Cell culture medium for eukaryotic cells

Abstract

To provide cell culture processes, recombinant polypeptide expression, titer, and a method for improving a cell survival rate that can lead to higher production levels, thereby reducing the costs associated with manufacturing protein therapeutics.SOLUTION: Cell culture media containing 5-methylthioadenosine or nicotinamide are provided, as are methods of using the media for cell culture and methods of protein production from cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] Field The present invention relates generally to cell culture media for eukaryotic cells and the production of natural products and recombinant products derived therefrom. [Background technology]

[0002] background Cell culture manufacturing technology is widely used for the production of biopharmaceuticals. As the demand for biopharmaceuticals increases, the demand for increased cell growth, viability, and protein production also increases significantly. Currently, much effort is being placed on methods and strategies for growing, feeding, and maintaining cell cultures.

[0003] Given the challenges and costs of large-scale cell culture processes, and the growing demand for large quantities and low-cost biological products, new cell culture methods that provide even incremental improvements in recombinant protein production would be beneficial. Improvements in cell culture processes, recombinant polypeptide expression, titer, and cell viability are needed that could result in higher production levels, thereby reducing the costs associated with the manufacture of protein therapeutics. Summary of the Invention

[0004] overview The increasing development, production, and sale of protein-based biopharmaceuticals has created a growing demand for production methods that can improve the production of biopharmaceuticals.

[0005] The embodiments disclosed herein address the above-mentioned needs by providing methods and media for the production of such biopharmaceuticals.

[0006] The present disclosure provides, at least in part, a cell culture medium for eukaryotic cells.

[0007] In an exemplary embodiment, the cell culture medium for eukaryotic cells may include a basal medium. In one aspect of this embodiment, the cell culture medium may further include 5-methylthioadenosine. In another aspect of this embodiment, the cell culture medium may include at least about 10 nM 5-methylthioadenosine. In yet another aspect of this embodiment, the cell culture medium may include about 10 nM to about 200 nM 5-methylthioadenosine. In one aspect of this embodiment, the cell culture medium may further include nicotinamide. In another aspect of this embodiment, the cell culture medium may include at least about 50 nM nicotinamide. In yet another aspect of this embodiment, the cell culture medium may include about 2000 nM 5-nicotinamide. In one aspect of this embodiment, the titer of the protein grown in the cell culture medium is at least about 2% greater than another cell culture medium that does not include at least about 10 nM 5-methylthioadenosine. In another aspect of this embodiment, the titer of the protein grown in the cell culture medium is at least about 2% greater than another cell culture medium that does not contain at least about 50 nM nicotinamide. In one aspect of this embodiment, the cell culture medium may further optionally include one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. In one aspect of this embodiment, the cell culture medium may further optionally include sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this embodiment, the cell culture medium may include a salt or ester of 5-methylthioadenosine. In another aspect of this embodiment, the cell culture medium may include a salt or ester of nicotinamide. In one aspect of this embodiment, the cell culture medium may have a pH of about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium is free of animal-derived proteins. In one aspect of this embodiment, the cell culture medium can be a serum-free medium. In one aspect of this embodiment, the cell culture medium can be a chemically defined medium.

[0008] In an exemplary embodiment, the cell culture medium for eukaryotic cells may comprise a feed medium. In one aspect of this embodiment, the cell culture medium may further comprise 5-methylthioadenosine. In another aspect of this embodiment, the cell culture medium may comprise at least about 10 nM 5-methylthioadenosine. In yet another aspect of this embodiment, the cell culture medium may comprise about 10 nM to about 200 nM 5-methylthioadenosine. In one aspect of this embodiment, the cell culture medium may further comprise nicotinamide. In another aspect of this embodiment, the cell culture medium may comprise at least about 50 nM nicotinamide. In yet another aspect of this embodiment, the cell culture medium may comprise about 50 nM to about 2000 nM 5-nicotinamide. In another aspect of this embodiment, the cell culture medium may further comprise 5-methylthioadenosine and nicotinamide. In one aspect of this embodiment, the cell culture medium may further optionally include one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. In one aspect of this embodiment, the cell culture medium may further optionally include sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this embodiment, the cell culture medium may include a salt or ester of 5-methylthioadenosine. In another aspect of this embodiment, the cell culture medium may include a salt or ester of nicotinamide. In one aspect of this embodiment, the cell culture medium may have a pH of about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium is free of animal-derived proteins. In one aspect of this embodiment, the cell culture medium may be a serum-free medium. In one aspect of this embodiment, the cell culture medium may be a chemically defined medium. In one aspect of this embodiment, the cell culture medium can optionally contain nicotinamide.In one aspect of this embodiment, the titer of the protein grown in the cell culture medium is at least about 2% greater than another cell culture medium that does not contain at least about 10 nM 5-methylthioadenosine.In another aspect of this embodiment, the titer of the protein grown in the cell culture medium is at least about 2% greater than another cell culture medium that does not contain at least about 50 nM nicotinamide.

[0009] The present disclosure provides, at least in part, a method for producing a protein.

[0010] In one exemplary embodiment, a method of producing a protein may include culturing eukaryotic cells having a nucleic acid encoding the protein in a cell culture production medium and feeding the eukaryotic cells with an enriched medium having 5-methylthioadenosine for a period of time. In one aspect of this embodiment, the enriched medium may have at least about 1 nM 5-methylthioadenosine. In one aspect of this embodiment, the cell culture production medium may include one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. In one aspect of this embodiment, the cell culture production medium may include sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this embodiment, the cell culture production medium may include salts or esters of 5-methylthioadenosine. In one aspect of this embodiment, the cell culture production medium may have a pH of about 6.5 to about 8. In one aspect of this embodiment, the cell culture production medium is free of animal-derived proteins. In one aspect of this embodiment, the cell culture production medium may be a serum-free medium. In one aspect of this embodiment, the cell culture production medium may be a chemically defined medium. In one aspect of this embodiment, the eukaryotic cells may be selected from a baby hamster kidney cell line, a Chinese hamster ovary cell line, a murine myeloma cell line, a mouse myeloma cell line, a human embryonic kidney cell line, a human retina-derived cell line, and / or an amniotic membrane cell line. In one aspect of this embodiment, the protein may be selected from the group consisting of an antibody or a fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein. In one aspect of this embodiment, the method may produce a titer of the protein that is at least about 2% greater than the titer of the protein in a cell culture production medium that does not have at least about 10 nM 5-methylthioadenosine. In one aspect of this embodiment, the enrichment medium may optionally include nicotinamide. In one aspect of this embodiment, the method for producing a protein can be a fed-batch method.

