Cell culture method for producing glycoproteins

Abstract

To provide a method for screening batches of soy hydrolysate for a desired amount of a component thereof, such as ornithine or putrescine, and selecting only those batches of soy hydrolysate that have a desired amount of such component.SOLUTION: The present disclosure also sets forth methods for culturing cells in media supplemented with selected batches of soy to produce more consistent, high quality lots of a protein of interest. Further, the present disclosure provides a plurality of protein preparations that have each been produced by culturing cells in media supplemented with separate batches of soy hydrolysate containing a desired amount of ornithine or putrescine, whereby each batch of protein produced exhibits improved quality of the protein of interest or amount of quality protein produced.SELECTED DRAWING: Figure 6A

Description

Technical Field

[0001] Incorporation of Sequence Listing The content of the submitted text file named "REGE009P02US_SeqList.txt", created on February 1, 2018 and having a size of 11.2 KB, is incorporated herein by reference in its entirety.

[0002] The present invention relates to methods for culturing cells and methods for producing recombinant proteins. In particular, the present invention relates to methods for culturing cells in a soy hydrolysate-containing medium to achieve consistent production of high-quality recombinant proteins.

Background Art

[0003] Cell culture media containing protein hydrolysates, such as soy hydrolysate, are commonly used in the production of recombinant proteins from cultured cells. However, protein hydrolysates may contain compounds that adversely affect cell growth or recombinant protein production. Despite these drawbacks, protein hydrolysates are widely used as supplements in cell culture.

[0004] Human biotherapeutic agents (biopharmaceuticals) are generally produced in mammalian cell culture. However, the quality and performance of biotherapeutic agents are highly dependent on the manufacturing process. Tebbey, P. and Declerck, P., Generics and Biosimilars Initiative Journal (2016) 5:2, pp. 70-73, which is incorporated herein for the production of biological drugs having consistent glycosylation. Changes to cell culture methods for the production of glycoproteins can alter glycosylation patterns, the presence of acidic species (e.g., sialic acid) or the amount of glycans on the protein (ibid.). Such variation increases heterogeneity of protein isoforms in the resulting protein production, which can change the stability, efficacy or immunogenicity of the biotherapeutic agent and ultimately lead to rejection of a protein lot.

[0005] Therefore, cell culture methods that eliminate lot-to-lot variability in the yield and composition of drug products are highly desirable. This disclosure identifies certain components in plant protein hydrolysates (e.g., soybean hydrolysates) that vary between batches and can alter the composition and yield of high-quality glycoproteins produced in cultures using soybean hydrolysates. This disclosure addresses the need for improved cell culture methods by screening batches of plant protein hydrolysates and selecting those batches containing components at desirable concentrations for use in the production of biopharmaceuticals. [Overview of the Initiative]

[0006] This disclosure is based in part on the finding that the concentration of ornithine or putrescine in a batch of soy hydrolysates affects the quality and composition of the proteins produced in cell cultures using soy hydrolysates. This disclosure also provides that cells cultured in a medium containing soy hydrolysates with certain concentrations of ornithine or putrescine produce a greater quantity of high-quality proteins that exhibit more consistent glycosylation patterns, glycan content, and sialic acid profiles across lots.

[0007] In one embodiment, the present invention relates to a method for culturing a population of cells expressing recombinant heterologous glycoproteins in a cell culture medium containing a soybean hydrolysate, wherein the soybean hydrolysate contains ≤0.067% (w / w) ornithine or putrescine, in order to produce recombinant heterologous glycoproteins.

[0008] In some embodiments, the method includes culturing a population of cells expressing recombinant heterologous glycoproteins in a cell culture medium containing a soy hydrolysate containing less than 0.67 milligrams (mg) of ornithine (w / w) per gram (g) of soy, or about 0.003% to 0.067% (w / w) of ornithine. In one embodiment, the culture medium contains ≤5 mg / L of ornithine, or about 0.6 to 3 mg / L of ornithine. In some embodiments, the cell population is obtained by clonal proliferation of cells expressing recombinant heterologous glycoproteins.

