch culture with cholesterol nanoparticle supplementation, Biotechnology Progress 27(3):796-802 (2011)) or by any means suitable for cell culture generally known in the art.
  • a synthetic cholesterol such as but not limited to SyntheCholTM
  • cholesterol nanoparticles see, e.g., Wu et al., Enhanced Productivity of NS0 cells in fed-batch culture with cholesterol nanoparticle supplementation, Biotechnology Progress 27(3):796-802 (2011)
  • the medium may contain from about 1 to about 10 g/L of cholesterol. In another embodiment, the medium may contain from about 1 to about 5 g/l of cholesterol. In another embodiment, the medium may contain from about 1.5 to about 4 g/l of cholesterol. In another embodiment, the medium may contain from about 2 to about 3 g/l of cholesterol. In another embodiment, the medium may contain about 2.5 g/l of cholesterol. In another embodiment, the medium may contain at least about 1 g/L of cholesterol, at least about 2.5 g/L of cholesterol or at least about 5 g/L of cholesterol.
  • the medium may contain a lipid other than cholesterol. In one embodiment, the medium may contain a phospholipid. In another embodiment, the medium may contain phosphatidylcholine. In one embodiment, the medium may contain from about 1 to about 10 g/L of phosphatidylcholine. In another embodiment, the medium may contain from about 1 to about 5 g/l of phosphatidylcholine. In another embodiment, the medium may contain from about 1.5 to about 4 g/l of phosphatidylcholine. In another embodiment, the medium may contain from about 2 to about 3 g/l of phosphatidylcholine. In another embodiment, the medium may contain about 2.5 g/l of phosphatidylcholine.
  • the cell culture medium or cell culture methods described herein further comprise a salt.
  • exemplary salts suitable for use in the cell culture medium or cell culture methods described herein include at least one of calcium chloride, magnesium chloride, potassium chloride, sodium chloride, potassium nitrate, any other salt suitable for cell culture generally known in the art, or any combinations thereof.
  • the cell culture medium or cell culture methods described herein do not include calcium chloride or magnesium chloride. This embodiment has advantages when cell dissociation or release is desired as calcium and magnesium promote cell adhesion.
  • the cell culture medium or cell culture methods described herein further comprise a vitamin.
  • exemplary vitamins suitable for use in the cell culture medium or cell culture methods described herein include fat-soluble vitamins, vitamins A, D, E, K, B1 (thiamine), B2 (riboflavin), B3 (nicotinamide), B5 (pantothenic acid), B6 (pyridoxal, pyridoxamine, and/or pyridoxine), B9 (folic acid), any other vitamin suitable for cell culture generally known in the art, or any combinations thereof.
  • At least one hormone may be added to the cell culture medium or cell culture methods described herein.
  • the hormone may be chosen from at least one of dexamethasone, erythropoietin, estradiol, hydrocortisone, insulin, progesterone, somatostatin, thyroxine (T4), triiodothyronine (T3), activin, BMP4, BMP7, BMPR1A, Cripto, FLT3 ligand, HGF, IGF, EGF, FGF, PDGF, IGFBP4, kallekrein, LEFTY-A, NGF, TGF ⁇ , VEGF, or any other hormone or growth factor suitable for cell culture generally known in the art.
  • At least one trace element may be added to the cell culture medium or cell culture methods described herein.
  • the trace element may be at least one of zinc, iron, copper, selenium, magnesium, manganese, molybdenum, tin, nickel, or any other trace element suitable for cell culture generally known in the art.
  • the cell culture medium or cell culture methods described herein further comprise at least one surfactant.
  • exemplary surfactants suitable for use in the cell culture medium or cell culture methods described herein include Tween-80, pluronic F-68, or any other surfactant suitable for cell culture generally known in the art.
  • the cell culture medium or cell culture methods described herein further comprise at least one pH buffering agent.