[0011] In one exemplary embodiment, a method of producing a protein may include culturing eukaryotic cells having a nucleic acid encoding the protein in a cell culture production medium and feeding the eukaryotic cells with an enriched medium having nicotinamide for a specified period of time. In one aspect of this embodiment, the enriched medium may have at least about 5 nM nicotinamide. In one aspect of this embodiment, the cell culture production medium may include one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. In one aspect of this embodiment, the cell culture production medium may include sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this embodiment, the cell culture production medium may include salts or esters of nicotinamide. In one aspect of this embodiment, the cell culture production medium may have a pH of about 6.5 to about 8. In one aspect of this embodiment, the cell culture production medium is free of animal-derived proteins. In one aspect of this embodiment, the cell culture production medium can be a serum-free medium. In one aspect of this embodiment, the cell culture production medium can be a chemically defined medium. In one aspect of this embodiment, the eukaryotic cells can be selected from a baby hamster kidney cell line, a Chinese hamster ovary cell line, a murine myeloma cell line, a mouse myeloma cell line, a human embryonic kidney cell line, a human retina-derived cell line, and / or an amniotic membrane cell line. In one aspect of this embodiment, the protein can be selected from the group consisting of an antibody or a fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein. In one aspect of this embodiment, the method can produce a titer of the protein that is at least about 2% greater than the titer of the protein in a cell culture production medium that does not have at least about 50 nM nicotinamide. In one aspect of this embodiment, the enrichment medium can optionally include 5-methylthioadenosine. In one aspect of this embodiment, the method of producing the protein can be a fed-batch method.

[0012] The present disclosure provides, at least in part, methods for increasing production of a protein.

[0013] In an exemplary embodiment, a method for increasing production of a protein may include culturing eukaryotic cells in a cell culture medium, supplementing the cell culture medium with 5-methylthioadenosine, and expressing the protein. In one aspect of this embodiment, the concentration of 5-methylthioadenosine may be at least about 10 nM. In one aspect of this embodiment, the concentration of 5-methylthioadenosine may be from about 10 nM to about 200 nM. In one aspect of this embodiment, the cell culture medium may include one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. In one aspect of this embodiment, the cell culture medium may include sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this embodiment, the cell culture medium may include a salt or ester of 5-methylthioadenosine. In one aspect of this embodiment, the cell culture medium may have a pH of about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium is free of animal-derived proteins. In one aspect of this embodiment, the cell culture medium may be a serum-free medium. In one aspect of this embodiment, the cell culture medium may be a chemically defined medium. In one aspect of this embodiment, the eukaryotic cells may be selected from a baby hamster kidney cell line, a Chinese hamster ovary cell line, a murine myeloma cell line, a mouse myeloma cell line, a human embryonic kidney cell line, a human retina-derived cell line, and / or an amniotic cell line. In one aspect of this embodiment, the protein may be selected from the group consisting of an antibody or a fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein. In one aspect of this embodiment, supplementation with 5-methylthioadenosine increases the titer of the recombinant protein by at least about 2%. In one aspect of this embodiment, the cell culture medium may be optionally supplemented with nicotinamide.

[0014] In an exemplary embodiment, a method for increasing production of a protein may include culturing eukaryotic cells in a cell culture medium, supplementing the cell culture medium with nicotinamide, and expressing the protein. In one aspect of this embodiment, the concentration of nicotinamide may be at least about 50 nM. In one aspect of this embodiment, the concentration of 5-methylthioadenosine may be from about 50 nM to about 2000 nM. In one aspect of this embodiment, the cell culture medium may include one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. In one aspect of this embodiment, the cell culture medium may include sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this embodiment, the cell culture medium may include salts or esters of nicotinamide. In one aspect of this embodiment, the cell culture medium may have a pH of about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium is free of animal-derived proteins. In one aspect of this embodiment, the cell culture medium may be a serum-free medium. In one aspect of this embodiment, the cell culture medium may be a chemically defined medium. In one aspect of this embodiment, the eukaryotic cells may be selected from a baby hamster kidney cell line, a Chinese hamster ovary cell line, a murine myeloma cell line, a mouse myeloma cell line, a human embryonic kidney cell line, a human retina-derived cell line, and / or an amniotic cell line. In one aspect of this embodiment, the protein may be selected from the group consisting of an antibody or a fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein. In one aspect of this embodiment, supplementation with nicotinamide increases the titer of the recombinant protein by at least about 2%. In one aspect of this embodiment, the cell culture medium may be optionally supplemented with 5-methyladenosine.

[0015] The present disclosure provides, at least in part, methods for producing proteins.