[0009] In one embodiment, the present invention relates to a method for producing glycoproteins. In one embodiment, the method comprises the step of culturing a population of cells expressing recombinant heterologous glycoprotein in a culture medium containing a soy hydrolysate containing less than 0.67 milligrams (w / w) of putrescine per gram (g) of soy, or about 0.003% to 0.067% (w / w) of putrescine. In one embodiment, the culture medium contains ≤ 5 mg / L of putrescine or about 0.6 to 3 mg / L of putrescine. In some embodiments, the population of cells is obtained by clonal proliferation of cells expressing recombinant heterologous glycoprotein.

[0010] In one embodiment, the glycoprotein is a trap molecule, such as lilonacept (e.g., IL1-trap, disclosed in U.S. Patent No. 6,927,004), aflibercept (e.g., VEGF-trap, disclosed in U.S. Patent No. 7,087,411), convert (e.g., VEGF-trap, disclosed in U.S. Patents No. 7,750,138 and 8,216,575), and etanercept (e.g., TNF-trap, disclosed in U.S. Patent No. 5,610,279). In one embodiment, ≥10% (w / w) of the total amount of all N-glycan species in the glycoprotein is A1 N-glycan.

[0011] In one embodiment, the present invention relates to a method for producing glycoproteins. In another embodiment, the present invention relates to a method for using soy hydrolysates in the production of glycoproteins. In yet another embodiment, the present invention relates to a method for selecting soy hydrolysates for use in producing glycoproteins by evaluating the quality of the produced glycoproteins. In one embodiment, the method comprises culturing cells expressing glycosylated proteins in cell culture medium to produce glycoproteins, purifying the glycosylated proteins, subjecting the purified glycosylated proteins to oligosaccharide fingerprint analysis, determining the relative amount of A1 N-glycans compared to the total amount of N-glycan species in the glycoproteins, and selecting soy hydrolysates that provide at least 10% (w / w) of A1 N-glycans compared to the total amount of N-glycan species in the glycoproteins.

[0012] In one embodiment, the method includes the steps of preparing a cell culture medium containing a soybean hydrolysate, culturing cells expressing a glycoprotein in the cell culture medium, purifying the glycosylated protein, subjecting the purified glycosylated protein to oligosaccharide fingerprint analysis, determining the relative amount of A1 N-glycan by comparing it with the total amount of N-glycan species of the glycoprotein, and then selecting a soybean hydrolysate that results in the production of a glycoprotein having at least 10% (w / w) of A1 N-glycan by comparison with the total amount of N-glycan species of the glycoprotein.

[0013] In one embodiment, the selected soybean hydrolysate contains ≤0.67 mg of ornithine (w / w) or about 0.003% to 0.067% (w / w) of ornithine per gram of soybean. In one embodiment, the culture medium contains ≤5 mg / L of ornithine or about 0.6 to 3 mg / L of ornithine.

[0014] In one embodiment, the selected soybean hydrolysis product contains ≤0.67 mg of putrescine (w / w) or about 0.003% to 0.067% (w / w) of putrescine per gram of soybean. In one embodiment, the culture medium contains ≤5 mg / L of putrescine or about 0.6 to 3 mg / L of putrescine.

[0015] In one embodiment, the present invention relates to a method for selecting a soy hydrolysate for use in producing glycoproteins by measuring the amount of ornithine or putrescine in the soy hydrolysate. In one embodiment, the method includes the steps of: measuring the amount of ornithine in the soy hydrolysate; selecting a soy hydrolysate having ≤0.67 mg of ornithine or about 0.003% to 0.067% (w / w) of ornithine per gram of soy; and combining the selected soy hydrolysate with further components to form a cell culture medium having ≤5 mg / L of ornithine or about 0.6 to 3 mg / L of ornithine. In one embodiment, the method includes the steps of: measuring the amount of putrescine in a potentially useful soybean hydrolysate; selecting a soybean hydrolysate having ≤0.67 mg of putrescine or about 0.003% to 0.067% (w / w) of putrescine per gram of soybean; and combining the selected soybean hydrolysate with further components to form a cell culture medium having ≤5 mg / L of putrescine or about 0.6 to 3 mg / L of putrescine.