  • buffering agents suitable for use in the cell culture medium or cell culture methods described herein include sodium bicarbonate, boric acid, citric acid, dithiothreitol, ethanolamine, glycerophosphate, potassium citrate, potassium phosphate, sodium acetate, sodium chloride, sodium phosphate, starch from wheat, HEPES, calcium chloride, MOPS, or any other buffering agent cell suitable for cell culture generally known in the art, or any combination thereof.
  • the cell culture medium or cell culture methods described herein further comprise a non-animal-sourced hydrolysate.
  • a plant or yeast hydrolysate provides protein digests comprising amino acids, short peptides, carbohydrates, vitamins, nucleosides, and minerals, providing a variety of nutritional supplements to media.
  • yeastolate a yeast hydrolysate
  • Yeastolate is a mixture of peptides, amino acids, carbohydrates, lipids, metals and vitamins. It may be added in addition to or in lieu of those ingredients provided separately.
  • the cell culture medium comprises 1 g/L of yeastolate.
  • tropolone may also be added to the cell culture medium or cell culture methods described herein.
  • nucleosides may be added to the cell culture medium or cell culture methods described herein.
  • ⁇ -mercaptoethanol may be added to the cell culture medium or cell culture methods described herein.
  • antibiotics may be added to the cell culture media or cell culture methods described herein.
  • the cell culture medium or cell culture methods described herein contain NAC, an amino acid, a vitamin, a lipid, a carbohydrate, a pH buffering agent, a trace metal, an inorganic salt, and a surfactant.
  • the lipid is cholesterol.
  • a variety of cell types such as cholesterol auxotrophic cells, myeloma cells, and hybridoma cells, may be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine.
  • the cells being cultured are derived from a mammal, including, but not limited to, cells derived from a mouse, rat, human, monkey, hamster, rabbit, etc.
  • cholesterol auxotroph refers to a cell or cell line that requires cholesterol for growth but is unable to synthesize it.
  • a cholesterol auxotroph is NS0, NS1, U937, M19, SRD-12B, SRD-13A, CHO-215, X63 cells, cell lines derived from these cells lines, or any other cell engineered to be a cholesterol auxotroph.
  • the cells are NS0 cells.
  • the cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine are cells that are cholesterol auxotrophs. In one embodiment, the cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine are NS0 cells. In another embodiment, the cells are NS1, U937, M19, SRD-12B, SRD-13A, CHO-215, X63 cells, cells derived from these cell lines, or any other cell engineered to be a cholesterol auxotroph.
  • the cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine are murine myeloma cells that are cholesterol auxotrophs. In one embodiment, the cells are mammalian myeloma cells that are cholesterol auxotrophs. In one embodiment the cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine are human myeloma cells that are cholesterol auxotrophs.
  • the cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine are myeloma cells.
  • the cells are X63Ag8, Sp2/0, NS1, NS0, J558L, U266, U937, P3U1, XG-1, XG-2, XG-3, XG-4, XG-5, XG-6, XG-7, XG-8, XG-9, U266, RPM1-8226, LP1, L363, OPM1, OPM2, and NCLH929 cells or cell lines derived from these cells lines.
  • the cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine are hybridoma cells.
  • the hybridoma cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine express and/or secrete an antibody.
  • the cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine are cultured without inducing and/or expressing a recombinant or heterologous protein.
  • the cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine e.g.
  • a heterologous nucleic acid including, but not limited to cDNA, a plasmid, a vector, nucleic acids operably linked to a promoter, and/or nucleic acids that transiently express a heterologous nucleic acid or incorporate the heterologous nucleic acid into the genome of the cell line.
  • the cells express a recombinant or heterologous protein.
  • the cells overexpress the recombinant or heterologous protein.
  • Cell lines expressing a wide variety of heterologous sequences may benefit from the present cell culture medium and method.
  • the heterologous protein is transiently expressed. In another embodiment, the heterologous protein is stably expressed.