[0016] In an exemplary embodiment, a method for producing a protein may include introducing a nucleic acid comprising a sequence encoding the protein into a cell(s) and culturing the cell(s) in a cell culture medium. In one aspect of this embodiment, the cell culture medium may be enriched with at least about 10 nM 5-methylthioadenosine. In another aspect of this embodiment, the cell culture medium may be enriched with at least about 50 nM nicotinamide. In yet another aspect of this embodiment, the cell culture medium may be enriched with at least about 10 nM 5-methylthioadenosine and at least about 50 nM nicotinamide. In one aspect of this embodiment, the method for producing a protein may further include maintaining the cell culture medium to express higher titers of the protein in the cell(s). In one aspect of this embodiment, the method for producing a protein may further include harvesting the protein. In one aspect of this embodiment, the cell(s) can be selected from a baby hamster kidney cell line, a Chinese hamster ovary cell line, a murine myeloma cell line, a mouse myeloma cell line, a human embryonic kidney cell line, a human retina-derived cell line, and / or an amniotic membrane cell line. In one aspect of this embodiment, the cell culture medium can further comprise one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. In one aspect of this embodiment, the cell culture medium can further comprise sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this embodiment, the cell culture medium can have a pH of about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium is free of animal-derived proteins. In one aspect of this embodiment, the cell culture medium can be a serum-free medium. In one aspect of this embodiment, the cell culture medium can be a chemically defined medium. In one aspect of this embodiment, the protein can be selected from the group consisting of an antibody or a fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein.In one aspect of this embodiment, the method may express a 2% higher titer of protein in the cell(s) compared to the cell(s) grown in a cell culture medium lacking at least about 10 nM 5-methylthioadenosine. In another aspect of this embodiment, the method for producing a protein may further include maintaining the cell culture medium to express a 2% higher titer of protein in the cell(s) compared to the cell(s) grown in a cell culture medium lacking at least about 50 nM nicotinamide. [The present invention 1001] A cell culture medium for a eukaryotic cell expressing aflibercept, comprising: a basal or feed medium, and 5-Methylthioadenosine or Nicotinamide The cell culture medium comprising: [The present invention 1002] 1001. The cell culture medium of claim 10, wherein the concentration of 5-methylthioadenosine in said cell culture medium is at least about 10 nM. [The present invention 1003] The cell culture medium of claim 10, wherein the concentration of said nicotinamide in said cell culture medium is at least about 50 nM. [The present invention 1004] The cell culture medium of the present invention 1001 further comprising one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. [The present invention 1005] The cell culture medium of claim 1002, wherein the titer of aflibercept produced in the cell culture medium is at least about 2% greater than in another cell culture medium that does not have at least about 10 nM 5-methylthioadenosine. [The present invention 1006] The cell culture medium of the present invention, wherein the potency of the aflibercept produced in the cell culture medium is at least about 2% greater than in another cell culture medium not having at least about 50 nM nicotinamide. [The present invention 1007] The cell culture medium of claim 10, wherein said 5-methylthioadenosine or said nicotinamide may be in the form of a salt or ester thereof. [The present invention 1008] The cell culture medium of the present invention, wherein the pH of the medium is maintained in the range of about 6.5 to about 8. [The present invention 1009] 1001. The cell culture medium of the present invention, which is free of animal-derived proteins. [The present invention 1010] The cell culture medium of the present invention, which is a serum-free medium. [The present invention 1011] The cell culture medium of the present invention 1001, which is a chemically defined medium. [The present invention 1012] 1. A method for culturing eukaryotic cells to enhance production of aflibercept, comprising: The method further comprising: Culturing eukaryotic cells in a cell culture medium; supplementing the cell culture medium with 5-methylthioadenosine, wherein the concentration of the 5-methylthioadenosine is about 10 nM to about 200 nM; and Producing aflibercept in said eukaryotic cell. Including, said supplementation with 5-methylthioadenosine increases the potency of said aflibercept; The method. [The present invention 1013] The method of claim 1012, wherein the potency of the aflibercept is at least about 2% greater than in another method using a cell culture medium that does not have at least about 10 nM 5-methylthioadenosine. [The present invention 1014] The method of claim 1012, wherein the cell culture medium further comprises one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. [The present invention 1015] The method of the present invention, wherein the eukaryotic cell comprises at least one selected from the group consisting of a baby hamster kidney cell line, a Chinese hamster ovary cell line, a murine myeloma cell line, a mouse myeloma cell line, a human embryonic kidney cell line, a human retina-derived cell line, and an amniotic cell line. [The present invention 1016] The method of claim 10, wherein said aflibercept is secreted into said medium. [The present invention 1017] The method of claim 1012, wherein said cell culture medium is free of animal-derived proteins. [The present invention 1018] The method of claim 1012, wherein the cell culture medium is a serum-free medium. [The present invention 1019] The method of claim 1012, wherein the cell culture medium is a chemically defined medium. [The present invention 1020] 1. A method for culturing eukaryotic cells to enhance production of aflibercept, comprising: The method further comprising: Culturing cells in a defined cell culture medium; supplementing the cell culture medium with nicotinamide, wherein the concentration of the nicotinamide is about 50 nM to about 2000 nM; and Producing aflibercept in said eukaryotic cell. Including, said supplementation with nicotinamide increases the potency of said aflibercept; The method. [The present invention 1021] The method of claim 1020, wherein the potency of the aflibercept is at least about 2% greater than in another method using a cell culture medium that does not have at least about 50 nM nicotinamide. [The present invention 1022] 1020. The method of claim 1020, wherein the cell culture medium further comprises one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. [The present invention 1023] The method of claim 1020, wherein the eukaryotic cell comprises at least one selected from the group consisting of a baby hamster kidney cell line, a Chinese hamster ovary cell line, a murine myeloma cell line, a mouse myeloma cell line, a human embryonic kidney cell line, a human retina-derived cell line, and an amniotic cell line. [The present invention 1024] The method of claim 1020, wherein said aflibercept is secreted into said medium. [The present invention 1025] The method of claim 1020, wherein said cell culture medium is free of animal-derived proteins. [The present invention 1026] The method of claim 1020, wherein said cell culture medium is a serum-free medium. [The present invention 1027] The method of claim 1020, wherein said cell culture medium is a chemically defined medium. [The present invention 1028] 1. A method for producing aflibercept, comprising: introducing into the cell a nucleic acid comprising a nucleotide sequence encoding aflibercept; culturing the cells in a cell culture medium containing at least about 50 nM nicotinamide or at least about 10 nM 5-methylthioadenosine; and Producing said aflibercept in said cell. The method comprising: [Brief description of the drawings]

[0017] [Figure 1-1] 1 shows the results of a multivariate analysis (MVA) of the investigation of components present in soy hydrolysates. [Figure 1-2]1 shows the results of a multivariate analysis (MVA) of the investigation of components present in soy hydrolysates. [Diagram 2] 1 shows correlation data obtained from studying the effect of 5-methylthioadenosine concentration in enrichment media used to supplement cell culture media on protein titer (g / L) according to an exemplary embodiment. [Diagram 3] 1 shows a regression line for variables (N=70) used to evaluate the effect of concentration of 5-methylthioadenosine in cell culture medium on protein titer (g / L), according to an exemplary embodiment. [Figure 4] 1 shows correlation data obtained from studying the effect of nicotinamide concentration in enrichment media used to supplement cell culture media on protein titer (g / L) according to an exemplary embodiment. [Diagram 5] 1 shows a regression line for variables (N=70) used to evaluate the effect of concentration of nicotinamide in cell culture medium on protein titer (g / L), according to an exemplary embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description Biopharmaceuticals are highly effective in diagnostic and therapeutic applications (Dawn M Ecker, Susan Dana Jones & Howard L Levine, The therapeutic monoclonal antibody market, 7 mAbs 9-14 (2014); Brian A. Baldo, Chimeric Fusion binding molecules Used for Therapy: Indications, Mechanisms, and Safety, 38 Drug Safety 455-479 (2015)). Several cell culture media used to culture cells that produce biopharmaceuticals are well described in the literature, and many media are commercially available. Typical components of cell culture media include amino acids, organic and inorganic salts, vitamins, trace metals, sugars, lipids, and nucleic acids, the type and amount of which may vary depending on the specific requirements of a given cell or tissue type.

[0019] One goal of protein production may be the optimization of cell culture to obtain the maximum amount of protein and the most efficient means of productivity. Any improvement, including incremental improvement, may have a great economic benefit. In the pharmaceutical industry, it is advantageous to optimize the protein production of biologics used in the treatment of disease, since any improvement may have a great impact when biologics are produced on a large scale. As such, there remains a need to maximize protein production from cell cultures that express biological proteins used in medical treatment.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, certain methods and materials are described herein. All publications mentioned above are incorporated herein by reference.

[0021] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variations understood by those of ordinary skill in the art, and when ranges are provided, the endpoints are included.

[0022] In some exemplary embodiments, the disclosure provides a method for producing a protein.

[0023] As used herein, the term "protein" includes any amino acid polymer having covalently linked amide bonds. A protein includes one or more amino acid polymer chains, commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and non-naturally occurring synthetic analogs thereof linked via peptide bonds, related naturally occurring structural variants, and non-naturally occurring synthetic analogs thereof. A "synthetic peptide or polypeptide" refers to a peptide or polypeptide that does not occur in nature. A synthetic peptide or polypeptide can be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid-phase peptide synthesis methods are known to those skilled in the art. A protein may contain one or more polypeptides to form a single functional biomolecule. Proteins may include biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion binding molecules, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In another exemplary embodiment, the protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Recombinant proteins may be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia species), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a recent review discussing biotherapeutic proteins and their production, see Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 Biotechnology and Genetic Engineering Reviews 147-176 (2012). In some embodiments, the proteins include modifications, additions, and other covalently attached moieties.These modifications, adducts and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels, and other dyes, etc. Proteins can be classified based on composition and solubility, and thus can include simple proteins, e.g., globular proteins and fibrous proteins; conjugated proteins, e.g., nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, e.g., primary derived proteins and secondary derived proteins.

[0024] In some exemplary embodiments, the protein or recombinant protein may be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, an Fc fusion binding molecule, an F(ab')2 fragment, an Fc fragment, or a combination thereof.

