Cell culture medium for eukaryotic cells.

Abstract

Cell culture media are provided herein, as well as methods for using the media for cell culture and protein production from cells.

Description

CELL CULTURE MEDIUM FOR EUKARYOTIC CELLS FIELD The present invention relates, in general, to a cell culture medium for eukaryotic cells and the production of natural and recombinant products derived from them. BACKGROUND Cell culture manufacturing technology is widely used for the production of biopharmaceuticals. As the demand for biopharmaceuticals increases, so does the demand for enhanced cell growth, viability, and protein production. Currently, significant efforts are being made in developing methods and strategies for the growth, feeding, and maintenance of cell cultures. Novel cell culture methods that provide even incremental improvements in recombinant protein production are valuable, given the challenges and cost of large-scale cell culture processes and the growing demand for higher yields and lower costs of biologics.Improvements are needed in cell culture processes, recombinant polypeptide expression, titration, and cell viability that can lead to higher production levels, thereby reducing the costs associated with manufacturing protein therapies. COMPENDIUM The growth in the development, manufacturing, and sale of protein-based biopharmaceutical products led to an increased demand for production methods that can improve the production of biopharmaceutical products. The modalities described in this document address the aforementioned demands by providing methods and means for the manufacture of such biopharmaceutical products. The description, at least in part, provides a cell culture medium for eukaryotic cells. In one example embodiment, the cell culture medium for eukaryotic cells may comprise a basal 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 approximately 10 nM of 5-methylthioadenosine. In a further aspect of this embodiment, the cell culture medium may comprise from approximately 10 nM to approximately 200 nM of 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 approximately 50 nM of nicotinamide. In a further aspect of this embodiment, the cell culture medium may comprise approximately 2000 nM of 5-nicotinamide.In one aspect of this embodiment, a protein cultured in the cell culture medium is at least approximately 2% higher than in another cell culture medium that does not comprise at least approximately 10 nM of 5-methylthioadenosine. In another aspect of this embodiment, a protein cultured in the cell culture medium is at least approximately 2% higher than in another cell culture medium that does not comprise at least approximately 50 nM of nicotinamide. In one aspect of this embodiment, the cell culture medium may additionally comprise, optionally, one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-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 optionally additionally comprise sugars, amino acids, vitamins, salts, trace metal ions, and / or pyrimidines. In one aspect of this embodiment, the cell culture medium may comprise 5-methylthioadenosine salts or esters. In another aspect of this embodiment, the cell culture medium may comprise nicotinamide salts or esters. In one aspect of this embodiment, the cell culture medium may have a pH of approximately 6.5 to approximately 8. In one aspect of this embodiment, the cell culture medium does not contain an animal-derived protein. 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 example embodiment, the cell culture medium for eukaryotic cells may comprise a free 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 approximately 10 nM of 5-methylthioadenosine. In a further aspect of this embodiment, the cell culture medium may comprise from approximately 10 nM to approximately 200 nM of 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 approximately 50 nM of nicotinamide. In a further aspect of this embodiment, the cell culture medium may comprise from approximately 50 nM to approximately 2000 nM of 5-nicotinamide.In an alternative embodiment of this embodiment, the cell culture medium may further comprise 5-methylthioadenosine and nicotinamide. In another embodiment of this embodiment, the cell culture medium may optionally further comprise one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-hydroxyisocaproic acid, 2-(4-hydroxyphenyllactic acid), or 2-hydroxy-3-methylvaleric acid, salts of these acids, esters of these acids, and combinations thereof. In another embodiment of this embodiment, the cell culture medium may optionally further comprise sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In another embodiment of this embodiment, the cell culture medium may comprise salts or esters of 5-methylthioadenosine. In yet another embodiment, the cell culture medium may comprise salts or esters of nicotinamide.In one aspect of this modality, the cell culture medium may have a pH of approximately 6.5 to approximately 8. In one aspect of this modality, the cell culture medium does not contain an animal-derived protein. In one aspect of this modality, the cell culture medium may be a serum-free medium. In one aspect of this modality, the cell culture medium may be a chemically defined medium. In one aspect of this modality, the cell culture medium may optionally comprise nicotinamide. In one aspect of this modality, a titration of a protein cultured in the cell culture medium is at least approximately 2% higher than in another cell culture medium that does not comprise at least approximately 10 nM of 5-methylthioadenosine.In another aspect of this modality, a titer of a protein grown in the cell culture medium is at least about 2% higher than another cell culture medium that does not comprise at least about 50 nM of nicotinamide. The description, at least in part, provides a method for producing a protein. In one example embodiment, the method for producing a protein may comprise culturing eukaryotic cells that have a nucleic acid encoding the protein in a cell culture production medium and feeding the eukaryotic cells using an enriched medium containing 5-methylthioadenosine for a period of time. In one aspect of this embodiment, the enriched medium may contain at least approximately 1 nM of 5-methylthioadenosine. In another aspect of this embodiment, the cell culture production medium may comprise one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-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 comprise sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this embodiment, the cell culture production medium may comprise salts or esters of 5-methylthioadenosine. In one aspect of this embodiment, the cell culture production medium may have a pH of approximately 6.5 to approximately 8. In one aspect of this embodiment, the cell culture production medium does not contain an animal-derived protein. 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 modality, eukaryotic cells may be selected from baby hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human embryonic kidney lA / a / ZUZl / UlZO^S cell lines, human retina-derived cell lines, and / or amniocyte cell lines. In one aspect of this modality, 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 protein other than an antibody. In one aspect of this modality, the method may produce a protein titration at least approximately 2% higher than a protein titration in a cell culture production medium that does not contain at least approximately 10 nM of 5-methylthioadenosine. In one aspect of this modality, the enriched medium may optionally comprise nicotinamide.In one aspect of this modality, the method of producing a protein can be a batch feeding method. In one example embodiment, the method for producing a protein may comprise culturing eukaryotic cells that have a nucleic acid encoding the protein in a cell culture production medium and feeding the eukaryotic cells using an enriched medium containing nicotinamide for a specified period of time. In one aspect of this embodiment, the enriched medium may contain at least approximately 5 nM of nicotinamide. In another aspect of this embodiment, the cell culture production medium may comprise one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-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 modality, the cell culture production medium may comprise sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this modality, the cell culture production medium may comprise nicotinamide salts or esters. In one aspect of this modality, the cell culture production medium may have a pH of approximately 6.5 to approximately 8. In one aspect of this modality, the cell culture production medium does not contain an animal-derived protein. In one aspect of this modality, the cell culture production medium may be a serum-free medium. In one aspect of this modality, the cell culture production medium may be a chemically defined medium.In one aspect of this modality, eukaryotic cells may be selected from baby hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human embryonic