  • the heterologous protein is not an antibody or antigen-binding fragment thereof.
  • the heterologous protein is a blood factor, anticoagulant, thrombolytic, erythropoietin, interferon, hormone, enzyme, vaccine, growth factor, and/or a fusion protein.
  • the cells suitable to be cultured with the presently-disclosed cell culture medium comprising N-acetylcysteine express a heterologous protein, wherein the heterologous protein is an antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof specifically binds IL-13 or specifically binds IL-9.
  • the antibody or antigen-binding fragment is an antibody or antigen-binding fragment disclosed in U.S. Pat. No. 7,947,273, U.S. Pat. No. 7,354,584 or U.S. Pat. No.
  • the antibody is BAK502G9 (comprising SEQ ID NOs 1 and 2).
  • the antibody or antigen-binding fragment thereof has the same CDRs as BAK502G9 (comprising heavy chain CDRS (SEQ ID NOs: 3-5) and light chain CDRs (SEQ ID NOs: 6-8)).
  • the antibody or antigen binding fragment has a heavy chain variable region comprising any one of SEQ ID NOs: 1, 9, 17, or 25 and a light chain variable region comprising any one of SEQ ID NOs: 2, 10, 18, or 26.
  • the antibody or antigen binding fragment has a heavy chain variable region comprising: (a) a HC CDR1 chosen from SEQ ID NOs: 3, 11, 19, and 27; (b) a HC CDR2 chosen from SEQ ID NOs: 4, 12, 20, and 28; and (c) a HC CDR chosen from SEQ ID NOs: 5, 13, 21, and 29; and a light chain variable region comprising: (a) a LC CDR1 chosen from SEQ ID NOs: 6, 14, 22, and 30; (b) a LC CDR2 chosen from SEQ ID NOs: 7, 15, 23, and 31; and (c) a LC CDR3 chosen from SEQ ID NOs: 8, 16, 24, and 32.
  • the antibody or antigen binding fragment comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) described in Table 1; or comprises a set of 6 of the CDRs described in Table 1.
  • the antibody or antigen binding fragment has a heavy chain variable region comprising a sequence that is about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 93%, about 94%, about 98%, or about 99% identical to any one of SEQ ID NOs: 1, 9, 17, or 25 and a light chain variable region comprising a sequence that is about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 93%, about 94%, about 98%, or about 99% identical to any one of SEQ ID NOs: 2, 10, 18, or 26.
  • the antibody or antigen binding fragment has a heavy chain variable region comprising: (a) a HC CDR1 that has one mutation compared to a sequence chosen from SEQ ID NOs: 3, 11, 19, or 27; (b) a HC CDR2 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 4, 12, 20, and 28; and (c) a HC CDR3 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 5, 13, 21, and 29; and a light chain variable region comprising: (a) a LC CDR1 that has one mutation compared to a sequence chosen from SEQ ID NOs: 6, 14, 22, and 30; (b) a LC CDR2 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 7, 15, 23, and 31; and (c) a LC CDR3 that has one or two mutations compared to a sequence chosen from SEQ ID NOs: 8, 16, 24, and 32.
  • the antibody or antigen binding fragment has a heavy chain variable region (VH) and/or a light chain variable region (VL) comprising a sequence that is about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 93%, about 94%, about 98%, or about 99% identical to any one of the VH and/or VL sequences described in Table 1.
  • the antibody or antigen binding fragment has one or two mutations in a CDR compared to any one of the CDRs described in Table 1.
  • the antibody or antigen binding fragment thereof specifically binds glycoprotein IIb/IIIa, IL-2 receptor (such as the IL-2 receptor a), TNF- ⁇ , RSV, F protein epitope of RSV, CD33, epidermal GF receptor, T-cell VLA4 receptor, complement protein C5, IL-1, IL-9, IL-12, IL-13, IL-23, CD-20, and/or BAFF.
