EA045820B1 - MEDIUM FOR CULTIVATION OF EUKARYOTIC CELLS - Google Patents

Description

Field of technology to which the invention relates

The present invention generally relates to a medium for culturing eukaryotic cells and the production thereof of natural and recombinant products.

State of the art

Cell culture technology is widely used for the manufacture of biopharmaceuticals. As the demand for biopharmaceuticals has increased, the need to increase levels of cell growth, viability and protein production has also increased significantly. Much effort is now being devoted to methods and strategies for growing, feeding, and maintaining cell cultures.

New cell culture methods that provide uniform, gradual increases in the level of production of recombinant proteins are valuable given the challenges and costs associated with large-scale cell culture processes, as well as the growing need for higher quantities and lower costs of biological products. Therefore, optimization of cell culture processes, recombinant polypeptide expression, titer, and cell viability is necessary, which can lead to higher product yields, thereby reducing the costs associated with the production of protein therapeutics.

The essence of the invention

The intensification of development, production and sales of protein-based biopharmaceutical products has led to increased demand for production methods that can optimize the production of biopharmaceutical products.

The embodiments described herein satisfy the above requirements by providing methods and environments for the production of such biopharmaceutical products.

This document, at least in part, describes a medium for culturing eukaryotic cells.

In one exemplary embodiment, the described eukaryotic cell culture medium may include 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 contain at least about 10 nM 5-methylthioadenosine. In yet another aspect of this embodiment, the cell culture medium may contain from about 10 nM to about 200 nM 5-methylthioadenosine. In one aspect of this embodiment, the cell culture medium may further contain nicotinamide. In another aspect of this embodiment, the cell culture medium may contain at least about 50 nM nicotinamide. In yet another aspect of this embodiment, the cell culture medium may contain about 2000 nM 5-nicotinamide. In one aspect of this embodiment, the titer of protein grown in the cell culture medium is at least about 2% greater than in another cell culture medium not containing at least about 10 nM 5-methylthioadenosine. In another aspect of this embodiment, the titer of protein grown in the cell culture medium is at least about 2% greater than in another cell culture medium not containing at least about 50 nM nicotinamide. In one aspect of this embodiment, the cell culture medium may further comprise one or more acids selected from lactic acid, phenyllactic acid, indolylacetic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-hydroxyisocaproic acid, 2-(4-hydroxyphenyl )lactic acid or 2-hydroxy-3methylvaleric acid, salts of these acids, esters of these acids and combinations thereof. In one aspect of this embodiment, the cell culture medium may further contain sugars, amino acids, vitamins, salts, trace metal ions, purines and/or pyrimidines. In one aspect of this embodiment, the cell culture medium may contain salts or esters of 5-methylthioadenosine. In another aspect of this embodiment, the cell culture medium may contain nicotinamide salts or esters. In one aspect of this embodiment, the cell culture medium may have a pH of from about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium does not contain animal 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 exemplary embodiment, the culture medium for eukaryotic cells may include a nutrient 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 contain at least about 10 nM 5-methylthioadenosine. In yet another aspect of this embodiment, the cell culture medium may contain from 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 contain

- 1 045820 at least about 50 nM nicotinamide. In yet another aspect of this embodiment, the cell culture medium may contain from about 50 nM to about 2000 nM 5-nicotinamide. In an alternative 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 comprise one or more acids selected from lactic acid, phenyllactic acid, indolylacetic 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 contain sugars, amino acids, vitamins, salts, trace metal ions, purines and/or pyrimidines. In one aspect of this embodiment, the cell culture medium may contain salts or esters of 5-methylthioadenosine. In another aspect of this embodiment, the cell culture medium may contain nicotinamide salts or esters. In one aspect of this embodiment, the cell culture medium may have a pH of from about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium does not contain animal 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 aspect of this embodiment, the cell culture medium may optionally contain nicotinamide. In one aspect of this embodiment, the titer of protein grown in the cell culture medium is at least about 2% greater than in another cell culture medium not containing at least about 10 nM 5-methylthioadenosine. In another aspect of this embodiment, the titer of protein grown in the cell culture medium is at least about 2% greater than in another cell culture medium not containing at least about 50 nM nicotinamide.

Presented herein, at least in part, is a method for producing protein.