kidney cell lines, human retina-derived cell lines, and / or amniocyte cell lines. In another aspect of this modality, 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 protein other than an antibody. In another aspect of this modality, the method may produce a protein titration at least approximately 2% higher than a protein titration in a cell culture production medium that does not contain at least approximately 50 nM nicotinamide. In another aspect of this modality, the enriched medium may optionally comprise 5-methylthioadenosine.In one aspect of this modality, the method of producing a protein can be a batch feeding method. The present description, at least in part, provides a method for increasing the production of a protein. In one example embodiment, the method for increasing the production of a protein may comprise culturing eukaryotic cells in a cell culture medium, supplementing the cell culture medium with 5-methylthioadenosine, and expressing a protein. In one aspect of this embodiment, the concentration of 5-methylthioadenosine may be at least approximately 10 nM. In another aspect of this embodiment, the concentration of 5-methylthioadenosine may be from approximately 10 nM to approximately 200 nM. In yet another aspect of this embodiment, the cell culture medium may comprise one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-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 modality, the cell culture medium may comprise sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this modality, the cell culture medium may comprise salts or esters of 5-methylthioadenosine. In one aspect of this modality, the cell culture medium may have a pH of approximately 6.5 to approximately 8. In one aspect of this modality, the cell culture medium does not contain an animal-derived protein. In one aspect of this modality, the cell culture medium may be a serum-free medium. In one aspect of this modality, the cell culture medium may be a chemically defined medium.In one aspect of this modality, eukaryotic cells may be selected from baby hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human embryonic kidney cell lines, human retina-derived cell lines, and / or amniocyte cell lines. In another aspect of this modality, 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 protein other than an antibody. In another aspect of this modality, supplementation with 5-methylthioadenosine increases the recombinant protein titer by at least approximately 2%. In another aspect of this modality, the cell culture medium may be optionally supplemented with nicotinamide. In one example embodiment, the method for increasing the production of a protein may comprise culturing eukaryotic cells in a cell culture medium, supplementing the cell culture medium with nicotinamide, and expressing a protein. In one aspect of this embodiment, the concentration of nicotinamide may be at least approximately 50 nM. In another aspect of this embodiment, the concentration of 5-methylthioadenosine may be from approximately 50 nM to approximately 2000 nM. In yet another aspect of this embodiment, the cell culture medium may comprise one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-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 modality, the cell culture medium may comprise sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In another aspect of this modality, the cell culture medium may comprise nicotinamide salts or esters. In one aspect of this modality, the cell culture medium may have a pH of approximately 6.5 to approximately 8. In one aspect of this modality, the cell culture medium does not contain an animal-derived protein. In one aspect of this modality, the cell culture medium may be a serum-free medium. In one aspect of this modality, the cell culture medium may be a chemically defined medium.In one aspect of this modality, eukaryotic cells may be selected from baby hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human embryonic kidney cell lines, human retina-derived cell lines, and / or amniocyte cell lines. In another aspect of this modality, 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 protein other than an antibody. In another aspect of this modality, nicotinamide supplementation increases the recombinant protein titer by at least approximately 2%. In yet another aspect of this modality, the cell culture medium may be optionally supplemented with 5-methyladenosine. The present description, at least in part, provides a method for producing a protein. In one example embodiment, the method for producing a protein may comprise introducing into one or more cells a nucleic acid comprising a sequence encoding a protein and culturing the cell or cells in a cell culture medium. In one aspect of this embodiment, the cell culture medium may be enriched with at least approximately 10 nM of 5-methylthioadenosine. In another aspect of this embodiment, the cell culture medium may be enriched with at least approximately 50 nM of nicotinamide. In yet another aspect of this embodiment, the cell culture medium may be enriched with at least approximately 10 nM of 5-methylthioadenosine and at least approximately 50 nM of nicotinamide.In one aspect of this modality, the method for producing a protein may further comprise maintaining a cell culture medium to express a higher titer of the protein in the cell(s). In another aspect of this modality, the method for producing a protein may further comprise harvesting the protein. In another aspect of this modality, the cell(s) may be selected from baby hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human embryonic kidney cell lines, human retina-derived cell lines, and / or amniocyte cell lines.In one aspect of this embodiment, the cell culture medium may further comprise one or more acids selected from lactic acid, phenyllactic acid, indolelactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-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 comprise sugars, amino acids, vitamins, salts, trace metal ions, purines, and / or pyrimidines. In one aspect of this embodiment, the cell culture medium may have a pH of approximately 6.5 to approximately 8. In one aspect of this embodiment, the cell culture medium does not contain an animal-derived protein. In one aspect of this embodiment, the cell culture medium may be a serum-free medium.In one aspect of this modality, the cell culture medium may be a chemically defined medium. In another aspect of this modality, 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 protein other than an antibody. In another aspect of this modality, the method may express a 2% higher protein titer in the cell(s) compared to the cell(s) cultured in a cell culture medium that does not contain at least approximately 10 nM of 5-methylthioadenosine. In another aspect of this modality, the method for producing a protein may further comprise maintaining the cell culture medium to express a 2% higher protein titer in the cell(s) compared to the cell(s) cultured in a cell culture medium that does not contain at least approximately 50 nM of nicotinamide. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the result of the multivariate analysis (MVA) of the investigation of the components present in the soybean hydrolysate. FIG. 2 shows the correlation data obtained from the study of the effect of 5-methylthioadenosine concentration in an enriched medium used to supplement a cell culture medium with respect to protein titration (g / L) according to an example modality. FIG. 3 shows a regression line with respect to the variables (N = 70) used to evaluate the effect of 5-methylthioadenosine concentration in a cell culture medium with respect to protein titration (g / L) according to an example modality. FIG. 4 shows the correlation data obtained from the study of the effect of nicotinamide concentration in an enriched medium used to supplement a cell culture medium with respect to protein titration (g / L) according to an example modality. FIG. 5 shows a regression line with respect to the variables (N = 70) used to evaluate the effect of nicotinamide concentration in a cell culture medium with respect to protein titration (g / L) according to an example modality. DETAILED DESCRIPTION Biopharmaceutical products have been highly effective in diagnostic and therapeutic applications (Dawn M Ecker, Susan Dana Jones, and 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)). Some cell culture media used to grow cells for the production of biopharmaceutical products have been extensively documented in the literature, and several 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 types and quantities of which may vary depending on the specific requirements of a given cell or tissue type. One of the goals of protein production is optimizing cell culture to obtain the highest protein yield and the most efficient production methods. Any improvement, including incremental improvements, can have enormous economic benefits. In the pharmaceutical industry, optimizing protein production for biologics used in disease