[0016] In one embodiment, the present invention relates to a glycoprotein comprising A1 N-glycan and at least one other N-glycan species, wherein the relative amount of A1 N-glycan is at least 10% (w / w) of the total amount of N-glycans in the glycoprotein. In one embodiment, the relative amount of A1 N-glycan is about 10% to 17% (w / w).

[0017] In one embodiment, the glycoprotein also includes A2 N-glycan, A2F N-glycan, A1F N-glycan, NGA2F N-glycan, NA2G1F N-glycan, NA2 N-glycan, and NA2F N-glycan.

[0018] In one embodiment, the glycoprotein contains 8 to 65 moles of sialic acid per mole of glycoprotein. In one embodiment where the glycoprotein is lilonacept, one of the asparagine residues N37, N98, N418, and N511 of SEQ ID NO: 1 contains A1 N-glycan. In one embodiment where the glycoprotein is aflibercept, one of the asparagine residues N123 and N196 of SEQ ID NO: 2 contains A1 N-glycan.

[0019] In one embodiment, the relative amount of A1 N-glycan in a glycoprotein is determined by comparing the area under the peak of A1 N-glycan obtained from the oligosaccharide fingerprint of the glycoprotein obtained by capillary electrophoresis with the total area under the peak relative to the total N-glycan.

[0020] In one embodiment, a method is provided for producing a soy hydrolysate product with reduced amounts of ornithine or putrescine. In one embodiment, the method comprises the steps of enzymatically digesting a soy extract in a residue-free reactor, measuring the amount of ornithine in the soy hydrolysate product, and selecting a lot of soy hydrolysate containing ≤0.067% (w / w) ornithine or putrescine for use in a cell culture medium. In one embodiment, the method comprises the steps of enzymatically digesting a soy extract in a residue-free reactor, measuring the amount of putrescine in the soy hydrolysate product, and selecting a soy hydrolysate product having ≤0.067% (w / w) ornithine or putrescine for use in a cell culture medium.

[0021] The term "about" can be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clearly indicated by the context, all numerical values ​​provided herein are qualified with the term "about."

[0022] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. References cited herein are not admitted to be prior art to the claimed disclosure. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the following detailed description and the claims.

[0023] Any of the above aspects and embodiments can be combined with any other aspects or embodiments disclosed herein in the Summary of the Invention and / or the Detailed Description of the Invention sections.

[0024] This patent or application file contains at least one drawing made in color. Copies of this patent or patent application publication with color drawings will be provided by the authority upon request and payment of the required fee.

[0025] Various objects and advantages of the present invention, as well as a more complete understanding thereof, will become apparent and more readily appreciated by reference to the following Detailed Description of the Invention and the appended claims, taken in conjunction with the accompanying drawings. Brief Description of the Drawings

[0026] [Figure 1] It is a diagram showing an elution profile of chromatography of ninhydrin-derived amino acids. The X-axis shows the elution time (retention time) from a chromatography column, and the Y-axis shows absorbance at 570 nm. Panel A shows a batch that does not meet the criteria for production of an N-glycan mixture permitted by FDA standards. Panel B shows an acceptable amino acid analysis of a soy protein hydrolysate. The peak corresponding to ornithine is circled in both chromatograms. [Figure 2]Shows a capillary electropherogram of oligosaccharides released from a glycoprotein by peptide:N-glycosidase F (PNGase F) digestion. The X-axis indicates elution time from the capillary, and the Y-axis indicates absorbance or fluorescence intensity. Peaks are numbered from 1 to 21. Peak 1 represents N-glycan A2, peak 4 represents N-glycan A2F, peak 11 represents N-glycan A1, peak 14 represents N-glycan A1F, peak 16 represents N-glycan NGA2F, peak 19 represents N-glycan NA2G1F, peak 20 represents N-glycan NA2, and peak 21 represents N-glycan NA2F. [Figure 3] Is a dot blot diagram showing the relative amount of A1 N-glycan as a function of ornithine and citrulline concentrations in soybean protein hydrolysate. The X-axis indicates the concentration of citrulline or ornithine in mg / L. The Y-axis indicates the relative area of peak 11 corresponding to A1 N-glycan. [Figure 4] Is a correlation plot showing the negative correlation between ornithine concentration in soybean hydrolysate (lower right quadrant) and the relative amount of peak 11 (A1 N-glycan, upper left quadrant) in aflibercept. [Figure 5] Is a correlation plot showing (i) the negative correlation between ornithine concentration in soybean hydrolysate (lower left quadrant) and the final titer of rilonacept (upper right quadrant), and (ii) the positive correlation between ornithine concentration in soybean hydrolysate (lower left quadrant) and lactate accumulation in the medium (lower left quadrant). [Figure 6A] Is a set of graphs showing the amount of polyamine synthesized from CHO cell culture. It is shown as either IVCD × 106 cells-day / ml or titer (grams / ml) as a function of batch day under various conditions including control, high and low ornithine concentrations, putrescine, MFC and IPC. [Figure 6B] Is a table showing the experimental conditions for each study group presented in Figure 6A. Mode for Carrying Out the Invention