In one exemplary embodiment, a method for producing a protein may include culturing eukaryotic cells containing a nucleic acid encoding the protein in cell culture production medium and feeding the eukaryotic cells using enriched medium containing 5-methylthioadenosine for a specified period of time. In one aspect of this embodiment, the enrichment medium may contain at least about 1 nM 5-methylthioadenosine. In one aspect of this embodiment, the cell culture production medium may contain one or more acids selected from lactic acid, phenyllactic acid, indolylacetic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-hydroxyisocaproic acid, 2-(4-hydroxy -phenyl)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 contain sugars, amino acids, vitamins, salts, trace metal ions, purines and/or pyrimidines. In one aspect of this embodiment, the cell culture production medium may contain salts or esters of 5-methylthioadenosine. In one aspect of this embodiment, the cell culture production medium may have a pH of from about 6.5 to about 8. In one aspect of this embodiment, the cell culture production medium does not contain animal 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 embodiment, the eukaryotic cells may be selected from neonatal hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human fetal kidney cell lines, human retina derived cell lines, and/or amniocyte cell lines. In one aspect of this embodiment, the protein may be selected from the group consisting of an antibody or fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein. In one aspect of this embodiment, the method can provide a protein titer that is at least about 2% greater than the protein titer in a cell culture production medium not containing at least about 10 nM 5-methylthioadenosine. In one aspect of this embodiment, the enriched medium may optionally contain nicotinamide. In one aspect of this embodiment, the protein production method may be a fed-batch culture method.

In one exemplary embodiment, a method for producing a protein may include culturing eukaryotic cells containing a nucleic acid encoding the protein in cell culture production medium and feeding the eukaryotic cells using enriched medium containing nicotinamide for a specified period of time. In one aspect of this embodiment, the enriched medium may contain at least about 5 nM nicotinamide.

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In one aspect of this embodiment, the cell culture production medium may contain one or more acids selected from lactic acid, phenyllactic acid, indolylacetic acid, succinic acid, alpha-hydroxyisovaleric acid, alpha-hydroxyisocaproic acid, 2-(4-hydroxyphenyl )lactic acid or 2-hydroxy-3methylvaleric acid, salts of these acids, esters of these acids and combinations thereof. In one aspect of this embodiment, the cell culture production medium may contain sugars, amino acids, vitamins, salts, trace metal ions, purines and/or pyrimidines. In one aspect of this embodiment, the cell culture production medium may contain nicotinamide salts or esters. In one aspect of this embodiment, the cell culture production medium may have a pH of from about 6.5 to about 8. In one aspect of this embodiment, the cell culture production medium does not contain animal 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 embodiment, the eukaryotic cells may be selected from neonatal hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human fetal kidney cell lines, human retina derived cell lines, and/or amniocyte cell lines. In one aspect of this embodiment, the protein may be selected from the group consisting of an antibody or fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein. In one aspect of this embodiment, the method can provide a protein titer that is at least about 2% greater than the protein titer in a cell culture production medium not containing at least about 50 nM nicotinamide. In one aspect of this embodiment, the enrichment medium may optionally contain 5-methylthioadenosine. In one aspect of this embodiment, the protein production method may be a fed-batch culture method.

Disclosed herein, at least in part, is a method for increasing protein production levels.

In one exemplary embodiment, the method of increasing protein production may include culturing eukaryotic cells in a cell culture medium, adding 5-methioadenosine to the cell culture medium, 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 can be from about 10 nM to about 200 nM. In one aspect of this embodiment, the cell culture medium may contain one or more acids selected from lactic acid, phenyllactic acid, indolylacetic 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 agent may contain sugars, amino acids, vitamins, salts, trace metal ions, purines and/or pyrimidines. In one aspect of this embodiment, the cell culture medium may contain salts or esters of 5-methylthioadenosine. In one aspect of this embodiment, the cell culture medium may have a pH of from about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium does not contain animal 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 aspect of this embodiment, the eukaryotic cells may be selected from neonatal hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human fetal kidney cell lines, human retina derived cell lines, and/or amniocyte cell lines. In one aspect of this embodiment, the protein may be selected from the group consisting of an antibody or fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein. In one aspect of this embodiment, the addition of 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 optionally be supplemented with nicotinamide.

In one exemplary embodiment, the method of increasing protein production may include culturing eukaryotic cells in a cell culture medium, adding nicotinamide to the cell culture medium, 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 can be from about 50 nM to about 2000 nM. In one aspect of this embodiment, the environment

- 3 045820 for cell culture may contain one or more acids selected from lactic acid, phenyllactic acid, indolylacetic 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 agent may contain sugars, amino acids, vitamins, salts, trace metal ions, purines and/or pyrimidines. In one aspect of this embodiment, the cell culture medium may contain nicotinamide salts or esters. In one aspect of this embodiment, the cell culture medium may have a pH of from about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium does not contain animal 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 aspect of this embodiment, the eukaryotic cells may be selected from neonatal hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human fetal kidney cell lines, human retina derived cell lines, and/or amniocyte cell lines. In one aspect of this embodiment, the protein may be selected from the group consisting of an antibody or fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein. In one aspect of this embodiment, the addition of nicotinamide increases the titer of the recombinant protein by at least about 2%. In one aspect of this embodiment, the cell culture medium may optionally be supplemented with 5-methyladenosine.

Presented herein, at least in part, is a method for producing protein.