therapies is advantageous, as any improvement can have a significant impact when the biologic is manufactured on a large scale. Therefore, there remains a need to maximize protein production from cell cultures expressing biological proteins for use in medicine. Unless otherwise specified, all technical and scientific terms used herein have the same meaning commonly assigned to them by those skilled in the art to which this invention pertains. While any method and material similar or equivalent to those described herein may be used for implementation or evaluation, particular methods and materials are described below. All publications mentioned herein are incorporated herein by reference. It should be understood that the term “one” means “at least one”. Likewise, it should be understood that the expressions “around” and “approximately” allow for standard variation, as will be understood by those skilled in the art, and where ranges are provided, extreme values ​​are included. In some illustrative forms, the description provides a method for producing a protein. As used herein, the term “protein” includes any amino acid polymer having covalently linked amide bonds. Proteins comprise one or more polymer chains of amino acids, generally known in the art as “polypeptides.” “Polypeptide” refers to a polymer composed of amino acid residues; related structural variants of natural origin and synthetic and non-natural analogues thereof, linked by peptide bonds; related structural variants of natural origin and synthetic and non-natural analogues thereof. “Synthetic peptides or polypeptides” refers to a peptide or polypeptide of non-natural origin. Synthetic peptides or polypeptides can be synthesized, for example, by an automated polypeptide synthesizer. Those skilled in the art are familiar with various methods for solid-phase peptide synthesis.A protein can contain one or multiple polypeptides to form a single functional biomolecule. A protein can include any of the following: biotherapeutic proteins, recombinant proteins used in research or therapy, TRAP proteins and other chimeric Fe receptor fusion-binding molecules, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In another illustrative sense, a protein can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Recombinant proteins can be produced using recombinant cell-based production systems, such as the insect baculovirus system, yeast systems (e.g., Pichia sp.), and mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells).For a review of 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 1 47–176 (2012). In some modalities, the proteins comprise modifications, adducts, and other covalently linked residues. These modifications, adducts, and residues include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tag, maltose-binding protein (MBP), chitin-binding protein (CBP), glutathione-S-transferase (GST) myc epitope, fluorescent markers and other dyes, and the like.Proteins can be classified on the basis of composition and solubility and can therefore include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins. In some example modalities, the protein or the recombinant protein or the The recombinant protein ΊΛ / a / ZUZ l / UI ZD4EI can be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, an Fe fusion-binding molecule, an F(ab')2 fragment, an Fe fragment, or combinations thereof. The term antibody, as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, Ch1, Ch2, and Ch3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (Cl1).The Vh and Vl regions can be further subdivided into hypervariability regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL can be composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the invention, the FRs of the anti-big-ET-1 antibody (or its antigen-binding portion) can be identical to human germline sequences or can be naturally or artificially modified. A consensus amino acid sequence can be defined based on a comparative analysis of two or more CDRs. The term “antibody,” as used herein, also includes antigen-binding fragments of complete antibody molecules.The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and similar terms, as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically altered glycoprotein or polypeptide that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from whole antibody molecules by any suitable standard technique, such as proteolytic digestion or recombinant genetic alteration techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of antibodies. Such DNA is known and / or readily available, for example, from commercial sources, DNA banks (including, for example, antibody-phage banks), or may be synthesized.It is possible to sequence and manipulate DNA chemically or by molecular biology techniques, for example, to order one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or remove amino acids, etc. As used herein, the term “monoclonal antibody” is not limited to antibodies produced using hybridoma technology. A monoclonal antibody may be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, using any means available or known in the art. Useful monoclonal antibodies as described herein may be prepared using a wide variety of techniques known in the industry, including hybridoma, recombinant, and phage expression technologies, or a combination thereof. As used herein, the term fusion protein or Fe fusion protein comprises part or all of two or more proteins, one of which may be an Fe portion or an immunoglobulin molecule, not naturally fused. The preparation of the fusion protein may comprise certain heterologous polypeptides fused to various antibody-derived polypeptide portions (including the Fe domain) and is described in, for example, 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).The “Fe receptor fusion protein” comprises one or more extracellular domains of a receptor coupled to an Fe residue, which, in some embodiments, may comprise a hinge region followed by a Ch2 and Ch3 domain of an immunoglobulin. In some embodiments, the Fe fusion protein may contain two or more different receptor chains that bind to a single ligand or more than one ligand. For example, the Fe fusion protein may be a trap, such as, for example, an IL-1 trap (e.g., Rilonacept, which contains the IL-1 ligand-binding region RAcP fused to the extracellular IL-1R1 region fused to the Fe of hlgG1; see U.S. Patent No.e6,927,004, which is incorporated herein in its entirety by this reference), or a VEGF trap (for example, Aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused with the Ig domain 3 of the VEGF receptor Flk1 fused with the Fe of hlgG1; see U.S. Patents n.e7,087,411 and 7,279,159, which are incorporated herein in their entirety by this reference). As used herein, an “antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, a Fab fragment, a Fab' fragment, an F(abj2) fragment, an scFv fragment, an Fv fragment, a dsFv diabody, a dAb fragment, an Fd' fragment, an Fd fragment, and an isolated complementarity-determining region (CDR), as well as tribodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Fv fragments are combinations of the variable regions of immunoglobulin heavy and light chains, and ScFv proteins are recombinant single-chain polypeptide molecules in which the variable regions of immunoglobulin light and heavy chains are linked by a peptide linker.In some examples, an antibody fragment contains sufficient amino acid sequence from the original antibody from which it is fragmented to bind to the same antigen as the original antibody. In some examples, a fragment binds to the antigen with an affinity comparable to that of the original antibody and / or competes with the original antibody for antigen binding. An antibody fragment can be produced by various means. For example, an antibody fragment can be produced enzymatically or chemically by fragmentation of an intact antibody and / or it can be produced recombinantly from a gene encoding a partial sequence of the antibody. Alternatively, an antibody fragment can be produced by totally or partially synthetic means. An antibody fragment may optionally comprise a single-chain antibody fragment.Alternatively or additionally, an antibody fragment may comprise multiple linked chains, for example, via disulfide bonds. An antibody fragment may optionally comprise a multimolecular complex. A functional antibody fragment typically comprises at least approximately 50 amino acids and, more typically, at least approximately 200 amino acids. In some example modalities, the protein or recombinant protein or recombinant protein may be an antibody variant or a binding molecule variant. A variant or variant of a binding molecule, as used herein, may include a binding molecule that differs from a target binding molecule by virtue of at least one amino acid modification or post-translational modification. The variant may refer to the binding molecule itself, a composition comprising the binding molecule, or the amino acid sequence encoding it. Preferably, the variant of the binding molecule has at least one