[0027] Please understand that the scope of this disclosure is not limited to such methods and conditions, as the specific methods and experimental conditions described may vary. Also, please understand that the terminology used herein is intended to describe, and not limit, specific embodiments.

[0028] Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as those generally understood by those skilled in the art to which this invention pertains. Any methods and materials similar to or equivalent to those described in this application may be used in the practice or testing of the invention, but certain specific methods and materials are described herein. Units, prefixes and symbols may be expressed in their standard, industry-recognized form. Numerical ranges described in the specification are in open brackets, meaning they include the number defining the range. Unless otherwise specified, the terms “a” or “an” should be interpreted as meaning “at least one of.”

[0029] Section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. Methods and techniques described herein are generally carried out in accordance with prior methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); Julio E. Celis, Cell Biology: A Laboratory Handbook, 2nd ed., Academic Press, New York, NY (1998); and Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1995). All publications referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0030] definition Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0031] The phrase "relative quantity" refers to the amount of a particular molecular species relative to the total amount of all molecular species of a common type. For example, the relative quantity of A1 glycan (i.e., (GlcNAc)2(Man)3(GlcNAc)2(Gal)2(SA)1) is calculated as the amount of A1 divided by the sum of the amounts of all N glycans. Relative quantities can be expressed as absolute mass versus mass (i.e., grams per gram) or as a percentage, i.e., %(w / w).

[0032] Ornithine is a non-protein-coding amino acid involved in the urea cycle, polyamine synthesis, and arginine metabolism. Ornithine is also known to affect the glycoform content of recombinant proteins. See PCT / US2014 / 069378. Ornithine is subject to the action of several enzymes. For example, ornithine decarboxylase catalyzes the conversion of ornithine to putrescine in the polyamine biosynthesis pathway. See Pegg A, J. of Biol. Chem. (2006) 281:21 pp. 14532. Furthermore, the conversion of ornithine to citrulline is catalyzed by ornithine transcarbamylase as part of the urea cycle. Ornithine metabolism occurs in both the cytosol and mitochondria of cultured cells. While the presence of putrescine or ornithine is considered important for the proliferation and productivity of cells cultured in synthetic media, its influence on key quality attributes of proteins produced by such cells remains unexplained.

[0033] Putrescine is a non-protein-coding amino acid, a polyamine, that is involved in the urea cycle. 12 (also known as 1,4-diaminobutane, with the chemical formula N2) is produced by the decarboxylation of ornithine and acts as a precursor to gamma-aminobutyric acid (γ-aminobutyric acid).

[0034] As used herein, “peptide,” “polypeptide,” and “protein” are used interchangeably throughout and refer to molecules containing two or more amino acid residues linked to each other by peptide bonds. Peptides, polypeptides, and proteins may also include modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation, alkylation, hydroxylation, and ADP-ribosylation of glutamate residues. Peptides, polypeptides, and proteins may be of scientific or commercial interest, including protein-based drugs (biotherapeutic agents). Among the many examples of peptides, polypeptides, and proteins are antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins can be produced by recombinant animal cell lines, such as mammalian cell lines, using cell culture methods.