In one exemplary embodiment, a method for producing a protein may include introducing into a cell(s) a nucleic acid containing a protein coding sequence 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 of producing a protein may further include maintaining a cell culture medium to express a higher titer of protein in the cell(s). In one aspect of this embodiment, the method for producing a protein may further comprise collecting the protein. In one aspect of this embodiment, the cells may be selected from neonatal hamster kidney cell lines, Chinese hamster ovary cell lines, murine myeloma cell lines, mouse myeloma cell lines, human fetal 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, indolylacetic 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 agent may further contain 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 from about 6.5 to about 8. In one aspect of this embodiment, the cell culture medium does not contain animal 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 aspect of this embodiment, the protein may be selected from the group consisting of an antibody or fragment or derivative thereof, a fusion protein, and a physiologically active non-antibody protein. In one aspect of this embodiment, the method can exhibit a 2% higher protein titer in the cell(s) compared to cell(s) grown in a cell culture medium that does not contain at least about 10 nM 5-methylthioadenosine. In one aspect of this embodiment, said protein production method may further comprise maintaining a cell culture medium to produce a 2% higher protein titer in the cell(s) compared to cell(s) grown in a cell culture medium that does not contain at least about 50 nM nicotinamide.

Brief description of graphic materials

Fig. 1 shows the result of multivariate analysis (MVA) of the study of components present

- 4 045820 present in soybean hydrolyzate.

Fig. 2 shows correlation data obtained from studying the effect of the concentration of 5-methylthioadenosine in the enrichment medium used to feed the cell culture medium on protein titer (g/L), according to one exemplary embodiment.

Fig. 3 shows a regression line relating to variables (N=70) used to evaluate the effect of 5-methylthioadenosine concentration in cell culture medium on protein titer (g/L) according to one exemplary embodiment.

Fig. 4 shows correlation data obtained from studying the effect of nicotinamide concentration in enrichment medium used to feed cell culture media on protein titer (g/L), according to one exemplary embodiment.

Fig. 5 shows a regression line relating to variables (N=70) used to evaluate the effect of nicotinamide concentration in cell culture medium on protein titer (g/L) according to one exemplary embodiment.

Detailed description

Biopharmaceuticals have proven to be very effective for diagnostic and therapeutic purposes (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 useful for culturing cells for the manufacture of biopharmaceutical products are well described in the literature, and a number of 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 amounts of which may vary depending on the specific requirements of a given cell or tissue type.

One goal of protein production may be to optimize cell culture to obtain the maximum amount of protein and the most efficient means of productivity. Any optimization, including incremental optimization, can have huge economic benefits. In the pharmaceutical industry, optimizing protein production for biologics used in therapeutics to treat diseases is an advantage because any optimization can have a significant impact when the biologic is produced on a large scale. Thus, there remains a need to maximize protein production from cell cultures expressing biological proteins for medical applications.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in practice or testing, specific methods and materials are described below. All publications mentioned are incorporated herein by reference.

Singular forms should be understood to mean at least one; and the terms about and approximately should be understood to be subject to standard variations as will be understood by those skilled in the art; and if ranges are specified, endpoints are included.

In some exemplary embodiments of the present invention, a method for producing protein is provided.

As used herein, the term protein includes any amino acid polymer with covalently linked amide bonds. Proteins contain one or more amino acid polymer chains, commonly known in the art as polypeptides.

The term polypeptide refers to a polymer consisting of amino acid residues, corresponding naturally occurring structural variants and their synthetic non-naturally occurring analogues, linked through peptide bonds, corresponding naturally occurring structural variants, and their synthetic non-naturally occurring analogues. Synthetic peptides or polypeptides refer to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automatic polypeptide synthesizer. Various methods for solid phase peptide synthesis are known to those skilled in the art. A protein may contain one or more polypeptides to form a single functioning biomolecule. The protein may include any of biotherapeutic proteins, recombinant proteins used in research or therapy, decoy proteins and other Fc fragment and chimeric receptor-based binding molecules, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies and bispecific antibodies. In another exemplary aspect, the protein may 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 insect baculovirus systems, yeast systems (eg Pichia sp.), mammalian systems (eg CHO cells and CHO cell derivatives such as CHO-K1 cells). For a recent 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, 147-176 (2012). In some embodiments, the proteins contain modifications, adducts, and other covalently linked moieties. These modifications, adducts and fragments include, for example, avidin, streptavidin, biotin, glycans (for example, N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose and other monosaccharides), PEG, polyhistidine, FLAG tag, maltose binder protein (MBP), chitin binding protein (CBP), glutathione S-transferase (GST), myc epitope, fluorescent tags and other dyes, etc. Proteins can be classified based on composition and solubility and thus can include simple proteins such as globular proteins and fibrillar 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 exemplary embodiments, the protein or protein or recombinant protein or recombinant protein may be an antibody, bispecific antibody, multispecific antibody, antibody fragment, monoclonal antibody, Fc fusion binding molecule, F(ab')2 fragment, Fc fragment, or their combination.