amino acid modification compared to the original binding molecule, for example, from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the original.The variant sequence of the binding molecule of the present invention will preferably have at least about 80% homology with a parental binding molecule sequence, and more preferably at least about 90% homology, more preferably at least about 92% homology. In some example modalities, the protein or recombinant protein or recombinant protein may be a protein with a specific post-translational modification. As used herein, the general term “post-translational modifications” or “PTMs” refers to covalent modifications that polypeptides undergo either during (co-translational modification) or after (post-translational modification) their ribosomal synthesis. PTMs are generally introduced by specific enzymes or enzymatic pathways. Many occur at the site of a specific characteristic protein sequence (e.g., a distinctive sequence) within the protein's main structure. Several hundred PTMs have been recorded, and these modifications invariably influence some aspect of a protein's structure or function (Walsh, G. “Proteins” (2014) second edition, published by Wiley and Sons, ISBN: 9780470669853). The various post-translational modifications include, but are not limited to, cleavage, N-terminus extensions, protein degradation, and N-terminus acylation.biotinylation (acylation of lysine residues with biotin), C-terminal amidation, glycosylation, iodination, covalent attachment of prosthetic groups, acetylation (the addition of an acetyl group, usually at the N-terminus of the protein), alkylation (the addition of an alkyl group (e.g., methyl, ethyl, propyl) usually to arginine or lysine residues), methylation, adenylation, ADP-ribosylation, covalent cross-linking between, or within, polypeptide chains, sulfonation, prenylation, vitamin C-dependent modifications (proline and lysine hydroxylations and carboxy-terminal amidation), vitamin K-dependent modification where vitamin K is a cofactor in the carboxylation of glutamic acid residues resulting in the formation of a γ-carboxyglutamate (a glu residue), glutamylation (covalent linkage of glutamic acid residues), glycylation (covalently bonded glycine residues),Glycosylation (addition of a glycosyl group to asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein), isoprenelylation (addition of an isoprenoid group such as farnesol and geranylgeraniol), lipoylation (attachment of a lipoate functional group), phosphopantetheynylation (addition of a 4'-phosphopantetheynyl moiety to coenzyme A, as in the biosynthesis of fatty acids, polyketides, nonribosomal peptides, and leucine), 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 amino acids include, but are not limited to, citrullination (e.g., the conversion of arginine to citrulline by deimination) and deamidation (e.g.,the conversion of glutamine to glutamic acid or asparagine to aspartic acid). Post-translational modifications involving structural changes include, but are not limited to, the formation of disulfide bridges (covalent bonding of two cysteine ​​amino acids) and proteolytic cleavage (cleavage of a protein at a peptide bond). Certain modifications, Post-translational modifications (PTMs) involve the addition of other proteins or peptides, such as ISGylation (covalent bonding to the ISG15 protein (interferon-stimulated gene)), SUMOylation (covalent bonding to the SUMO protein (small ubiquitin-related modifier)), and ubiquitination (covalent bonding to the ubiquitin protein). See http: / / www.uniprot.org / docs / ptmlist for a more detailed controlled vocabulary of PTMs selected by UniProt. In some example modalities, the description provides a cell culture medium for eukaryotic cells comprising a basal medium or a feeding medium and 5-methylthioadeosine and / or nicotinamide. As used in this document, the term cell culture medium refers to cells grown in an artificial environment (e.g., in vitro). However, it should be understood that the term cell culture is a generic term and can be used to encompass the cultivation not only of individual prokaryotic (e.g., bacterial) or eukaryotic (e.g., animal, plant, and fungal) cells, but also of tissues, organs, organ systems, or whole organisms, for which the terms tissue culture, organ culture, organ system culture, or organotypic culture may occasionally be used interchangeably with cell culture. Suitable culture conditions for eukaryotic cells can be found in the technical literature, for example, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992).Cell culture media can be optimized for a specific cell culture use, including, for example, cell culture growth medium that can be formulated to promote cell growth, or cell culture production medium that can be formulated to promote the production of recombinant proteins. The terms nutrient, ingredient, and component are used interchangeably in this document to refer to the constituents that make up a cell culture medium. As used in this document, the term basal medium can refer to any medium capable of supporting cell growth. Basal medium may comprise various ingredients, including amino acids, vitamins, organic and inorganic salts, and carbohydrate sources, each present in an amount that supports in vitro cell culture. The medium may also contain auxiliary substances, such as buffers like sodium bicarbonate, oxidation stabilizers, stabilizers to counteract mechanical stress, or protease inhibitors.Examples of basal media include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), DME / F12, Minimum Essential Medium (MEM), Eagle Basal Medium (BME), Medium 199, RPMI 1640, F-10, F-12, Minimum Essential Medium a (a-MEM), Glasgow Minimum Essential Medium (G-MEM), PF CHO (SAFC Biosciences), Iscove Modified Dulbecco Medium, or combinations thereof, and others known in the literature or commercially available. As used in this document, the term feeding medium includes a medium containing one or more nutrients that can be added to the culture beginning some time after inoculation. The feeding medium may also be a combination feed comprising a basal medium and at least one type of hydrolysate, for example, a soybean-based hydrolysate, a yeast-based hydrolysate, or a combination of the two. In addition, the feeding medium may also include only a basal medium, such as a concentrated basal medium, or it may include only hydrolysates or concentrated hydrolysates. As used in this document, the term eukaryotic cells may include individual cells, tissues, organs, insect cells, bird cells, mammalian cells, primary cells, continuous cell lines, stem cells, and / or genetically modified cells, such as recombinant cells expressing a heterologous polypeptide or protein. Some mammalian cells suitable for culture in 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 strains (e.g., embryonic kidney 293 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 VA13 cells, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, M-3 clone cells, 1-10 cells, RAG cells, TCMK-1 cells, Yl 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 cells, B1 cells, BSC-1 cells, RAf cells, RK-c cells, PK-15 cells or derivatives thereof), fibroblast cells from any tissue or organ (including, but not limited to, heart, liver, kidney, colon, intestines, esophagus, stomach, neural tissue (brain, spinal cord), lung, tissue (artery, vein, capillary), lymphoid tissue (lymph gland, 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, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, cells WI-26, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSVBALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, cells HSDMÍC3, cells KLN205, McCoy cells, mouse L cells, strain Cells 2071 (mouse L), ΊΛ / a / ZUZ l / UI LM strain cells (mouse L), L-MTK' cells (mouse L), NCTC clones 2472 and 2555, SCCPSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, Cn cells and Jensen cells, Sp2 / 0 cells, NSO, NS1 or derivatives thereof). As used in this document, 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 CeHeNsO. As used in this document, 5-methylthioadenosine may also be referred to as 5-deoxy-5'-methylthioadenosine, CAS number 2457-80-9, CnHuN-ChS, MTA, MeSAdo, NSC 335422, Vitamin L2, 5-methylthioadenosine salts, or 5-methylthioadenosine esters. Non-exhaustive examples of suitable salts for 5-methylthioadenosine include acid addition salts. Acid addition salts may be addition salts of organic and inorganic acids, such as hydrochlorides, sulfates, nitrates, carbonates, phosphates, formates, oxalates, citrates, ascorbic acid, methanesulfonic acid, 1,4-butane sulfonate, 1,5-pentanesulfonate, and ptoluenesulfonate salts. In some example modalities, the description provides a method for producing a protein that comprises growing eukaryotic cells that have a nucleic acid encoding the protein in a cell culture production medium. The cell culture medium or cell culture production medium can be supplemented with enriched media containing components, such as nutrients and amino acids, that are