  • IL-2 receptor such as the IL-2 receptor a
  • TNF- ⁇ such as the IL-2 receptor a
  • RSV F protein epitope of RSV
  • CD33 epidermal GF receptor
  • T-cell VLA4 receptor T-cell VLA4 receptor
  • the antibody or antigen binding fragment thereof is ofatumumab, belimumab, gemtuzumab ozogamicin, palivizumab, natalizumab, cetuximab, canakinumab, infliximab, abciximab, basiliximab, daclizumab, eculizumab, or ustekinumab.
  • a cell culture method comprises providing a cell culture medium sufficient to support cell growth, wherein the cell culture medium comprises N-acetylcysteine and culturing a cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.
  • the method is a method of increasing cell viability, increasing cell growth rate, and/or reducing cell doubling time of a cell by providing a cell culture medium sufficient to support cell growth comprising N-acetylcysteine; and culturing the cell in the cell culture medium, wherein the cell is a cholesterol auxotroph, a myeloma, or a hybridoma.
  • the cells are cultured at 37° C. at 5% CO 2 and 85% relative humidity.
  • the pH may be from 6.8 to 7.4.
  • Other acceptable conditions generally known in the art may also be used.
  • the cell culture occurs while scaling up production from a frozen stock to a large bioreactor.
  • the improved medium allows for at least a four-fold split ratio when passaging the cells (namely 1 ⁇ of cell culture medium containing the cells to be passaged is mixed with 3 ⁇ of fresh cell culture medium not containing the cells). In another aspect, it allows for a five-fold split ratio, six-fold split ratio, or a seven-fold split ratio.
  • the cells are cultured in a 100 L bioreactor, then transferred to a 500 L bioreactor, and then transferred to a 2500 L bioreactor.
  • the cells are thawed from a frozen stock.
  • the cells e.g. cholesterol auxotrophs, myelomas, or hybridomas
  • the cells are in an expansion phase.
  • the cells e.g. cholesterol auxotrophs, myelomas, or hybridomas
  • the present cell culture method and/or media may provide multiple advantages.
  • it reduces the time required for cells (e.g. cholesterol auxotrophs, myelomas, or hybridomas such as NS0 cells) to grow in a vessel (such as a bioreactor) by at least about 10% to at least about 50% (e.g. at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%) compared to a cell culture medium without N-acetylcysteine. In one embodiment, this is determined by calculating the period of time necessary to get to a desired cell count per volume.
  • cells e.g. cholesterol auxotrophs, myelomas, or hybridomas such as NS0 cells
  • a vessel such as a bioreactor
  • this is determined by calculating the period of time necessary to get to a desired cell count per volume.
  • the cell culture method and/or media reduce the average cell doubling time of a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured using a cell culture method and/or media comprising N-acetylcysteine compared to a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured in a cell culture with a control medium excluding N-acetylcysteine.
  • the average doubling time of a cholesterol auxotroph, a myeloma, or a hybridoma e.g.
  • NS0 cells cultured using a cell culture method and/or media comprising N-acetylcysteine is shorter than a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured in a cell culture with a control medium excluding N-acetylcysteine.
  • the average and/or median cell doubling time of a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured using a cell culture method and/or media comprising N-acetylcysteine is reduced by at least about 10% to at least about 50% (e.g.
  • the average and/or median cell doubling time of a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured using a cell culture method and/or media comprising N-acetylcysteine is less than or equal to 60 hours to less than or equal to about 29 hours (e.g. less than or equal to about 60 hours, about 42 hours, about 34 hours, about 30 hours, or about 29 hours) or about less than or about equal to any of the average cell doubling times reported in FIGS. 1-12 .
  • the cell culture method and/or media increase the cell growth rate of a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured using a cell culture method and/or media comprising N-acetylcysteine compared to a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured in a cell culture with a control medium excluding N-acetylcysteine.
  • the cell growth rate of a cholesterol auxotroph, a myeloma, or a hybridoma e.g.