As used herein, the term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy chains (H) and two light chains (L), linked by disulfide bonds, as well as multimers thereof (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains _CH1 , CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and _VL may consist of three CDRs and four FRs, located from the amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, FR antibodies against big-ET-1 (or an antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs. As used herein, the term antibody also includes antigen-binding portions of complete antibody molecules.

The terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like as used herein include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding antibody fragments can be prepared, for example, from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and processed chemically or using molecular biology techniques, for example, to place one or more variable and/or constant domains in 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 by hybridoma technology. A monoclonal antibody can be produced from a single clone, including any eukaryotic, prokaryotic or phage clone, by any methods available or known in the art. Monoclonal antibodies useful in the present invention can be produced using a wide range of methods known in the art, including the use of hybridoma, recombinant and phage display technologies, or combinations thereof.

As used herein, the term fusion protein or Fc fusion protein includes part or all of two or more proteins, one of which may be part of an Fc immunoglobulin molecule, that are not fused in their natural state. The production of the fusion protein may include certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain), as described, for example, in A. Ashkenazi et al., Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin. , 88 PROCEEDINGS OF THE

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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). Receptor-Fc fragment fusion proteins include one or more extracellular domains of the receptor linked to an Fc fragment, which in some embodiments may comprise a hinge region followed by an immunoglobulin C _H 2 C _H 3 domain. In some embodiments, the -Fc fusion protein may comprise two or more different receptor chains that bind to one or more ligands. For example, the Fc fusion protein may be a decoy, such as an IL-1 decoy (eg, rilonocept, which contains the RAcP ligand binding region of IL-1 fused to the extracellular region of IL-1R1 fused to the Fc of hIgG1; see patent US No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., aflibercept, which contains VEGF receptor Flt1 Ig domain 2 fused to VEGF receptor Flk1 Ig domain 3 fused to hIgG1 Fc; for example, see US Pat. Nos. 7,087,411 and 7,279,159, which are incorporated herein by reference in their entirety).

As used herein, the term antibody fragment includes a portion of an intact antibody, such as, for example, an antigen binding or antibody variable region. Examples of antibody fragments include, but are not limited to, Fab fragment, Fab' fragment, F(ab')2 fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd' fragment, Fd fragment and selected complementarity determining region region (CDR), as well as tribodies, tetrabodies, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Fv fragments are a combination of immunoglobulin heavy and light chain variable regions, and ScFv proteins are recombinant single-chain polypeptide molecules in which the immunoglobulin light and heavy chain variable regions are connected by a peptide linker. In some exemplary embodiments, the antibody fragment contains sufficient amino acid sequence of the parent antibody of which it is a fragment that binds to the same antigen as the parent antibody; in some exemplary embodiments, the fragment binds to an antigen with comparable affinity to that of the parent antibody, and/or competes with the parent antibody for binding to the antigen. The antibody fragment can be obtained by any method. For example, an antibody fragment may be produced enzymatically or chemically by fragmentation of an intact antibody, and/or it may be produced recombinantly from a gene encoding a partial sequence of the antibody. Alternatively or additionally, the antibody fragment may be produced in whole or in part synthetically. The antibody fragment may optionally comprise a single chain antibody fragment. Alternatively or additionally, an antibody fragment may contain multiple chains that are linked together, for example, by disulfide bonds. The antibody fragment may optionally contain a multimolecular complex. A functional antibody fragment typically contains at least about 50 amino acids, and more typically contains at least about 200 amino acids.

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

A variant or variation of a binding molecule as used herein may include a binding molecule that differs from the target binding molecule based on at least one amino acid modification or post-translational modification. The variant may refer to the binding molecule itself, to a composition containing the binding molecule, or to the amino acid sequence that encodes it. Preferably, the variant binding molecule has at least one amino acid modification compared to the parent binding molecule, for example, from about one to about seventy amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent protein. The variant binding molecule sequence of the present invention will preferably have at least about 80% homology to the sequence of the parent binding molecule, more preferably at least about 90% homology, and most preferably at least about 92% homology.

In some exemplary embodiments, the protein, or 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 usually introduced by specific enzymes or enzymatic pathways. Many of them arise at the location of a specific characteristic protein sequence (eg, signature sequence) within the protein scaffold. Several hundred PTMs have been reported, and these modifications invariably affect some aspect of protein structure or function (Walsh, G. Proteins (2014), second edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853). Various post-broadcast