consumed during the course of the cell culture production phase. As used in this document, the expression "cell culture production medium" may include a cell culture medium designed for use during the production phase of a cell culture. In some example modalities, the cell culture production medium or cell culture medium may be a serum-free medium. As used herein, the term serum-free medium includes a cell culture medium that does not contain animal serum, such as fetal bovine serum. Serum-free media may or may not contain hydrolysates, growth factors, hormones, carrier proteins, and binding factors. Examples of known serum-free media include CHO-S-SFM II (Gibco) and 293 SFM II (Gibco). In some example modalities, the cell culture production medium or cell culture medium may be a medium without animal protein. As used herein, the term "animal-protein-free medium" may refer to a medium that does not contain proteins and protein components from higher, non-plant multicellular eukaryotes (i.e., vertebrates), which possess the secondary, tertiary, and quaternary structures characteristic of proteins as found 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 that can be obtained from plants (typically about 10–30 amino acids in length), such as soybeans, and lower eukaryotes, such as yeast.When cells are brought into contact or inoculated with an animal-derived protein medium, the medium will contain animal proteins shed or secreted by those cells, including any recombinant protein expressed by genetically modified cells if such cells are cultured. Therefore, the term "animal protein-free medium," and biological materials and preparations produced with it, should not be interpreted as requiring the absence of proteins shed or secreted by cells propagated in the medium, but rather as referring to the lack of direct complementation of the medium with animal proteins and protein components obtained from animal sources or similar recombinantly produced sources. In some example modalities, the cell culture production medium or cell culture medium may be a chemically defined medium. As used in this document, a chemically defined medium may include a medium composed of pure ingredients in measured concentrations. A chemically defined medium may contain a simple sugar as the carbon and energy source, an inorganic nitrogen source, various mineral salts, and, if necessary, growth factors (purified amino acids, vitamins, purines, and pyrimidines). Several tissue culture media, including defined culture media, are commercially available. For example, any one or a combination of the following cell culture media may be used: RPMI-1640 Medium, Medium 199, RPMI-1641 Medium, Dulbecco's Modified Eagle Medium (DMEM), Eagle's Essential Minimal Medium, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco 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. In some example modalities, the method of producing a protein may be a batch feeding method. As used herein, the term batch-fed method refers to a method by which a batch-fed cell culture can be supplied with additional nutrients. For example, such a method may comprise the addition of supplemental media according to a predetermined feeding schedule within a given time period. Batch-fed cell culture, as used herein, refers to a cell culture where cells and culture medium are initially supplied to the culture vessel, and additional culture nutrients are fed, either continuously or in discrete increments, to the culture during growth, with or without periodic harvesting of cells and / or product before the termination of the culture. It is understood that the present invention is not limited to any of the aforementioned eukaryotic cells, protein, basal medium, feeding medium, cell culture medium, cell culture production medium, cell propagation method, method of protein expression, method of protein collection, method for introducing into a cell a nucleic acid comprising a protein-coding sequence and time period for adding or supplementing components in the medium, and that any suitable medium may be selected in any suitable way. As used herein, the terms “include”, “includes” and “that includes” are not intended to be exhaustive and are understood to mean “comprising”, “comprises” and “that comprises”, respectively. In some example modalities, the description provides a cell culture medium for eukaryotic cells. In some example modalities, the description provides a method for producing a protein. In some example modalities, the description provides a method for growing eukaryotic cells to increase the production of a protein. In some example embodiments, the cell culture medium may comprise 5-methylthioadenosine at a concentration of at least around 0.05 nM, at least around 1 nM, at least around 2 nM, at least around 3 nM, at least around 4 nM, at least around 5 nM, at least around 6 nM, at least around 7 nM, at least around 8 nM, at least around 9 nM, at least around 10 nM, at least around 15 nM, at least around 20 nM, at least around 25 nM, at least around 30 nM, at least around 35 nM, at least around 40 nM, at least around 45 nM, at least around 50 nM, at least around 65 nM, at least around 70 around 80 nM, at least around 85 around 95 nM, at least around 100 around 110 nM, at least around 115 around 125 nM, at least around 130 around 140 nM, at least around 145 around 155 nM,at least around 160 nM, at least around 170 nM, at least around 175 nM, at least around 60 nM, at least nM, at least around 75 nM, at least nM, at least around 90 nM, at least nM, at least around 105 nM, at least nM, at least around 120 nM, at least nM, at least around 135 nM, at least nM, at least around 150 nM, at least nM, at least around 165 nM, at least nM, at least around 180 nM, at least around 195 nM, at least around 200 nM, around 205 nM, at least around 210 nM, at least around 215 nM, at least around 220 nM, at least around 225 nM, at least around 230 nM, at least around 235 nM, at least around 240 nM, at least around 245 nM, at least around 250 nM, at least around 255 nM, at least around 260 nM, at least around 265 nM, at least around 270 nM, at least around 275 nM, at least around 280 nM,at least around 295 nm or at least around 300 nm. In some example modalities, the cell culture medium may comprise 5-nicotinamide at a concentration of at least around 0.05 nM, at least around 1 nM, at least around 2 nM, at least around 3 nM, at least around 4 nM, at least around 5 nM, at least around 6 nM, at least around 7 nM, at least around 8 nM, at least around 9 nM, at least around 10 nM, at least around 15 nM, at least around 25 nM, at least around 30 nM, at least 35 nM, at least around 40 nM, at least around 45 nM, at least 50 nM, at least around 55 nM nM, at least around 60 nM, at least 65 nM, at least around 70 nM, at least around 75 nM, at least 80 nM, at least around 85 nM, at least around 90 nM, at least 95 nM,at least around 100 nM, at least around 105 nM, at least 110 nM, at least around 115 nM, at least around 120 nM, at least 125 nM, at least around 130 nM, at least around 135 nM, at least 140 nM, at least around 145 nM, at least around 150 nM, at least 155 nM, at least around 160 nM, at least around 165 nM, at least 170 nM, at least around 175 nM, at least around 180 nM, at least around 195 nM, at least around 200 nM, around 205 nM, at least around 210 nM, at least around 215 nM, at least around 220 nM, at least around 225 nM, at least around 230 nM, at least around 235 nM, at least around 240 nM, at least around 245 nM, at least around 250 nM, at least around 255 nM, at least around 260 nM, at least around 275 nM, at least around 300 nM, at least around 315 nM, at least around 330 nMat least around 345 nM, at least around 360 nM, at least around 375 nM, at least around 265 nM, at least around 280 nM, at least around 305 nM, at least around 320 nM, at least around 335 nM, at least around 350 nM, at least around 365 nM, at least around 380 nM, at least around 270 nM, at least around 295 nM, at least around 310 nM, at least around 325 nM, at least around 340 nM, at least around 355 nM, at least around 370 nM, at least around 395 nM, at least around 400 nM, around 405 nM, at least around 410 nM, at least around 515 nM, at least around 420 nM, less around 425 nM, at least around 430 nM, at least around 435 nM, less around 440 nM, at least around 445 nM, at least around 450 nM, less around 455 nM, at least around 460 nM, around 475 nM, around 500 nM,around 530 around 560 around 590 around 620 around 650 around 680 around 710 around 740 around 770 around 800 around 830 around 860 around 890 around 920 around 950 at least less less around 465 around 480 around 510 nm, nm, nm, less less less around 470 around 495 around 520 nm, nm, nm, nm, nm, nm, nm, nm, nm, nm, nm, nm, nm, nm, nm, nm, nm, nm, around 980 nm, at least less less less less less less less less less less less less less less less less less less minus minus around 540 around 570 around 600 around 630 around 660 around 690 around 720 around 750 around 780 around 810 around 840 around 870 around 900 around 930 