[0025] As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, inter-connected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V H The heavy chain constant region comprises a C H 1. C H 2, and C H Each light chain comprises three domains: a light chain variable region (herein referred to as LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H and V LThe regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L can be composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In various embodiments of the present invention, the FRs of the anti-big ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. The term "antibody" as used herein also includes antigen-binding fragments of a complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived, for example, from a complete antibody molecule using any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding the antibody variable domains and, optionally, the constant domains. Such DNA is known and / or readily available, e.g., from commercial sources, DNA libraries (including, e.g., phage antibody libraries), or can be synthesized. The DNA may be manipulated using sequencing and chemical or molecular biology techniques, e.g., to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids.

[0026] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display techniques, or a combination thereof.

[0027] As used herein, the term "fusion protein" or "Fc fusion protein" includes parts or all of two or more proteins, one of which may be the Fc portion of an immunoglobulin molecule that is not naturally fused. The preparation of fusion proteins may include a specific heterologous polypeptide fused to various portions of an antibody-derived polypeptide (including an Fc domain), and is described, for example, in A. Ashkenazi et al., Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin., 88 Proceedings of the National Academy of Sciences 10535-10539 (1991); Randal A. Byrn et al., Biological properties of a CD4 immunoadhesin, 344 Nature 667-670 (1990); Diane Hollenbaugh & Alejandro Aruffo, Construction of Immunoglobulin Fusion binding molecules, Current Protocols in Immunology (2002). A "receptor-Fc fusion protein" comprises one or more of one or more extracellular domain(s) of a receptor coupled to an Fc portion, and in some embodiments, an immunoglobulin hinge region followed by a C H 2 and C H In some embodiments, the nucleotide sequence may comprise three domains: TIFF2025028979000001.tif5136 may contain two or more separate receptor chains that bind to a single or multiple ligand(s). For example, the Fc fusion protein may be a trap, such as an IL-1 trap (e.g., rilonacept, which contains the IL-1 RAcP ligand binding domain fused to the IL-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159, which are incorporated herein by reference in their entirety).

[0028] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the heavy and light chain variable regions of an immunoglobulin, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the light and heavy chain variable regions of an immunoglobulin are linked by a peptide linker. In some exemplary embodiments, an antibody fragment contains sufficient amino acid sequence of its parent antibody such that it binds to the same antigen as the parent antibody; in some exemplary embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Antibody fragments may be produced by any means. For example, they may be enzymatically or chemically produced by fragmentation of an intact antibody and / or recombinantly produced from a gene encoding a partial antibody sequence. Alternatively or additionally, they may be wholly or partially synthetically produced. Antibody fragments may optionally include single chain antibody fragments. Alternatively or additionally, they may include multiple chains linked together, for example, by disulfide bonds. Antibody fragments may optionally include multimolecular complexes. Functional antibody fragments usually contain at least about 50 amino acids, more usually at least about 200 amino acids.

[0029] In some exemplary embodiments, the protein or recombinant protein may be an antibody variant or a binding molecule variant.

[0030] A "variant" or "binding molecule variant" as used herein may include a binding molecule that differs from a target binding molecule by at least one amino acid modification or post-translational modification. A variant may refer to the binding molecule itself, a composition comprising the binding molecule, or the amino sequence encoding it. Preferably, a binding molecule variant has at least one amino acid modification compared to the parent binding molecule, e.g., about 1 to about 10 amino acid modifications, preferably about 1 to about 5 amino acid modifications, compared to the parent. A binding molecule variant sequence herein will preferably have at least about 80% homology with the parent binding molecule sequence, more preferably at least about 90% homology, and most preferably at least about 92% homology.

[0031] In some exemplary embodiments, the protein or recombinant protein may be a protein with a particular post-translational modification.

[0032] As used herein, the general term "post-translational modification" or "PTM" refers to a covalent modification that a polypeptide undergoes either during ribosomal synthesis (co-translational modification) or after synthesis (post-translational modification). PTMs are generally introduced by specific enzymes or enzymatic pathways. Many occur at the site of a specific characteristic protein sequence (e.g., signature sequence) within the protein backbone. Hundreds of PTMs have been documented, and these modifications always affect some aspect of protein structure or function (Walsh, G. "Proteins" (2014) second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853).Various post-translational modifications include, but are not limited to, truncation, N-terminal extension, proteolysis, N-terminal acylation, biotinylation (acylation of lysine residues with biotin), C-terminal amidation, glycosylation, iodination, covalent attachment of prosthetic groups, acetylation (addition of an acetyl group, usually at the N-terminus of a protein), alkylation (addition of an alkyl group (e.g., methyl, ethyl, propyl) usually at a lysine or arginine residue), methylation, adenylation, ADP-ribosylation, covalent crosslinks within or between polypeptide chains, sulfonation, prenylation, vitamin C-dependent modifications (hydroxylation of proline and lysine and amidation of the carboxy terminus), vitamin K-dependent modifications in which vitamin K inhibits the synthesis of glutamate, resulting in the formation of gamma-carboxyglutamate (glu residues). Vitamin K-dependent modifications include glutamylation (covalent attachment of a glutamic acid residue), glycylation (covalently attached glycine residues), glycosylation (addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine to produce glycoproteins), isoprenylation (addition of an isoprenoid group such as farnesol and geranylgeraniol), lipoylation (attachment of a lipoate functional group), phosphopantetheinylation (addition of the 4'-phosphopantetheinyl moiety of coenzyme A, such as in fatty acid, polyketide, nonribosomal peptide, and leucine biosynthesis), phosphorylation (addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine), and sulfation (addition of a sulfate group, usually to a tyrosine residue). Post-translational modifications that change the chemical nature of an amino acid include, but are not limited to, citrullination (e.g., conversion of arginine to citrulline by deimination) and deamidation (e.g., conversion of glutamine to glutamic acid or asparagine to aspartic acid). Post-translational modifications that involve structural changes include, but are not limited to, the formation of disulfide bridges (covalent bonding of two cysteine ​​amino acids) and proteolytic cleavage (breaking of a protein at a peptide bond).Specific post-translational modifications include the addition of other proteins or peptides, e.g., ISGylation (covalent attachment to the ISG15 protein (interferon stimulated gene)), SUMOylation (covalent attachment to the SUMO protein (small ubiquitin-related modifier)), and ubiquitination (covalent attachment to the protein ubiquitin). For a more detailed controlled vocabulary of PTMs curated by UniProt, see http: / / www.uniprot.org / docs / ptmlist.

[0033] In some exemplary embodiments, the present disclosure provides a cell culture medium for a eukaryotic cell comprising a basal or feed medium and 5-methylthioadenosine and / or nicotinamide.

[0034] As used herein, the term "cell culture" refers to cells grown in an artificial (e.g., in vitro) environment. However, it should be understood that the term "cell culture" is a general term and may be used to encompass not only the culture of individual prokaryotic (e.g., bacterial) or eukaryotic (e.g., animal, plant, and fungal) cells, but also the culture of tissues, organs, organ systems, or whole organisms, and that "tissue culture," "organ culture," "organ system culture," or "organotypic culture" may be used interchangeably, as the case may be. Suitable culture conditions for eukaryotic cells can be found in the art, for example, in Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Cell culture media may be optimized for a particular cell culture application, including, for example, cell culture growth media, which may be formulated to promote cell growth, or cell culture production media, which may be formulated to promote recombinant protein production. The terms nutrients, ingredients, and components are used interchangeably herein to refer to the components that make up a cell culture medium.