- 7 045820 tion modifications include, but are not limited to, elimination, N-terminal extensions, protein degradation, N-terminal acylation, biotinylation (acylation of lysine residues with biotin), C-terminal amidation, glycosylation, iodination, covalent attachment of prosthetic groups, acetylation (adding acetyl group, usually at the N-terminus of a protein), alkylation (adding an alkyl group (e.g., methyl, ethyl, propyl) usually at lysine or arginine residues), methylation, adenylation, ADP-ribosylation, covalent cross-linking within, or between, polypeptide chains , sulfonation, prenylation, vitamin C-dependent modifications (proline and lysine hydroxylations and carboxy-terminal amidation), vitamin K-dependent modification, with vitamin K being a cofactor in the carboxylation of glutamic acid residues to form γ-carboxyglutamate (glu residue) , glutamylation (covalent bonding of glutamic acid residues), glycylation (covalent bonding of glycine residues), glycosylation (adding a glycosyl group to either asparagine, hydroxylysine, serine or threonine, providing a glycoprotein), isoprenylation (adding an isoprenoid group such as farnesol and geranylgeraniol), lipoylation (attachment of a lipoate functional group), phosphopantetheinylation (adding a 4'-phosphopantetheinyl moiety from coenzyme A, as in fatty acid, polyketide, nonribosomal peptide and leucine biosynthesis), phosphorylation (adding a phosphate group, usually to serine, tyrosine, threonine or histidine) and sulfation (adding 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 (eg, converting arginine to citrulline via deimination), and deamidation (eg, converting 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 (a covalent bond between two cysteine amino acids) and proteolytic cleavage (the removal of a protein from a peptide bond). Certain post-translational modifications include the addition of other proteins or peptides, such as ISGylation (covalently linked to the ISG15 (interferon-stimulated gene) protein), SUMOylation (covalently linked to the SUMO (small ubiquitin-like modifier) protein), and ubiquitination (covalent connection with the protein ubiquitin). For a more detailed PTM normative dictionary recommended by UniProt, see http://www.uniprot.org/docs/ptmlist.

In some exemplary embodiments, the present invention provides a eukaryotic cell culture medium comprising basal medium or growth medium, as well as 5-methylthioadenosine and/or nicotinamide.

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

The terms nutrient, ingredient, and component are used interchangeably herein to refer to the components that make up the cell culture medium.

As used herein, the term basal medium can refer to any medium that can support cell growth. The basal medium may contain a number of ingredients, including amino acids, vitamins, organic and inorganic salts, and carbohydrate sources, each ingredient being present in an amount that supports in vitro culture of the cell. The medium may contain auxiliary substances such as buffers such as 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's Medium (DMEM), DME/F12, Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), Medium 199, RPMI 1640, F-10, F-12, α -minimal essential medium (α-MEM), Glasgow minimum essential medium (G-MEM), PF CHO (SAFC Biosciences), Iscove's modified Dulbecco's medium or combinations thereof, as well as other media known from the literature or commercially available.

As used herein, the term growth medium includes a medium containing one or more nutrients that can be added to the culture starting some time after inoculation. The nutrient medium can also be a combined feed containing

- 8 045820 compressing a base medium and at least one type of hydrolysate, for example a soy-based hydrolysate, a yeast-based hydrolysate or a combination of two types of hydrolysates. In addition, the culture medium may also include only basal medium, such as concentrated basal medium, or may include only hydrolysates or concentrated hydrolysates.

As used herein, the term eukaryotic cells may include individual cells, tissues, organs, insect cells, avian cells, mammalian cells, primary cells, continuous cell lines, stem cells and/or genetically engineered cells such as recombinant cells, expressing a heterologous polypeptide or protein. Some mammalian cells suitable for culture in cell culture media may be of human or non-human origin and may include primary epithelial cells (eg, keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal cells), commonly used cell lines and their strains (eg, fetal 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-C-I cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, cells LLC-PKi cells, PK cells (15), 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 cells, PK-15 cells or derivatives thereof), fibroblast cells from any tissue or organ (including, but not limited to, heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord ), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, 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, WI-26 cells, 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-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H cells /IOTI/2, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L cells, strain 2071 cells (mouse L), strain L-M cells (mouse L), L-MTK cells (mouse L), NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, Cn cells and Jensen cells, Sp2/0, NS0, NS1 cells or their derivatives).

As used herein, the term 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 C ₆ H ₆ N ₂ O.

As used herein, the term 5-methylthioadenosine may also be referred to as 5-deoxy-5'-methylthioadenosine, CAS number 2457-80-9, SpH ₁₅ ^O^, MTA, MeSAdo, NSC 335422, vitamin L2, 5-methylthioadenosine salts or 5-methylthioadenosine esters. Non-limiting examples of salts compatible with 5-methylthioadenosine, for example, include acid addition salts. Acid addition salts can be acid addition salts of inorganic and organic acids, for example hydrochlorides, sulfates, nitrates, carbonates, phosphates, formates, oxalates, citrates, ascorbic acid, methanesulfonic acid, 1,4-butanesulfonate, 1,5-pentanesulfonate , as well as p-toluenesulfonate salts.

In some exemplary embodiments, the present invention provides a method for producing a protein, comprising culturing eukaryotic cells having a protein-encoding nucleic acid in a cell culture production medium.