[0035] As used herein, the terms “polynucleotide sequence” or “peptide sequence” refer to nucleic acid polymers encoding proteins of interest produced as the raw material for biopharmaceuticals, e.g., chimeric proteins (such as trap molecules), antibodies, or parts of antibodies (e.g., VH, VL, CDR3). Polynucleotide sequences can be produced by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, sequences without introns) and introduced into cells, where they may exist as episomes or be incorporated into the cell’s genome. Polynucleotide sequences may also be naturally occurring sequences introduced ectopically within the genome of a host cell. Peptide sequences may be heterologous, e.g., naturally occurring sequences from another organism, recombinant sequences, genetically modified sequences, or in particular sequences expressed under the control of a different promoter than the wild type, e.g., nucleotide sequences encoding human orthologues (where the host (producing) cell is a CHO cell).

[0036] The phrase “antigen-binding protein” includes proteins that have at least one CDR and are capable of selectively recognizing an antigen, i.e., can bind to an antigen with a KD of at least the micromolar range. Therapeutic antigen-binding proteins (e.g., therapeutic antibodies) often require a KD in the nanomolar or picomolar range. Typically, antigen-binding proteins contain two or more CDRs, e.g., two, three, four, five, or six CDRs. Examples of antigen-binding proteins include antibodies, antigen-binding fragments of antibodies, e.g., polypeptides containing the variable regions of the heavy and light chains of an antibody (e.g., Fab fragments, F(ab')2 fragments), and proteins containing the variable regions of the heavy and light chains of an antibody and additional amino acids derived from the constant regions of the heavy and / or light chains (e.g., one or more constant domains, i.e., one or more of the CL, CH1, hinge, CH2, and CH3 domains).

[0037] An "antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains, and two light (L) chains, interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain has a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The term “antibody” includes both glycosylated and nonglycosylated immunoglobulins of any isotype or subclass. The term “antibody” includes antibody molecules prepared, expressed, produced, or isolated by recombinant means, e.g., antibodies isolated from host cells transfected with a nucleotide sequence to express the antibody. The term “antibody” also includes bispecific antibodies, which include heterotetrameric immunoglobulins capable of binding to more than one epitope. Bispecific antibodies are generally described in U.S. Patent Application Publication 2010 / 0331527, which is incorporated herein by reference.

[0038] The term "antigen-binding portion" of an antibody (or antibody fragment) or the protein of interest refers to one or more fragments of the antibody or protein of interest that retain the ability to specifically bind to the antigen. Non-limiting examples of protein-binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of the VH domain (Ward et al., Nature (1989) 241:544-546); (vi) isolated CDRs; and (vii) scFv fragments consisting of two domains, VL and VH, of an Fv fragment linked by a synthetic linker so that the VL and VH regions pair to form a single protein chain that forms a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also encompassed under the term "antibody." For example, see Holliger et al., PNAS USA (1993) 90:6444-6448 and Poljak et al., Structure (1994) 2:1121-1123.

[0039] Furthermore, the antibody or its antigen-binding portion may also be part of a larger immunoadhesion molecule formed by the covalent or noncovalent association of the antibody or a portion of the antibody with one or more other proteins or peptides. Non-limiting examples of such immunoadhesion molecules include the use of the streptavidin core region for constructing tetrameric scFv molecules (Kipriyanov et al., Human Antibodies and Hybridomas (1995) 6:93-101) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags for producing divalent biotinylated scFv molecules (Kipriyanov et al., Mol.Immunol. (1994) 31:1047-1058). Portions of the antibody, e.g., Fab and F(ab')2 fragments, can be prepared from the whole antibody using conventional techniques, for example, by papain or pepsin digestion of the whole antibody. Furthermore, antibodies, some antibodies, and immunoadhesion molecules can generally be obtained using standard recombinant DNA methods known in the art (see Sambrook et al., 1989).

[0040] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of this disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo), for example, in the CDR, particularly in CDR3. As used herein, the term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinants, combinatorial human antibody libraries, antibodies isolated from animals transgenic for human immunoglobulin genes (e.g., mice) (see, e.g., Taylor et al. Nucl. Acids Res. (1992) 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal transgenic to the human Ig sequence is used, in vitro somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but which may not exist naturally in vivo within the human antibody germline repertoire.