  • NS0 cells cultured using a cell culture method and/or media comprising N-acetylcysteine is higher than a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured in a cell culture with a control medium excluding N-acetylcysteine.
  • cell growth rate of a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured using a cell culture method and/or media comprising N-acetylcysteine is increased by at least about 10% to at least about 50% (e.g.
  • a cholesterol auxotroph e.g. a myeloma, or a hybridoma (e.g. NS0 cells) cultured in a cell culture medium without N-acetylcysteine.
  • cell growth may be determined using a cell counting method.
  • a sample volume of cell culture medium is obtained and the cells counted in that volume.
  • Cell counting may be done in a hemocytometer or with a Coulter Counter. Another method plots the number of cells against time, with the slope of the graph stepper for cultures showing improved growth rates.
  • the cell culture method and/or media disclosed herein increase the cell viability of a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) over a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured in a cell culture with a control medium excluding N-acetylcysteine.
  • cell viability of a cholesterol auxotroph, a myeloma, or a hybridoma e.g.
  • NS0 cells cultured using a cell culture method and/or media comprising N-acetylcysteine is higher compared to a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured in a cell culture with a control medium excluding N-acetylcysteine.
  • cell viability of a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured using a cell culture method and/or media comprising N-acetylcysteine is increased by at least about 5% to at least about 15% (e.g.
  • the cell viability of a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured using a cell culture method and/or media comprising N-acetylcysteine is at least about 85% to at least about 95% (e.g. at least about 85%, about 88%, about 90%, about 92%, about 93%, about 94%, or about 95%).
  • cell viability may be determined by a trypan blue viability exclusion assay.
  • a cell suspension may be mixed with 0.4% trypan blue in phosphate buffered solution and cells counted using a hemocytometer. Live cells appear round and refractile without any blue-dye coloration, while dead cells absorb the dye and appear blue. Viability may be expressed as a percentage of viable cells over total cells counted, with a viable cell being a cell whose membrane integrity is still able to prevent the absorption of the trypan blue in a trypan blue exclusion viability assay.
  • increased protein yield is obtained using the cell culture method and/or media disclosed herein.
  • recombinant or heterologous protein expression is higher in a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured using a cell culture method and/or media comprising N-acetylcysteine compared to a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured in a cell culture with a control medium excluding N-acetylcysteine.
  • the cholesterol auxotroph, myeloma, or hybridoma (e.g. NS0 cells) cultured using a cell culture method and/or media comprising N-acetylcysteine shows at least 10% to at least 200% (e.g. at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 100%, 150%, 200%) higher protein expression compared to a cholesterol auxotroph, a myeloma, or a hybridoma (e.g. NS0 cells) cultured in a cell culture medium without N-acetylcysteine.
  • N-Acetylcysteine N-acetylcysteine
  • APF Animal Protein Free
  • One of the cell lines was a NS0 null host cell that had not been transformed to express a recombinant protein (NS0 Null Cell Line).
  • NS0 Cell Line 1 (expressing an anti-IL-9 antibody) and NS0 Cell Line 2 (expressing BAK502G9, an anti-IL-13 antibody).
  • NS0 Cell Line 1 was thawed and expanded in two different APF media (APF Medium 1 or APF Medium 2) supplemented with various concentrations of NAC ranging from 1.5 mM to 2.5 mM.
  • NS0 Cell Line 2 was thawed and expanded in three different APF media (APF Medium 1, APF Medium 2 or a commercially available NS0 cell culture media (CD Hybridoma+cholesterol from Invitrogen/Gibco)) supplemented with NAC ranging from 0.5 mM to 2.5 mM, while the untransformed host cell line (NS0 Null Cell Line) was thawed in APF medium 2 supplemented with NAC ranging from 0.5 mM to 1.5 mM.