The cell culture medium or cell culture production medium may be supplemented with a medium enriched with components such as nutrients and amino acids that are consumed during the cell culture production phase.

As used herein, the term cell culture production medium may include cell culture medium for use during the cell culture production phase.

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

As used herein, the term serum-free medium includes cell culture medium that does not contain animal serum, such as fetal bovine serum. The serum-free medium 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).

- 9 045820

In some exemplary embodiments, the cell culture production medium or cell culture medium may be a protein-free medium of animal origin.

As used herein, the term animal protein-free medium may refer to a medium that does not contain proteins and protein components from higher multicellular non-plant eukaryotes (i.e., vertebrates) that possess the secondary, tertiary, and quaternary structure characteristic of proteins found in nature. This medium does not contain proteins such as albumin, transferrin, insulin and other growth factors. However, animal proteins and protein components are different from non-animal proteins, small polypeptides and oligopeptides derived from plants (usually 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 a protein medium of animal origin, the medium will contain animal proteins secreted or secreted by the cells, including any recombinant proteins expressed by the genetically modified cells if such cells are cultured. Thus, the term animal protein-free medium, as well as biological materials and preparations derived therefrom, should not be construed to require the absence of proteins excreted or secreted by cells proliferating in the medium, but rather the term refers to the absence of direct addition to medium of animal proteins and protein components derived from animal sources, etc., produced recombinantly.

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

As used herein, the term chemically defined medium may include a medium consisting of pure ingredients in measured concentrations. The chemically defined medium may contain simple sugar as a source of carbon and energy, a source of inorganic nitrogen, various mineral salts and, if necessary, growth factors (purified amino acids, vitamins, purines and pyrimidines). Various tissue culture media, including specific culture media, are commercially available, for example, any one or combination of the following cell culture media may be used: RPMI-1640, 199, RPMI-1641, Dulbecco's Modified Eagle's Medium (DMEM) ), minimum essential Eagle's medium, F-12K medium, Ham's F12 medium, Iscove's modified Dulbecco's medium, McCoy's 5A medium, Leibowitz's L-15 medium, and serum-free media such as EX-CELL™ 300 Series (JRH Biosciences, Lenexa , Kansas) and others.

In some exemplary embodiments, the protein production method may be a fed-batch culture method.

As used herein, the term fed-batch culture method refers to a method by which additional nutrients can be supplied to a fed-batch cell culture. For example, the method may include adding additional media in accordance with a specified recharge schedule over a predetermined period of time. As used herein, the term fed-batch cell culture refers to a cell culture in which cells and culture medium are initially supplied to the culture vessel and additional culture nutrients are supplied to the culture continuously or in discrete portions during culture, with or without periodic collection cells and/or products before stopping culture.

It should be understood that the present invention is not limited to any of the above eukaryotic cells, protein, basal medium, nutrient medium, cell culture medium, cell culture production medium, cell propagation method, protein expression method, protein collection method, protein administration method. a nucleic acid cell containing a protein coding sequence and a period of time for adding or supplementing components in the medium, and that any suitable medium may be selected by any suitable means.

As used herein, the terms include, includes, and including are intended to be non-limiting and are understood to mean include, includes, and including, respectively.

In some exemplary embodiments of the present invention, a medium for culturing eukaryotic cells is provided.

In some exemplary embodiments of the present invention, a method for producing protein is provided.

Some exemplary embodiments of the present invention provide a method for culturing eukaryotic cells to increase protein production.

In some exemplary embodiments, the cell culture medium may contain 5-methylthioadenosine at a concentration of at least about 0.05 nM, at least about nM, at least about 2 nM, at least about 3 nM, at least about nM , at least about 5 nM, at least about 6 nM, at least about nM, at least about 8 nM, at least about 9 nM, at least about

- 10045820 nM, at least about 15 nM, at least about 20 nM, at least about nM, at least about 30 nM, at least about 35 nM, at least about nM, at least about 45 nM, at least about 50 nM, at least about nM, at least about 60 nM, at least about 65 nM, at least about nM, at least about 75 nM, at least about 80 nM, according to at least about 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, at least 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, or at least about 300 nM.

In some exemplary embodiments, the cell culture medium may contain 5-nicotinamide 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 nM, at least about 5 nM, at least about 6 nM, at least about 8 nM, at least about 9 nM, at least about 6 nM, at least about 15 nM, at least about 20 nM, at least about nM, at least about 30 nM, at least about 35 nM, at least about nM, at least about 45 nM, at least about 50 nM, at least about nM, at least about 60 nM, at least about 65 nM, at least about nM, at least about 75 nM, at least about 80 nM, at least about 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, at least 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, 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, at least 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, 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

- 11 045820

740 nM, at least about 750 nM, at least about 760 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, 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.

In some exemplary embodiments, the cell culture medium may have a pH of from about 6.5 to about 8.0. In some 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.