[0035] As used herein, the term "basal medium" may refer to any medium that may be capable of supporting the growth of cells. Basal media may contain a number of components, including amino acids, vitamins, organic and inorganic salts, carbohydrate sources, each component being present in an amount that supports the cultivation of cells in vitro. The medium may contain supplementary substances, for example, buffer substances such as sodium bicarbonate, oxidative stabilizers, stabilizers that counteract the effects of mechanical stress, or protease inhibitors. Examples of basal media include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), DME / F12, Minimum Essential Medium (MEM), Basal Medium Eagle (BME), Medium 199, RPMI 1640, F-10, F-12, α-Minimum Essential Medium (α-MEM), Glasgow Minimum Essential Medium (G-MEM), PF CHO (SAFC Biosciences), Iscove's Modified Dulbecco's Medium, or combinations thereof, and others known from the literature or commercially available.

[0036] As used herein, the term "feed medium" includes a medium containing one or more nutrients that can be added to a culture that has been started at some point after inoculation. A feed medium can also be a combination feed that includes a basal medium and at least one type of hydrolysate, such as a soy-based hydrolysate, a yeast-based hydrolysate, or a combination of two types of hydrolysates. Furthermore, a feed medium can also include only a basal medium, such as a concentrated basal medium, or it can include only a hydrolysate or a concentrated hydrolysate.

[0037] As used herein, the term "eukaryotic cell" can include individual cells, tissues, organs, insect cells, avian cells, mammalian cells, primary cells, continuous cell lines, stem cells, and / or genetically engineered cells, e.g., recombinant cells expressing a heterologous polypeptide or protein. Some mammalian cells suitable for culturing in the cell culture medium may be of human or non-human origin and may include: primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), established cell lines and their derivatives (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 cells, and the like). VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PKi cells, PK(15) cells, GHi cells, GH3 cells, L2 cells, LLC-RC 256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK-cells, PK-15 cells, or derivatives thereof), fibroblasts from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestine, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphatic tissue (lymph glands, adenoids, tonsils, bone marrow, and blood), spleen), and fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, D BS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells cells, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L cells, strain 2071 (mouse L) cells, LM strain (mouse L) cells, L-MTK' (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, Cn cells, as well as Jensen cells, Sp2 / 0, NS0, NS1 cells, or derivatives thereof).

[0038] As used herein, "nicotinamide" may also be referred to as niacinamide, nicotinic acid amide, pyridine-3-carboxylic acid amide, vitamin B3, vitamin PP, 3-pyridinecarboxamide, CAS number 98-92-0, or C6H6N2O.

[0039] As used herein, "5-methylthioadenosine" refers to 5-deoxy-5'-methyl-thioadenosine, CAS number 2457-80-9; TIFF2025028979000002.tif4128, MTA, MeSAdo, NSC 335422, Vitamin L2, can also be referred to as salts of 5-methylthioadenosine or esters of 5-methylthioadenosine. Non-limiting examples of suitable salts of 5-methylthioadenosine include, for example, acid addition salts. Acid addition salts can be inorganic and organic acid addition salts, such as hydrochloride, sulfate, nitrate, carbonate, phosphate, formate, oxalate, citrate, ascorbic acid, methanesulfonate, 1,4-butanesulfonate, 1,5-pentanesulfonate, and p-toluenesulfonate.

[0040] In some exemplary embodiments, the disclosure provides a method of producing a protein comprising culturing a eukaryotic cell having a nucleic acid encoding the protein in a cell culture production medium.

[0041] Cell culture media or cell culture production media can be supplemented with concentrated media that contain components such as nutrients and amino acids that are consumed during the production phase of the cell culture.

[0042] As used herein, the term "cell culture production medium" can include cell culture media designed to be used during the production stage of a cell culture.

[0043] In some exemplary embodiments, the cell culture production medium or cell culture medium may be a serum-free medium.

[0044] As used herein, the term "serum-free medium" includes cell culture media that do not contain animal serum, such as fetal bovine serum. Serum-free media may or may not contain hydrolysates, growth factors, hormones, carrier proteins, and attachment factors. Examples of known serum-free media include CHO-S-SFM II (Gibco) and 293 SFM II (Gibco).

[0045] In some exemplary embodiments, the cell culture production medium or cell culture medium may be an animal-derived protein-free medium.

[0046] As used herein, the term "animal-derived protein-free medium" may refer to a medium that does not contain proteins and protein components derived from higher multicellular non-plant eukaryotes (i.e., vertebrates) that have secondary, tertiary, and quaternary structures characteristic of proteins as they occur in nature. Such a medium does not contain proteins such as albumin, transferrin, insulin, and other growth factors. However, animal-derived proteins and protein components are distinct from non-animal proteins, small polypeptides and oligopeptides available from plants such as soybean (usually about 10-30 amino acids in length), and lower eukaryotes such as yeast. When cells are contacted or inoculated with animal-derived protein medium, the medium will contain animal proteins excreted or secreted by those cells, including any recombinant proteins expressed by genetically modified cells, when such cells are cultured. Thus, the term animal protein free media, and biological materials and preparations produced therewith, should not be construed to require the absence of proteins excreted or secreted by cells grown in the media, but rather refers to the absence of direct supplementation of the media with animal proteins and protein components obtained from animal sources, such as those produced by recombinant technology.

[0047] In some exemplary embodiments, the cell culture production medium or cell culture medium may be a chemically defined medium.

[0048] As used herein, "chemically defined media" may include media composed of pure components at measured concentrations. Chemically defined media may contain simple sugars as carbon and energy sources, inorganic nitrogen sources, various mineral salts, and growth factors (purified amino acids, vitamins, purines, and pyrimidines) as needed. A variety of tissue culture media, including synthetic media, are commercially available, for example, any one or combination of the following cell culture media may be used: RPMI-1640 medium, medium 199, RPMI-1641 medium, Dulbecco's modified Eagle's medium (DMEM), minimal essential medium Eagle, F-12K medium, Ham's F12 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as EX-CELL™ 300 series (JRH Biosciences, Lenexa, Kansas), among others.

[0049] In some exemplary embodiments, the method of producing a protein may be a fed-batch method.

[0050] As used herein, the term "fed-batch method" refers to a method in which a fed-batch cell culture can be supplied with additional nutrients. For example, the method can include adding supplemental medium according to a determined feeding schedule within a given period of time. As used herein, "fed-batch cell culture" refers to a cell culture in which cells and medium are initially supplied to the culture vessel, and additional culture nutrients are fed to the growing culture continuously or in discrete increments, with or without periodic cell and / or product harvesting prior to the end of the culture.

[0051] It is understood that the invention is not limited to any of the above-mentioned eukaryotic cells, proteins, basal medium, feed medium, cell medium, cell culture production medium, methods of growing cells, methods of expressing proteins, methods of harvesting proteins, methods of introducing nucleic acids comprising sequences encoding proteins into cells, and times of adding or replenishing components in the medium, and that any suitable medium can be selected by any suitable means.