  • NAC was obtained from Sigma and was either added to the medium directly or dissolved in water at 100 mM concentration before adding to the media at the appropriate concentration.
  • APF Medium 1 and APF Medium 2 contain standard cell culture components including: amino acids, vitamins, lipids, sugar, small peptides, pH buffer, trace metals, inorganic salts, nucleotides, nucleotide precursors, surfactants, reducing agents, cholesterol, lipids and antioxidants.
  • Viable cell densities during the first 3 to 4 days (i.e. exponential growth phase) at each passage were used to calculate population doubling times during both the first few days after the vial thaw while the cells were recovering from the thaw and during subsequent cell passages when the cells have fully recovered and reached a consistent doubling time from passage to passage.
  • NS0 cells i.e. NS0 Cell Line 1 expressing an anti-IL-9 monoclonal antibody were in a frozen stock.
  • Frozen NS0 Cell Line 1 cells were thawed in animal-protein free (APF) medium (i.e. APF media 1 or APF media 2) supplemented with various concentrations of N-acetylcysteine (NAC) and viable cell density during exponential growth phase was used to calculate average cell doubling time as described above.
  • APF animal-protein free
  • NAC N-acetylcysteine
  • FIG. 1 shows NS0 Cell Line 1 cell doubling time in control medium (APF media 1 without NAC) and APF media 1 supplemented with three increasing concentrations of N-acetylcysteine for cells thawed from a frozen stock.
  • Addition of 1.5 mM to 2.5 mM NAC (1.5 mM, 2.0 mM or 2.5 mM NAC) reduced average cell doubling time at vial thaw (55.6 hours, 48.4 hours, and 53.9 hours, respectively) compared to NS0 Cell Line 1 cells thawed in control medium (57.6 hours).
  • the slight increase in doubling time between 2 mM and 2.5 mM may be due to an increase in osmolality of the solution, especially as NS0 cells can be sensitive to osmolality during thawing.
  • FIG. 2 shows NS0 Cell Line 1 cell doubling time in control medium (APF media 2 without NAC) and APF media 2 supplemented with three increasing concentrations of N-acetylcysteine for cells thawed from a frozen stock. Similar to the results observed with APF Media 1, adding 1.5 mM to 2.5 mM NAC (1.5 mM, 2.0 mM or 2.5 mM NAC) to APF Media 2 also reduced cell doubling time at vial thaw (47.5 hours, 56.9 hours, and 47.7 hours, respectively) compared to NS0 Cell Line 1 cells thawed in control medium (109.8 hours).
  • NS0 cells i.e. NS0 Cell Line 2 expressing a monoclonal antibody (i.e. BAK502G9, an anti-IL-13 antibody) were in a frozen stock.
  • Frozen NS0 Cell Line 2 cells were thawed in animal-protein free (APF) medium (i.e. APF media 1, APF media 2 or CD Hybridoma medium (Gibco) supplemented with cholesterol (1 ⁇ Invitrogen Cholesterol Lipid Concentrate)) supplemented with various concentrations of N-acetylcysteine (NAC) and viable cell density during exponential growth phase was used to calculate average cell doubling time as described in Example 1.
  • APF animal-protein free
  • FIG. 3 shows NS0 Cell Line 2 cell doubling time in control medium (APF media 1 without NAC) and APF media 1 supplemented with three increasing concentrations of N-acetylcysteine for cells thawed from a frozen stock.
  • Addition of 0.5 mM to 2.5 mM NAC (0.5 mM, 1.0 mM or 2.5 mM NAC) reduced average cell doubling time at vial thaw (39.6 hours, 41.6 hours, and 37.5 hours, respectively) compared to NS0 Cell Line 2 cells thawed in control medium (44.6 hours), with 2.5 mM NAC showing the greatest reduction of cell doubling time at vial thaw.
  • FIG. 4 shows NS0 Cell Line 2 cell doubling time in control medium (APF media 2 without NAC) and APF media 2 supplemented with three increasing concentrations of N-acetylcysteine for cells thawed from a frozen stock.