In some exemplary embodiments, eukaryotic cells are grown at a temperature of from about 25 to about 40°C. In some exemplary embodiments, 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.

In some exemplary embodiments, the cell culture medium may contain at least about 10 nM 5-methylthioadenosine, wherein the titer of protein produced in the cell culture medium may be at least about 2% greater than in the other culture medium cells that does not contain at least about 10 nM 5-methylthioadenosine. In one aspect, the cell culture medium may contain 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, 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 aspect, the titer of protein produced in the cell culture medium may be greater by at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least by at least about 7%, by at least about 8%, by at least about 9%, by at least about 10%, by at least about 11%, by at least about 12%, by 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%.

In some exemplary embodiments, the cell culture medium may contain at least about 50 nM nicotinamide, wherein the titer of protein produced in the cell culture medium may be at least about 2% greater than in another cell culture medium, which does not contain at least about 50 nM nicotinamide. In one

- 12 045820 aspect, 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 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 aspect, the titer of protein produced in the cell culture medium may be greater by at least about 3%, at least about 4%, at least about 5%, at least about 6%, etc. 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 by about 13%, by at least about 14%, by at least about 15%, by at least about 16%, by at least about 17%, by at least about 18%, by 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%.

In some exemplary embodiments, the cell culture medium may include feeding the eukaryotic cells using enrichment medium containing 5-methylthioadenosine at a concentration of at least about 0.05 nM, at least about 1 nM, at least about 2 nM, at least 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 nM, at least about 12 nM, at least about 13 nM, at least about nM, at least about 15 nM, at least about 20 nM, at least about nM, at least about 30 nM, at least about 35 nM, at least about nM, or at least about 50 nM.

In some exemplary embodiments, the cell culture medium may include feeding the eukaryotic cells using an enriched medium containing an enriched medium containing nicotinamide at a concentration of at least about 1 nM, at least about 3 nM, at least about 3 nM, at least about 4 nM, at least about 6 nM, at least about 7 nM, at least about nM, at least about 9 nM, at least about 10 nM, at least about nM, at least about 20 nM, at least about 25 nM, at least about nM, at least about 35 nM, at least about 40 nM, at least about nM, at least about 55 nM, at least about 60 nM, at least about nM, at least about 70 nM, at least about 75 nM, at least about nM, at least about 85 nM, at least about 90 nM, at least about 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.

In some exemplary embodiments, the cell culture medium may include feeding the eukaryotic cells using enriched medium containing 5-methylthioadenosine at a concentration of at least about 1 nM, wherein the titer of protein produced in the cell culture medium may be at least about 2% more than other cell culture media that do not contain at least about 10 nM 5-methylthioadenosine.

In one aspect, the enrichment 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, 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

- 13 045820 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 aspect, the titer of protein produced in the cell culture medium may be greater by at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least by at least about 7%, by at least about 8%, by at least about 9%, by at least about 10%, by at least about 11%, by at least about 12%, by 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%.

In some exemplary embodiments, the cell culture medium may include feeding the eukaryotic cells using enrichment medium containing nicotinamide at a concentration of at least about 5 nM, wherein the titer of protein produced in the cell culture medium may be at least about 2% more than other cell culture media that do not contain at least about 50 nM nicotinamide. In one aspect, 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 aspect, the titer of protein produced in the cell culture medium may be greater by at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least by at least about 7%, by at least about 8%, by at least about 9%, by at least about 10%, by at least about 11%, by at least about 12%, by 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%.

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

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

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

In some exemplary embodiments, the protein may be a recombinant protein, wherein the recombinant protein may be a decoy protein, an Fc moiety and a chimeric receptor binding molecule, a chimeric protein, an antibody, a monoclonal antibody, a polyclonal antibody, a human antibody, a bispecific antibody, a fragment antibody, nanobody, recombinant antibody chimera, cytokine, chemokine or peptide hormone.

In some exemplary embodiments, the protein may contain modifications, adducts, and other covalently linked moieties.

In some exemplary embodiments, the protein may contain a post-translational modification.

The sequential labeling of steps in the methods proposed herein by numbers and/or letters is not intended to limit the method or any embodiments thereof to the particular order stated.

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

The present invention will be better understood with reference to the following examples, which are given

- 14 045820 for a more detailed description of the invention. They are intended to be illustrative and should not be construed as limiting the scope of the invention.

Examples

To study the content of cell culture medium and its effect on protein production, a cell culture containing soybean hydrolysate was selected to generate VEGFR-binding protein 1.

Example 1.

The soybean hydrolyzate content was analyzed using nine different batches obtained from three different shipments - 1, 2 and 3. Samples from these three different shipments were sent to Metabolon (Durham, NC, USA). The soybean hydrolyzate content was analyzed by Metabolon using liquid chromatography-mass spectrometry. More than three hundred components in soybean hydrolysate have been measured. Only results scaled to the median are shown.