[0052] As used herein, the terms "include", "includes", and "including" are meant to be open-ended and are understood to mean "comprise", "comprises", and "comprising", respectively.

[0053] In some exemplary embodiments, the present disclosure provides cell culture media for eukaryotic cells.

[0054] In some exemplary embodiments, the disclosure provides a method for producing a protein.

[0055] In some exemplary embodiments, the present disclosure provides methods for culturing eukaryotic cells to increase production of a protein.

[0056] In some exemplary embodiments, the cell culture medium contains at least about 0.05 nM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, at least about 100 nM, at least about 105 nM, at least about 110 nM, at least about 115 nM, at least about 120 nM, at least about 125 nM , at least about 130 nM, at least about 135 nM, at least about 140 nM, at least about 145 nM, at least about 150 nM, at least about 155 nM, at least about 160 nM, at least about 165 nM, at least about 170 nM, at least about 175 nM, at least about 180 nM, at least about 195 nM, at least about 200 nM, about 205 nM, at least about 210 nM, at least about 215 nM, at least about 22 In some embodiments, the 5-methyl-thioadenosine may be present in a concentration of 0 nM, at least about 225 nM, at least about 230 nM, at least about 235 nM, at least about 240 nM, at least about 245 nM, at least about 250 nM, at least about 255 nM, at least about 260 nM, at least about 265 nM, at least about 270 nM, at least about 275 nM, at least about 280 nM, at least about 295 nM, or at least about 300 nM.

[0057] In some exemplary embodiments, the cell culture medium contains at least about 0.05 nM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about Also about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, at least about 100 nM, at least about 105 nM, at least about 110 nM, at least about 115 nM, at least about 120 nM, at least about 125 nM, at least about 130 nM, at least about 135 nM, at least about 140 nM, at least about 145 nM, at least about 150 nM, at least about 155 nM, at least about 160 nM, at least about 165 nM, at least about 17 0 nM, at least about 175 nM, at least about 180 nM, at least about 195 nM, at least about 200 nM, about 205 nM, at least about 210 nM, at least about 215 nM, at least about 220 nM, at least about 225 nM, at least about 230 nM, at least about 235 nM, at least about 240 nM, at least about 245 nM, at least about 250 nM, at least about 255 nM, at least about 260 nM, at least about 265 nM, at least about 270 nM, at least about 275 nM, at least about 280 nM, at least about 295 nM M, at least about 300 nM, at least about 305 nM, at least about 310 nM, at least about 315 nM, at least about 320 nM, at least about 325 nM, at least about 330 nM, at least about 335 nM, at least about 340 nM, at least about 345 nM, at least about 350 nM, at least about 355 nM, at least about 360 nM, at least about 365 nM, at least about 370 nM, at least about 375 nM, at least about 380 nM, at least about 395 nM, at least about 400 nM, about 405 nM, at least about 410 nM,at least about 515 nM, at least about 420 nM, at least about 425 nM, at least about 430 nM, at least about 435 nM, at least about 440 nM, at least about 445 nM, at least about 450 nM, at least about 455 nM, at least about 460 nM, at least about 465 nM, at least about 470 nM, at least about 475 nM, at least about 480 nM, at least about 495 nM, at least about 500 nM, at least about 510 nM, at least about 520 nM, at least about 530 nM, at least about 540 nM, at least about 550 nM M, at least about 560 nM, at least about 570 nM, at least about 580 nM, at least about 590 nM, at least about 600 nM, at least about 610 nM, at least about 620 nM, at least about 630 nM, at least about 640 nM, at least about 650 nM, at least about 660 nM, at least about 670 nM, at least about 680 nM, at least about 690 nM, at least about 700 nM, at least about 710 nM, at least about 720 nM, at least about 730 nM, at least about 740 nM, at least about 750 nM, at least about 76 0 nM, at least about 770 nM, at least about 780 nM, at least about 790 nM, at least about 800 nM, at least about 810 nM, at least about 820 nM, at least about 830 nM, at least about 840 nM, at least about 850 nM, at least about 860 nM, at least about 870 nM, at least about 880 nM, at least about 890 nM, at least about 900 nM, at least about 910 nM, at least about 920 nM, at least about 930 nM, at least about 940 nM, at least about 950 nM, at least about 960 nM, at least about 970 nM, at least about 980 nM, at least about 990 nM, at least about 1000 nM, at least about 1050 nM, at least about 1100 nM, at least about 1150 nM, at least about 1200 nM, at least about 1250 nM, at least about 1300 nM, at least about 1350 nM, at least about 1400 nM, at least about 1450 nM, at least about 1500 nM, at least about 1550 nM, at least about 1600 nM, at least about 1650 nM, at least about 1700 nM, at least about 1750 nM, at least about 1800 nM,The 5-nicotinamide may comprise a concentration of at least about 1850 nM, at least about 1900 nM, at least about 1950 nM, at least about 2000 nM, at least about 2050 nM, at least about 2100 nM, at least about 2150 nM, at least about 2200 nM, at least about 2250 nM, at least about 2300 nM, at least about 2350 nM, at least about 2400 nM, at least about 2450 nM, at least about 2500 nM, at least about 2550 nM, at least about 2600 nM, at least about 2650 nM, at least about 2700 nM, at least about 2750 nM, at least about 2800 nM, at least about 2850 nM, at least about 2900 nM, at least about 2950 nM, or at least about 3000 nM.

[0058] In some exemplary embodiments, the cell culture medium may have a pH of about 6.5 to about 8.0. In some specific exemplary embodiments, the cell culture medium may have a pH of about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0059] In some exemplary embodiments, the eukaryotic cells are grown at a temperature of about 25° C. to about 40° C. In some specific exemplary embodiments, the eukaryotic cells are grown at a temperature of about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., or about 40° C.

[0060] In some exemplary embodiments, the cell culture medium may include at least about 10 nM 5-methylthioadenosine, and the titer of the protein produced in the cell culture medium may be at least about 2% greater than another cell culture medium that does not have at least about 10 nM 5-methylthioadenosine. In one aspect, the cell culture medium may include at least about 20 nM 5-methylthioadenosine, at least about 30 nM 5-methylthioadenosine, at least about 40 nM 5-methylthioadenosine, at least about 50 nM 5-methylthioadenosine, at least about 60 nM 5-methylthioadenosine, at least about 70 nM 5-methylthioadenosine, at least about 80 nM 5-methylthioadenosine, at least about 90 nM 5-methylthioadenosine, at least about 100 nM 5-methylthioadenosine, at least about 110 nM 5-methylthioadenosine. The methylthioadenosine may comprise at least about 120 nM 5-methylthioadenosine, at least about 130 nM 5-methylthioadenosine, at least about 140 nM 5-methylthioadenosine, at least about 150 nM 5-methylthioadenosine, at least about 160 nM 5-methylthioadenosine, at least about 170 nM 5-methylthioadenosine, at least about 180 nM 5-methylthioadenosine, at least about 190 nM 5-methylthioadenosine, or at least about 200 nM 5-methylthioadenosine. In one embodiment, the titer of the protein produced in the cell culture medium can be at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30% greater.