around 960 nM, nM, nM, nM, nM, nM, nM, nM, nM, nM, nM, nM, nM, nM, nM, minus around 990 nM, to around 1050 nM,at least around 1100 nM, around 1200 nM, at least around 1250 nM, around 1350 nM, at least around 1400 nM, around 1500 nM, at least around 1550 nM, around 1650 nM, at least around 1700 nM, around 1800 nM, at least around 1850 nM, around 1950 nM, at least around 2000 nM, around 2100 nM, at least around 2150 nM, around 2250 nM, at least around 2300 nM, around 2400 nM, at least around 2450 nM, around 2550 nM, at least around 2600 nM, around 2700 nM, at least around 2750 nM, less less less less less less less less less less less less less less less less less less less less less less less less around 550 around 580 around 610 around 640 around 670 around 700 around 730 around 760 around 790 around 820 around 850 around 880 around 910 around 940 around 970 nM, at least nM, nM, nM, nM, nM, nM, nM,nM, nM, nM, nM, nM, nM, nM, nM, nM, nM, less around 1000 nM, at least around 1150 nM, at least around 1300 nM, at least around 1450 nM, at least around 1600 nM, at least around 1750 nM, at least around 1900 nM, at least around 2050 nM, at least around 2200 nM, at least around 2350 nM, at least around 2500 nM, at least around 2650 nM, at least around 2800 nM, at least less ... less less less less less less less less less less less less around 2850 nm, at least around 2900 nm, at least around 2950 nm or at least around 3000 nm. In some example modalities, the culture medium may have a pH of around 6.5 to around 8.0. In some specific example modalities, the cell culture medium may have a pH of around 6.5, around 6.6, around 6.7, around 6.8, around 6.9, around 7.0, around 7.1, around 7.2, around 7.3, around 7.4, around 7.5, around 7.6, around 7.7, around 7.8, around 7.9, or around 8.0. In some example modalities, eukaryotic cells are cultured at a temperature of around 25°C to around 40°C. In some specific example modalities, eukaryotic cells are cultured at a temperature of around 25°C, around 26°C, around 27°C, around 28°C, around 29°C, around 30°C, around 31°C, around 32°C, around 33°C, around 34°C, around 35°C, around 36°C, around 37°C, around 38°C, around 39°C, or around 40°C. In some example modalities, the cell culture medium may comprise at least around 10 nM of 5-methylthioadenosine, where a titer of a protein produced in the cell culture medium may be at least around 2% higher than another cell culture medium that does not have at least around 10 nM of 5-methylthioadenosine. In one aspect, the cell culture medium may comprise at least around 20 nM of 5-methylthioadenosine, at least around 30 nM of 5-methylthioadenosine, at least around 40 nM of 5-methylthioadenosine, at least around 50 nM of 5-methylthioadenosine, at least around 60 nM of 5-methylthioadenosine, at least around 70 nM of 5-methylthioadenosine, at least around 80 nM of 5-methylthioadenosine, at least around 90 nM of 5-methylthioadenosine, at least around 100 nM of 5-methylthioadenosine, at least around 110 nM of 5-methylthioadenosine, at least around 120 nM of 5-methylthioadenosine,at least around 130 nM of 5-methylthioadenosine, at least around 140 nM of 5-methylthioadenosine, at least around 150 nM of 5-methylthioadenosine, at least around 160 nM of 5-methylthioadenosine, at least around 170 nM of 5-methylthioadenosine, at least around 180 nM of 5-methylthioadenosine, at least around 190 nM of 5-methylthioadenosine or at least around 200 nM of 5-methylthioadenosine. In one respect, the titer of a protein produced in the cell culture medium may be greater than at least around 3%, at least around 4%, at least around 5%, at least around 6%, at least around 7%, at least around 8%, at least around 9%, at least around 10%, at least around 11%, at least around 12%, at least around 13%, at least around 14%, at least around 15%, at least around 16%, at least around 17%, at least around 18%, at least around 19%,at least around 20%, at least around 21%, at least around 22%, at least around 23%, at least around 24%, at least around 25%, at least around 26%, at least around 27%, at least around 28%, at least around 29%, or at least around 30%. In some example configurations, the cell culture medium may contain at least approximately 50 nM of nicotinamide, where a protein titer produced in the cell culture medium may be at least approximately 2% higher than in another cell culture medium that does not contain at least approximately 50 nM of nicotinamide. In one aspect, the cell culture medium may contain at least approximately 50 nM of nicotinamide, at least approximately 100 200 300 400 nM of nicotinamide, nM of nicotinamide, nM of nicotinamide, nM of nicotinamide, at at least less less less around 150 nM of nicotinamide, around 250 nM of nicotinamide, around 350 nM of nicotinamide, around 450 nM of nicotinamide, at at least less less less less around around around around around 500 nM nicotinamide at least 600 nM of nicotinamide, at least around 550 nM of nicotinamide, around 650 nM of nicotinamide, at least less around around 700 nM of nicotinamide, at least around 750 nM of nicotinamide, at least around 800 nM of nicotinamide, at least around 850 nM of nicotinamide, at least around 900 nM of nicotinamide at least around 1000 nM of nicotinamide. In one respect, the titer of a protein produced in the cell culture medium may be greater than at least around 3%, at least around 4%, at least around 5%, at least around 6%, at least around 7%, at least around 8%, at least around 9%, at least around 10%, at least around 11%, at least around 12%, at least around 13%, at least around 14%, at least around 15%, at least around 16%, at least around 17%, at least around 18%, at least around 19%, at least around 20%, at least around 21%, at least around 22%, at least around 23%, at least around 24%, at least around 25%, at least around 26%, at least around 27%, at least around 28%, at least around 29%, or at least around 30%. In some example modalities, the cell culture medium may comprise feeding the eukaryotic cells by using an enriched medium that has 5-methylthioadenosine at a concentration of at least around 0.05 nM, at least around 1 nM, at least around 2 nM, at least around 3 nM, at least around 4 nM, at least around 5 nM, at least around 6 nM, at least around 7 nM, at least around 8 nM, around 9 nM, at least around 10 nM, at least around 11 nM, around 12 nM, at least around 13 nM, at least around 14 nM, to at least around 15 nM, at least around 20 nM, at least around 25 nM, at least around 30 nM, to less around 35 nM, at least around 40 nM or at least around 50 nM. In some example modalities, the cell culture medium may comprise feeding the eukaryotic cells by using an enriched medium that has nicotinamide at a concentration of at least around 1 nM, at least around 2 nM, at least around nM, at least around 4 nM, at least around 5 nM, at least around 6 nM, at least around 7 nM, at least around 8 nM, at least around 9 nM, at least around 10 nM, at least around 15 nM, at least around 20 nM, at least around 25 nM, at least around 30 nM, at least around 35 nM, at least around 40 nM, at least around 50 nM, at least around 55 nM, at least around 60 nM, at least around 65 nM nM, at least around 70 nM, at least around 75 nM, at least around 80 nM, at least around 85 nM, at least around 90 nM, at least around 95 nM, at least around 100 nM, at least around 105 nM,at least around 110 nM, at least around 115 nM, at least around 120 nM, at least around 125 nM, at least around 130 nM, at least around 135 nM, at least around 140 nM, at least around 145 nM, at least around 150 nM, at least around 155 nM, at least around 160 nM, at least around 165 nM, at least around 170 nM, at least around 175 nM, at least around 180 nM, at least around 195 nM or at least around 200 nM. In some example embodiments, the cell culture medium may comprise feeding eukaryotic cells by using an enriched medium that has 5-methylthioadenosine at a concentration of at least about 1 nM, wherein a titration of a protein produced in the cell culture medium may be at least about 2% higher than another cell culture medium that does not have at least about 10 nM of 5-methylthioadenosine. In one aspect, the enriched medium may have at least about 2 nM of 5-methylthioadenosine, at least about 3 nM of 5-methylthioadenosine, at least about 4 nM of 5-methylthioadenosine, at least about 5 nM of 5-methylthioadenosine, at least about 6 nM of 5-methylthioadenosine, at least about 7 nM of 5-methylthioadenosine, at least about 8 nM of 5-methylthioadenosine, at least about 9 nM of 5-methylthioadenosine, at least about 10 nM of 5-methylthioadenosine,at least around 11 nM of 5-methylthioadenosine, at least around 12 nM of 5-methylthioadenosine, at least around 13 nM of 5-methylthioadenosine, at least around 14 nM of 5-methylthioadenosine, at least around 15 nM of 5-methylthioadenosine, at least around 16 nM of 5-methylthioadenosine, at least around 17 nM of 5-methylthioadenosine, at least around 18 nM of 5-methylthioadenosine, at least around 19 nM of 5-methylthioadenosine, or at least around 20 nM of 