  • APF media 1 Similar to the results observed with APF media 1, 0.5 mM to 2.0 mM NAC (0.5 mM, 1.0 mM or 2.0 mM NAC) reduced average cell doubling time at vial thaw (57.7 hours, 37.0 hours, and 39.7 hours, respectively) compared to NS0 Cell Line 2 cells thawed in control medium (77.0 hours), with 1.0 mM and 2.0 mM showing the greatest reduction of cell doubling time at vial thaw.
  • FIG. 5 shows NS0 Cell Line 2 cell doubling time in control medium (CD Hybridoma medium (Gibco) supplemented with 1 ⁇ Invitrogen Cholesterol Lipid Concentrate without NAC) and control medium supplemented with three increasing concentrations of N-acetylcysteine for cells thawed from a frozen stock.
  • control medium CD Hybridoma medium (Gibco) supplemented with 1 ⁇ Invitrogen Cholesterol Lipid Concentrate without NAC)
  • control medium supplemented with three increasing concentrations of N-acetylcysteine for cells thawed from a frozen stock.
  • NS0 cells i.e. NS0 Null Cell Line
  • Frozen NS0 Null Cell Line cells were thawed in animal-protein free (APF) medium (i.e. APF media 2) supplemented with various concentrations of N-acetylcysteine (NAC) and viable cell density during exponential growth phase was used to calculate average cell doubling time as described in Example 1.
  • APF animal-protein free
  • NAC N-acetylcysteine
  • FIG. 6 shows NS0 Null Cell Line cell doubling time in control medium (APF media 2 without NAC) and APF media supplemented with three increasing concentrations of N-acetylcysteine for cells thawed from a frozen stock. Similar to the results reported in Examples 1 and 2, addition of 0.5 mM to 1.5 mM NAC (0.5 mM, 1.0 mM or 1.5 mM NAC) reduced cell doubling time at vial thaw (36.8 hours, 38.4 hours and 57.9 hours, respectively) compared to NS0 Null Cell Line cells thawed in control medium (67.8 hours), with 0.5 mM and 1.0 mM showing the greatest reduction of cell doubling time at vial thaw.
  • control medium APF media 2 without NAC
  • NS0 cells i.e. NS0 Cell Line 1 expressing an anti-IL-9 monoclonal antibody were cultured in animal-protein free (APF) medium (i.e. APF media 1 or APF media 2).
  • APF media 1 or APF media 2 supplemented with various concentrations of N-acetylcysteine
  • FIG. 7 and Table 2 show NS0 Cell Line 1 cell doubling time in control medium (APF media 1 without NAC) and APF media 1 supplemented with three increasing concentrations of N-acetylcysteine during cell expansion.
  • Addition of 1.5 mM to 2.0 mM NAC (1.5 mM or 2.0 mM) reduced average cell doubling time during cell expansion compared to NS0 Cell Line 1 cells cultured in control medium. Error bars represent 1 standard deviation (1 S.D.) of average doubling time.
  • FIG. 8 and Table 3 show NS0 Cell Line 1 cell doubling time in control medium (APF media 2 without NAC) and APF media 2 supplemented with three increasing concentrations of N-acetylcysteine during expansion. Similar to the results observed with APF media 1, addition of 1.5 mM to 2.5 mM NAC (1.5 mM, 2.0 mM or 2.5 mM NAC) reduced average cell doubling time during expansion compared to NS0 Cell Line 1 cells cultured in control medium. Error bars represent 1 standard deviation (1 S.D.) of average doubling time.
  • N-acetylcysteine (about 1.5 mM to about 2.5 mM; or about 1.5 mM, about 2.0 mM or about 2.5 mM NAC) while NS0 cells are expanding increases cell viability, cell growth and reduces cell doubling time.