[0061] In some exemplary embodiments, the cell culture medium may include at least about 50 nM nicotinamide, and the titer of a protein produced in the cell culture medium may be at least about 2% greater than another cell culture medium that does not have at least about 50 nM nicotinamide. In one embodiment, the cell culture medium may contain at least about 50 nM nicotinamide, at least about 100 nM nicotinamide, at least about 150 nM nicotinamide, at least about 200 nM nicotinamide, at least about 250 nM nicotinamide, at least about 300 nM nicotinamide, at least about 350 nM nicotinamide, at least about 400 nM nicotinamide, at least about 450 nM nicotinamide, at least about 500 nM nicotinamide, at least about 550 nM nicotinamide, at least about 600 nM nicotinamide, at least about 650 nM nicotinamide, at least about 700 nM nicotinamide, at least about 750 nM nicotinamide, at least about 800 nM nicotinamide, at least about 850 nM nicotinamide, at least about 900 nM nicotinamide, or at least about 1000 nM nicotinamide. In one embodiment, the titer of the protein produced in the cell culture medium can be at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30% greater.

[0062] In some exemplary embodiments, the cell culture medium may include feeding the eukaryotic cells using an enriched medium having 5-methylthioadenosine at a concentration of at least about 0.05 nM, at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 11 nM, at least about 12 nM, at least about 13 nM, at least about 14 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, or at least about 50 nM.

[0063] In some exemplary embodiments, the cell culture medium contains at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, The method may include feeding the eukaryotic cells with an enriched medium having a nicotinamide concentration of at least about 95 nM, at least about 100 nM, at least about 105 nM, at least about 110 nM, at least about 115 nM, at least about 120 nM, at least about 125 nM, at least about 130 nM, at least about 135 nM, at least about 140 nM, at least about 145 nM, at least about 150 nM, at least about 155 nM, at least about 160 nM, at least about 165 nM, at least about 170 nM, at least about 175 nM, at least about 180 nM, at least about 195 nM, or at least about 200 nM.

[0064] In some exemplary embodiments, the cell culture medium may include feeding eukaryotic cells with an enriched medium having a concentration of at least about 1 nM 5-methylthioadenosine, and the titer of the protein produced in the cell culture medium may be at least about 2% greater than another cell culture medium that does not have at least about 10 nM 5-methylthioadenosine. In one aspect, the enriched medium may contain at least about 2 nM 5-methylthioadenosine, at least about 3 nM 5-methylthioadenosine, at least about 4 nM 5-methylthioadenosine, at least about 5 nM 5-methylthioadenosine, at least about 6 nM 5-methylthioadenosine, at least about 7 nM 5-methylthioadenosine, at least about 8 nM 5-methylthioadenosine, at least about 9 nM 5-methylthioadenosine, at least about 10 nM 5-methylthioadenosine, at least about 11 nM 5-methylthioadenosine. The methylthioadenosine may have at least about 12 nM 5-methylthioadenosine, at least about 13 nM 5-methylthioadenosine, at least about 14 nM 5-methylthioadenosine, at least about 15 nM 5-methylthioadenosine, at least about 16 nM 5-methylthioadenosine, at least about 17 nM 5-methylthioadenosine, at least about 18 nM 5-methylthioadenosine, at least about 19 nM 5-methylthioadenosine, or at least about 20 nM 5-methylthioadenosine. In one embodiment, the titer of the protein produced in the cell culture medium can be at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30% greater.

[0065] In some exemplary embodiments, the cell culture medium may include feeding eukaryotic cells with an enriched medium having a nicotinamide concentration of at least about 5 nM, and the titer of the protein produced in the cell culture medium may be at least about 2% greater than an alternative cell culture medium that does not have at least about 50 nM nicotinamide. In one embodiment, the cell culture medium may contain at least about 5 nM nicotinamide, at least about 10 nM nicotinamide, at least about 15 nM nicotinamide, at least about 20 nM nicotinamide, at least about 25 nM nicotinamide, at least about 30 nM nicotinamide, at least about 35 nM nicotinamide, at least about 400 nM nicotinamide, at least about 45 nM nicotinamide, at least about 50 nM nicotinamide, at least about 55 nM nicotinamide, at least about 60 nM nicotinamide, at least about 65 nM nicotinamide, at least about 70 nM nicotinamide, at least about 75 nM nicotinamide, at least about 80 nM nicotinamide, at least about 85 nM nicotinamide, at least about 90 nM nicotinamide, at least about 95 nM nicotinamide, or at least about 100 nM nicotinamide. In one embodiment, the titer of the protein produced in the cell culture medium can be at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, or at least about 30% greater.

[0066] In some exemplary embodiments, the protein may be a naturally occurring protein.

[0067] In some exemplary embodiments, the protein may be a recombinant protein.

[0068] In some exemplary embodiments, the protein may be a biotherapeutic protein.

[0069] In some exemplary embodiments, the protein may be a recombinant protein, which may be a trap protein, a chimeric receptor Fc fusion binding molecule, a chimeric protein, an antibody, a monoclonal antibody, a polyclonal antibody, a human antibody, a bispecific antibody, an antibody fragment, a nanobody, a recombinant antibody chimera, a cytokine, a chemokine, or a peptide hormone.

[0070] In some exemplary embodiments, proteins may include modifications, additions, and other covalently attached moieties.

[0071] In some exemplary embodiments, the protein may include a post-translational modification.

[0072] The sequential labeling of method steps provided herein by numbers and / or letters does not limit the method, or any embodiment thereof, to the particular order presented.

[0073] Various publications, including patents, patent applications, published patent applications, accession numbers, technical articles, and scholarly articles, are cited throughout the specification. Each of these cited references is incorporated herein by reference in its entirety for all purposes.

[0074] The present disclosure may be more fully understood by reference to the following examples, which are provided to more fully illustrate the present disclosure, and are for illustrative purposes and should not be construed as limiting the scope of the disclosure. EXAMPLES

[0075] To study the contents of cell medium and its effect on protein production, cell cultures containing soybean hydrolysate were selected to produce VEGFR-binding protein 1.

[0076] Example 1. Content analysis of soy hydrolysates was performed using nine different lots 1, 2, and 3 obtained from three different shipments. Samples from these three different shipments were sent to Metabolon (Durham, NC, USA). Content analysis of soy hydrolysates was performed at Metabolon using liquid chromatography-mass spectrometry. Over 300 components of soy hydrolysates were measured. Only median scaled results were provided.

[0077] Orthogonal partial least squares (OPLS) model was used for multivariate analysis (MVA) to study the effect of biochemicals on the titer of VEGFR (vascular endothelial growth factor receptor) binding protein 1. Seventy soy hydrolysate lots were included in the study.

[0078] The dependencies between the components and potencies were evaluated by studying correlations and covariances. A positive dependency suggests an increase in potency with increasing biochemical, whereas a negative dependency suggests a decrease in potency with increasing biochemical. For the final potency evaluation of the major components, R 2 X, R 2 Y and Q 2 were found to be 0.315, 0.672, and 0.493, respectively. The components that showed a positive dependency are shown in Table 1, and the components that showed a negative dependency are shown in Table 2.

[0079] [Table 1]

[0080] [Table 2]

[0081] The MVA results normalized to unit length are shown in Figure 1. Based on these findings in Example 2, nicotinamide and 5-methylthioadenosine were selected as positive markers for further analysis.