An orthogonal partial least squares (OPLS) multivariate analysis (MVA) model was used to study the effect of biochemicals on VEGFR-binding protein 1 (vascular endothelial growth factor receptor) titer. The study included 70 batches of soybean hydrolysates.

The relationship between components and titer was assessed by examining correlation and covariance. A positive relationship implies an increase in titer with an increase in the biochemical indicator, and a negative relationship implies a decrease in titer with an increase in the biochemical indicator. For the final assessment of the principal component titer, the values of R2X, R2Y and Q ² were found to be 0.315, 0.672 and 0.493, respectively. The components that demonstrated a positive relationship are shown in Table. 1, and the components that demonstrated a negative dependence are given in table. 2.

Table 1

Positive dependence

Nicotinamide

Lactate phenyllactate (PLA)

5-methylthioadenosine (MTA) indolelacate succinate alpha-hydroxyisocaproate

3-(4-hydroxyphenyl) lactate (HPLA) alpha-hydroxyisovalerate

2-Hydroxy-3-methylvalerate

table 2

Negative dependence nicotinate sucrose uracil phenylalanine valileucine maltose digalactosyl glycerol pantothenate (vitamin B5) xanthine serine

The MVA result normalized per unit length is shown in Fig. 1. Based on these data from Example 2, nicotinamide and 5-methylthioadenosine were selected as positive markers for further analysis.

Example 2.

The effect of 5-methylthioadenosine on the production of recombinant protein (VEGFR-binding protein 1) was studied by examining the protein titer (g/L) at different concentrations of 5-methioadenosine present in the cell culture medium.

Enriched medium (soybean hydrolysate) containing 5-methylthioadenosine was added to the cell culture medium. Fig. 2 demonstrates the correlation between the concentration of 5-methylthioadenosine present in soybean hydrolysate added to cell culture medium with titer

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Claims (10)

VEGFR binding protein 1. As can be seen in FIG. 2, the regression line relating to the variables (N=70) extrapolates that the titer varied linearly with the concentration of 5-methylthioadenosine in soybean hydrolyzate added to the cell culture medium, suggesting that a higher titer may be obtained by increasing the concentration of 5-methylthioadenosine in the cell culture medium. Example 3. Based on the correlation data obtained by studying the effect of 5methylthioadenosine concentrations in enriched medium (soybean hydrolysate) added to the cell culture medium (Example 2, Fig. 2), the optimal concentration of 5-MTA in cell culture was assessed. As can be seen in FIG. 3, the regression line relating to the variables (N=70) extrapolates that the titer can vary linearly with the concentration of 5-methylthioadenosine in the cell culture medium, demonstrating that as the concentration of 5-methylthioadenosine in the cell culture medium increases cells, a higher titer can be obtained. Example 4. The effect of nicotinamide on the recombinant protein titer (VEGFR-binding protein 1) was also studied by examining the protein titer (g/L) at different concentrations of nicotinamide present in the cell culture medium. Enriched medium (soybean hydrolysate) containing nicotinamide was added to the cell culture medium at various concentrations. Fig. 4 shows the correlation between the concentration of nicotinamide present in soy hydrolysate added to the cell culture medium with the titer of VEGFR binding protein 1. As seen in FIG. 4, the regression line relating to the variables (N=70) extrapolated to indicate that the titer varied linearly with the concentration of nicotinamide in the soybean hydrolysate, suggesting that by increasing the concentration of nicotinamide in the cell culture medium, a higher titer. Example 5. Based on the correlation data obtained by studying the effect of nicotinamide concentration in enriched medium (soybean hydrolysate) added to the cell culture medium (Example 4, Fig. 4), the optimal concentration of nicotinamide in cell culture was assessed. As can be seen in FIG. 5, the regression line relating to the variables (N=70) extrapolates that the titer may vary linearly with the concentration of nicotinamide in the cell culture medium, suggesting that as the concentration of nicotinamide in the cell culture medium increases, there may be a higher titer was obtained. CLAIM 1. Medium for culturing eukaryotic cells expressing aflibercept, containing: basal medium or nutrient medium; and 5-methylthioadenosine. 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. Cell culture medium according to claim 1, further containing one or more acids selected from lactic acid, phenyllactic acid, indolylacetic 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. 4. The cell culture medium of claim 2, 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 contain at least about 10 nM 5- methylthioadenosine. 5. Cell culture medium according to claim 1, in which 5-methylthioadenosine may be in the form of its salts or esters. 6. 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. 7. Cell culture medium according to claim 1, wherein the medium does not contain protein of animal origin. 8. Cell culture medium according to claim 1, wherein the medium is a serum-free medium. 9. Cell culture medium according to claim 1, wherein the medium is a medium of a certain chemical composition. 10. A method for culturing eukaryotic cells to increase the level of aflibercept production, including the following steps: -