5-methylthioadenosine. In one respect, the titer of a protein produced in the cell culture medium may be greater than at least around 3%, at least around 4%, at least around 5%, at least around 6%, at least around 7%, at least around 8%, at least around 9%, at least around 10%, at least around 11%, at least around 12%, at least around 13%, at least around 14%, at least around 15%,at least around 16%, at least around 17%, at least around 18%, at least around 19%, at least around 20%, at least around 21%, at least around 22%, at least around 23%, at least around 24%, at least around 25%, at least around 26%, at least around 27%, at least around 28%, at least around 29%, or at least around 30%. In some example modalities, the cell culture medium may comprise feeding the eukaryotic cells by using an enriched medium that has nicotinamide at a concentration of at least around 5 nM, 50 nM of nicotinamide.In one aspect, the cell culture medium may comprise at least approximately 5 nM nicotinamide, at least approximately 10 nM nicotinamide, at least approximately 15 nM nicotinamide, at least approximately 20 nM nicotinamide, at least approximately 25 nM nicotinamide, at least approximately 30 nM nicotinamide, at least approximately 35 nM nicotinamide, at least approximately 400 nM nicotinamide, at least approximately 45 nM nicotinamide, at least approximately 50 nM nicotinamide, at least approximately 55 nM nicotinamide, at least approximately 60 nM nicotinamide, at least approximately 65 nM nicotinamide, at least approximately 70 nM nicotinamide, at least approximately 75 nM of nicotinamide, at least around 80 nM of nicotinamide, at least around 85 nM of nicotinamide, at least around 90 nM of nicotinamide, at least around 95 nM of nicotinamide or at least around 100 nM of nicotinamide.In one respect, the titer of a protein produced in the cell culture medium may be greater than at least around 3%, at least around 4%, at least around 5%, at least around 6%, at least around 7%, at least around 8%, at least around 9%, at least around 10%, at least around 11%, at least around 12%, at least around 13%, at least around 14%, at least around 15%, at least around 16%, at least around 17%, at least around 18%, at least around 19%, at least around 20%, at least around 21%, at least around 22%, at least around 23%, at least around 24%, at least around 25%, at least around 26%, at least around 27%, at least around 28%, at least around 29%, or at least around 30%. In some example modalities, the protein may be a naturally occurring protein. In some example modalities, the protein may be a recombinant protein. In some example modalities, the protein may be a biotherapeutic protein. In some example modalities, the protein may be a recombinant protein, where the recombinant protein may be a trap protein, a chimeric Fe receptor 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. In some example modalities, the protein may comprise modifications, adducts, and other covalently bound residues. In one example, the protein may involve a post-translational modification. The sequential numbering of the steps of the method as provided herein with numbers and / or letters is not intended to limit the method or any modality thereof to the order indicated in particular. Throughout this descriptive report, various publications are cited, including patents, patent applications, published patent applications, accession numbers, technical articles, and academic articles. Each of these cited references is incorporated herein in its entirety by reference for all purposes. The description will be more fully understood by referring to the following examples, which are provided to illustrate the description in more detail. These are intended to be illustrative and should not be interpreted as limiting the scope of the description. EXAMPLES To study the content of a cell culture medium and its effect on the production of a protein, a cell culture comprising soy hydrolysate was selected to generate a VEGFR 1 binding protein. Example 1. Soybean hydrolysate content analysis was performed using nine different lots obtained from three separate shipments: 1, 2, and 3. Samples from these three shipments were sent to Metabolon (Durham, NC, USA). Soybean hydrolysate content analysis was performed at Metabolon using liquid chromatography-mass spectrometry. More than three hundred components were measured in the soybean hydrolysate. Only results scaled to the median are provided. To study the effect of biochemicals on the titration of VEGFR (vascular endothelial growth factor receptor) binding protein 1, an orthogonal partial least squares (OPLS) model was used for multivariate analysis (MVA). The study ΊΛ / a / ZUZ l / UI included seventy lots of soybean hydrolysates. The relationship between the components and the titration was assessed by studying the correlation and covariance. A positive relationship suggests an increase in titration with an increase in the biochemical concentration, and a negative relationship suggests a decrease in titration with an increase in the biochemical concentration. For the final titration assessment of a principal component, the R²X, R²Y, and Q² values ​​were found to be 0.315, 0.672, and 0.493, respectively. The components that showed a positive relationship are shown in Table 1, and the components that showed a negative relationship are shown in Table 2. Table 1. Positive dependence Nicotinamide Lactate phenyl lactate (PLA) 5-methylthioadenosine (MTA) indolelacate succinate alpha-hydroxyisocaproate 3-(4-hydroxyphenyl)lactate (HPLA) alpha-hydroxyisovalerate 2-hydroxy-3-methylvalerate ΊΛ / a / ZUZ l / UI ZD4U Table 2. Negative dependence on nicotinate, sucrose, uracil, phenylalanine, valyleucine, maltose, digalactosylglycerol, pantothenate (vitamin B5), xanthine, serine The MVA result normalized with respect to unit length is shown in FIG. 1. Based on these findings from example 2, nicotinamide and 5-methylthioadenosine were selected as positive markers for further analysis. Example 2. The impact of 5-methylthioadenosine on the production of a recombinant protein (VEGFR 1 binding protein) was studied by investigating the protein titration (g / L) at different concentrations of 5-methylthioadenosine present in the cell culture medium. An enriched medium (soybean hydrolysate) containing 5-methylthioadenosine was added to the cell culture medium. Figure 2 shows the correlation between the concentration of 5-methylthioadenosine present in the soybean hydrolysate added to the cell culture medium and the titer of VEGFR-binding protein 1. As observed in FIG. 2, the regression line related to the extrapolated variables (N = 70) suggests that the titration varied linearly with the concentration of 5-methylthioadenosine in the soybean hydrolysate added to the cell culture medium, suggesting that a higher titration can be obtained by increasing the concentration of 5-methylthioadenosine in the cell culture medium. Example 3. Based on the correlation data obtained from studying the effect of 5-methylthioadenosine concentrations in the enriched medium (soybean hydrolysate) added to the cell culture medium (Example 2, FIG. 2), an estimate of the optimal 5-MTA concentration in cell culture was made. FIG. 3 shows the regression line related to the extrapolated variables (N=70), suggesting that the titration may vary linearly with respect to the 5-methylthioadenosine concentration in the cell culture medium, indicating that higher titrations can be obtained by increasing the 5-methylthioadenosine concentration in the cell culture medium. Example 4. The impact of nicotinamide on a recombinant protein titration (VEGFR 1 binding protein) was also studied by investigating the protein titration (g / L) at different concentrations of nicotinamide present in the cell culture medium. An enriched medium (soybean hydrolysate) containing nicotinamide was added to the cell culture medium at varying concentrations. Figure 4 shows the correlation between the concentration of nicotinamide present in the soybean hydrolysate added to the cell culture medium and the titration of VEGFR-binding protein 1. As observed in Figure 4, the regression line relative to the extrapolated variables (N = 70) suggests that the titration varied linearly with the concentration of nicotinamide in the soybean hydrolysate, suggesting that a higher titration can be obtained by increasing the concentration of nicotinamide in the cell culture medium. Example 5. Based on the correlation data obtained from studying the effect of nicotinamide concentration in the enriched medium (soybean hydrolysate) added to the cell culture medium (Example 4, FIG. 4), an estimation of the optimal nicotinamide concentration in a cell culture was carried out. FIG. 5 shows that the regression line relative to the extrapolated variables (N = 70) suggests that the titration may vary linearly with respect to the nicotinamide concentration in the cell culture medium, suggesting that a higher titration can be obtained by increasing the nicotinamide concentration in the cell culture medium.