[0082] Example 2. The effect of 5-methylthioadenosine on the production of a recombinant protein (VEGFR binding protein 1) was studied by investigating the protein titer (g / L) at different concentrations of 5-methylthioadenosine present in the cell culture medium.

[0083] A concentrated medium (soybean hydrolysate) containing 5-methylthioadenosine was added to the cell culture medium. Figure 2 shows the correlation of the concentration of 5-methylthioadenosine present in the soybean hydrolysate added to the cell culture medium with the titer of VEGFR binding protein 1.

[0084] As seen in FIG. 2, the regression line associated with the extrapolated variables (N=70) suggests that the titer varied linearly with the concentration of 5-methylthioadenosine in the soy hydrolysate added to the cell culture medium, suggesting that higher titers can be obtained with increasing concentrations of 5-methylthioadenosine in the cell culture medium.

[0085] Example 3. Based on correlation data (Example 2, Figure 2) obtained from studying the effect of 5-methylthioadenosine concentration in enrichment medium (soy hydrolysate) supplemented to the cell culture medium, an estimate was made regarding the optimal concentration of 5-MTA in the cell culture. Figure 3 shows that the regression line associated with the extrapolated variables (N=70) suggests that the titer can vary linearly with the concentration of 5-methylthioadenosine in the cell culture medium, indicating that higher titers can be obtained with increasing 5-methylthioadenosine concentration in the cell culture medium.

[0086] Example 4. The effect of nicotinamide on recombinant protein (VEGFR binding protein 1) titer was also studied by investigating the protein titer (g / L) at different concentrations of nicotinamide present in the cell culture medium.

[0087] Concentrated medium (soy hydrolysate) containing nicotinamide was added to the cell culture medium at various concentrations. Figure 4 shows the correlation of the concentration of nicotinamide present in the soy hydrolysate added to the cell culture medium with the titer of VEGFR binding protein 1. As seen in Figure 4, the regression line associated with the extrapolated variables (N=70) suggests that the titer changed linearly with respect to the concentration of nicotinamide in the soy hydrolysate, suggesting that higher titers can be obtained with increasing nicotinamide concentration in the cell culture medium.

[0088] Example 5. Based on correlation data (Example 4, Figure 4) obtained from studying the effect of nicotinamide concentration in enrichment medium (soy hydrolysate) supplemented to the cell culture medium, an estimate was made regarding the optimal concentration of nicotinamide in the cell culture. Figure 5 shows that the regression line associated with the extrapolated variables (N=70) suggests that the titer can vary linearly with the concentration of nicotinamide in the cell culture medium, suggesting that higher titers can be obtained with increasing nicotinamide concentration in the cell culture medium.

Claims

1. A cell culture medium for a eukaryotic cell expressing aflibercept, comprising: a basal or feed medium, and 5-Methylthioadenosine or Nicotinamide The cell culture medium comprising:

2. 2. The cell culture medium of claim 1, wherein the concentration of 5-methylthioadenosine in the cell culture medium is at least about 10 nM.

3. 2. The cell culture medium of claim 1, wherein a concentration of said nicotinamide in said cell culture medium is at least about 50 nM.

4. 2. The cell culture medium of claim 1, further comprising one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof.

5. 3. The cell culture medium of claim 2, wherein a titer of the aflibercept produced in the cell culture medium is at least about 2% greater than in another cell culture medium that does not have at least about 10 nM 5-methylthioadenosine.

6. 4. The cell culture medium of claim 3, wherein a titer of the aflibercept produced in the cell culture medium is at least about 2% greater than in another cell culture medium that does not have at least about 50 nM nicotinamide.

7. 2. The cell culture medium of claim 1, wherein the 5-methylthioadenosine or the nicotinamide is in the form of a salt or ester thereof.

8. 10. The cell culture medium of claim 1, wherein the pH of the medium is maintained in the range of about 6.5 to about 8.

9. 10. The cell culture medium of claim 1, which is free of animal-derived proteins.

10. 2. The cell culture medium of claim 1, which is a serum-free medium.

11. 2. The cell culture medium of claim 1, which is a chemically defined medium.

12. 1. A method for culturing eukaryotic cells to enhance production of aflibercept, comprising: The method further comprising: Culturing eukaryotic cells in a cell culture medium; supplementing the cell culture medium with 5-methylthioadenosine, wherein the concentration of the 5-methylthioadenosine is about 10 nM to about 200 nM; and Producing aflibercept in said eukaryotic cell. Including, said supplementation with 5-methylthioadenosine increases the potency of said aflibercept; The method.

13. 13. The method of claim 12, wherein the potency of the aflibercept is at least about 2% greater than an alternative method using a cell culture medium that does not have at least about 10 nM 5-methylthioadenosine.

14. 13. The method of claim 12, wherein the cell culture medium further comprises one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof.

15. 13. The method of claim 12, wherein the eukaryotic cells comprise at least one selected from the group consisting of a baby hamster kidney cell line, a Chinese hamster ovary cell line, a murine myeloma cell line, a mouse myeloma cell line, a human embryonic kidney cell line, a human retina-derived cell line, and an amniotic membrane cell line.

16. 13. The method of claim 12, wherein the aflibercept is secreted into the medium.

17. 13. The method of claim 12, wherein the cell culture medium is free of animal-derived proteins.

18. 13. The method of claim 12, wherein the cell culture medium is a serum-free medium.

19. 13. The method of claim 12, wherein the cell culture medium is a chemically defined medium.

20. 1. A method for culturing eukaryotic cells to enhance production of aflibercept, comprising: The method further comprising: Culturing cells in a defined cell culture medium; supplementing the cell culture medium with nicotinamide, wherein the concentration of the nicotinamide is from about 50 nM to about 2000 nM; and Producing aflibercept in said eukaryotic cell. Including, said supplementation with nicotinamide increases the potency of said aflibercept; The method.

21. 21. The method of claim 20, wherein the potency of the aflibercept is at least about 2% greater than another method using a cell culture medium that does not have at least about 50 nM nicotinamide.

22. 21. The method of claim 20, wherein the cell culture medium further comprises one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, α-hydroxyisovaleric acid, α-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid, or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof.

23. 21. The method of claim 20, wherein the eukaryotic cells comprise at least one selected from the group consisting of a baby hamster kidney cell line, a Chinese hamster ovary cell line, a murine myeloma cell line, a mouse myeloma cell line, a human embryonic kidney cell line, a human retina-derived cell line, and an amniotic membrane cell line.

24. 21. The method of claim 20, wherein the aflibercept is secreted into the medium.

25. 21. The method of claim 20, wherein the cell culture medium is free of animal-derived proteins.

26. 21. The method of claim 20, wherein the cell culture medium is a serum-free medium.

27. 21. The method of claim 20, wherein the cell culture medium is a chemically defined medium.

28. 1. A method for producing aflibercept, comprising: introducing into the cell a nucleic acid comprising a nucleotide sequence encoding aflibercept; culturing the cells in a cell culture medium containing at least about 50 nM nicotinamide or at least about 10 nM 5-methylthioadenosine; and Producing said aflibercept in said cell. The method comprising:

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