  • N-acetylcysteine concentrations of 1.5 mM and 2.0 mM consistently increased cell viability, cell growth and reduced cell doubling time of NS0 cell line 1 cells undergoing cell expansion in two different media.
  • NS0 cells i.e. NS0 Cell Line 2 expressing a monoclonal antibody (i.e. BAK502G9, an anti-IL-13 antibody) were cultured in animal-protein free (APF) medium (i.e. APF media 1, APF media 2 or CD Hybridoma medium (Gibco) supplemented with cholesterol (1 ⁇ Invitrogen Cholesterol Lipid Concentrate)).
  • APF media 1 APF media 2 or CD Hybridoma medium (Gibco) supplemented with cholesterol (1 ⁇ Invitrogen Cholesterol Lipid Concentrate)
  • the cells used in the vial thaw studies described in Example 2 were split into the same media (APF media 1, APF media 2 or CD Hybridoma medium+cholesterol) supplemented with various concentrations of N-acetylcysteine and allowed to recover in subsequent passages until a consistent doubling time from passage to passage was achieved.
  • Viable cell density during exponential growth phase after recovery was used to calculate average cell doubling time as described
  • FIG. 9 and Table 4 show NS0 Cell Line 2 cell doubling time in control medium (APF media 1 without NAC) and APF media 1 supplemented with three increasing concentrations of N-acetylcysteine during expansion.
  • Addition of 0.5 mM to 2.5 mM NAC (0.5 mM, 1.0 mM or 2.5 mM) reduced average cell doubling time during expansion compared to NS0Cell Line 2 cells cultured in control medium, with 1.0 mM and 2.5 mM NAC showing the greatest reduction of cell doubling time during expansion.
  • Error bars represent 1 standard deviation (1 S.D.) of average doubling time.
  • FIG. 10 and Table 5 show NS0 Cell Line 2 cell doubling time in control medium (APF media 2 without NAC) and APF media 2 supplemented with three increasing concentrations of N-acetylcysteine during expansion. Similar to the results observed with APF media 1, addition of 0.5 mM to 2.0 mM NAC (0.5 mM, 1.0 mM, or 2.0 mM) reduced average cell doubling time during expansion compared to NS0 Cell Line 2 cells cultured in control medium. Error bars represent 1 standard deviation (1 S.D.) of average doubling time.
  • FIG. 11 and Table 6 show NS0 Cell Line 2 cell doubling time in control medium (CD Hybridoma medium (Gibco) supplemented with 1 ⁇ Invitrogen Cholesterol Lipid Concentrate without NAC) and control medium containing three increasing concentrations of N-acetylcysteine during cell expansion. Similar to the results observed with APF media 1 and APF media 2, addition of 1.0 mM to 2.0 mM NAC reduced average cell doubling time compared to NS0 Cell Line 2 cells cultured in control medium. Error bars represent 1 standard deviation (1 S.D.) of average doubling time.
  • N-acetylcysteine (about 0.5 mM to about 2.5 mM; or about 0.5 mM, about 1.0 mM, about 2.0 mM or about 2.5 mM NAC) to the cell culture media of NS0 cells during expansion increases cell viability, cell growth and reduces cell doubling time.
  • NS0 cells i.e. NS0 Null Cell Line
  • APF media 2 animal-protein free medium
  • Viable cell density during exponential growth phase after recovery was used to calculate average cell doubling time as described in Example 1.
  • FIG. 12 and Table 7 show NS0 Null Cell Line cell doubling time in control medium (APF medium 2 without NAC) and APF media 2 supplemented with three increasing concentrations of N-acetylcysteine during expansion.
  • Addition of 0.5 mM to 1.5 mM NAC 0.5 mM. 1.0 mM or 1.5 mM NAC
  • Error bars represent 1 standard deviation (1 S.D.) of average doubling time.
  • the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term about generally refers to a range of numerical values (e.g., +/ ⁇ 5% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • the term about may include numerical values that are rounded to the nearest significant figure.

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