Claims

CLAIMS The following is claimed:

1. A cell culture medium for eukaryotic cells expressing Aflibercept, comprising: a basal medium or a feeding medium; and 5-methylthioadenosine or nicotinamide.

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

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

4. The cell culture medium of claim 1 further comprising one or more acids selected from lactic acid, phenyl lactic acid, indole lactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-hydroxyisocaproic acid, 2-(4-hydroxyphenyl)lactic acid or 2-hydroxy3-methylvaleric acid, salts of these acids, esters of these acids and combinations thereof.

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

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

7. The cell culture medium of claim 1, wherein the 5-methylthioadenosine or nicotinamide may be in the form of its salts or esters.

8. 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. The cell culture medium of claim 1, wherein the medium does not have a protein derived from an animal.

10. The cell culture medium of claim 1, wherein the medium is a serum-free medium.

11. The cell culture medium of claim 1, wherein the medium is a chemically defined medium.

12. A method for growing eukaryotic cells to increase the production of Aflibercept, comprising the steps of: growing eukaryotic cells in a cell culture medium; supplementing the cell culture medium with 5-methylthioadenosine, wherein the concentration of 5-methylthioadenosine is from about 10 nM to about 200 nM; and producing Aflibercept in the eukaryotic cells, wherein supplementation with 5-methylthioadenosine increases a titer of Aflibercept.

13. The method of claim 12, wherein the titration of Aflibercept is at least about 2% higher than another method with a cell culture medium that does not have at least about 10 nM of 5-methyl-thioadenosine.

14. The method of claim 12, wherein the cell culture medium further comprises one or more acids selected from lactic acid, phenyl lactic acid, indole lactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-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. The method of claim 12, wherein the eukaryotic cells include at least one selected from the group consisting of: baby hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human embryonic kidney cell lines, human retina-derived cell lines, and amniocyte cell lines.

16. The method of claim 12, wherein Aflibercept is segregated in the middle.

17. The method of claim 12, wherein the cell culture medium does not have an animal-derived protein.

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

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

20. A method for growing eukaryotic cells to increase the production of Aflibercept, comprising the steps of: growing cells in a defined cell culture medium; supplementing the cell culture medium with nicotinamide, wherein the concentration of nicotinamide is from about 50 nM to about 2000 nM; and producing Aflibercept in the eukaryotic cells, wherein supplementation with nicotinamide increases an Aflibercept titration.

21. The method of claim 20, wherein the titration of Aflibercept is at least about 2% higher than another method with a cell culture medium that does not have at least 50 nM nicotinamide.

22. The method of claim 20, wherein the cell culture medium further comprises one or more acids selected from lactic acid, phenyl lactic acid, indole-lactic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-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. The method of claim 20, wherein the eukaryotic cells include at least one selection from the group consisting of: baby hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human embryonic kidney cell lines, human retina-derived cell lines, and amniocyte cell lines.

24. The method of claim 20, wherein the Aflibercept are segregated in the medium.

25. The method of claim 20, wherein the cell culture medium does not have a protein derived from an animal.

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

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

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