[0041] An "Fc fusion protein" comprises part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, and these proteins are not otherwise found together in nature. The preparation of fusion proteins, including certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain), is described, for example, by Ashkenazi et al., PNAS USA (1991) 88:10535, Byrn et al., Nature (1990) 344:677, and Hollenbaugh et al., Current Protocols in Immunology (1992) Suppl. 4, pp. 10.19.1-10.19.11. In some embodiments, a "receptor Fc fusion protein" comprises a hinge region, followed by one or more extracellular domains of a receptor bound to the Fc portion containing the CH2 and CH3 domains of the immunoglobulin. In some embodiments, the Fc-fusion protein comprises two or more different receptor chains bound to one or more ligands.

[0042] In certain embodiments, the “Fc-fusion protein” is a “trap” molecule, which is a decoy receptor molecule containing two different receptor components and an antibody’s Fc portion that mimic the binding domain of the corresponding endogenous receptor. Non-limiting examples of trap molecules include IL-1 traps (e.g., lilonacept, which contains an IL-1RAcP ligand-binding domain fused to the IL-1R1 extracellular domain (which then fuses to the Fc of hIgG1)) (e.g., SEQ ID NO: 1) (see U.S. Patent No. 6,927,004), or VEGF traps (e.g., aflibercept, which contains Ig domain 2 of VEGF receptor Flt1 fused to Ig domain 3 of VEGF receptor Flk1 (which then fuses to the Fc of hIgG1); see U.S. Patents No. 7,087,411 and 7,279,159; also see U.S. Patent No. 5,610,279 for etanercept (TNF trap)).

[0043] "Glycosylation" includes the formation of glycoproteins in which an oligosaccharide is bound to either an asparagine (Asn) residue (i.e., N-linked) or a serine (Ser) or threonine (Thr) residue (i.e., O-linked) side chain of a protein. "Glycoprotein" includes any protein containing either an O-linked glycan or an N-linked glycan. The glycan may be linear or branched, and may be a homo or heteropolymer of monosaccharide residues. N-linked glycosylation is known to initiate primarily in the endoplasmic reticulum, while O-linked glycosylation has been shown to initiate in either the ER or the Golgi apparatus. The term "N-glycan" is used interchangeably with "N-linked oligosaccharide." The term "O-glycan" is used interchangeably with "O-linked oligosaccharide."

[0044] "N-glycan proteins" include proteins that contain or can accept N-linked oligosaccharides. N-glycans may consist of N-acetylgalactosamine (GalNAc), mannose (Man), fucose (Fuc), galactose (Gal), neuraminic acid (NANA), and other monosaccharides, but N-glycans typically have a common core pentasaccharide structure containing three mannose and two N-acetylglucosamine (GlcNAc) sugars. Proteins having a continuous amino acid sequence, Asn-X-Ser or Asn-X-Thr (where X is any amino acid other than proline), can provide a binding site for N-glycans.

[0045] N-glycans include N-linked oligosaccharides listed in Table 1. The abbreviations for the listed oligosaccharides are used herein as simplified names for describing oligosaccharides. For example, A1 N-glycan contains arginine linked to an oligosaccharide consisting of (SA)(Gal)2(GlcNAc)2(Man)3(GlcNAc)3. [Table 1]

[0046] screening "Hydrolyzed products" are composite materials derived from the hydrolysis of plant materials, animal materials, whey, yeast, etc. The term "hydrolyzed product" is used interchangeably with "protein hydrolyzed product." "Plant hydrolyzed products" (plant protein hydrolyzed products) are hydrolyzed plant materials, such as rice flour, wheat flour, corn flour, and soybean flour. Protein hydrolyzed products can be produced by three common methods: acid hydrolysis, alkali hydrolysis, and enzymatic hydrolysis. For biological applications, including the manufacture of biotherapeutic agents, protein hydrolyzed products are usually produced by enzymatic hydrolysis. For example, soybean hydrolyzed products produced by pepsin digestion can be called "soybean peptones," and yeast hydrolyzed products produced by trypsin digestion can be called "yeast tryptones." Franek et al., Biotechnol. Prog. 16(5):688-92 (2000) are incorporated herein by reference to plant protein hydrolyzed products and methods for producing them.

[0047] In some embodiments, the hydrolyzed product in question is a plant hydrolyzed product. In specific embodiments, the protein hydrolyzed product in question is a soybean hydrolyzed product. "Soybean hydrolyzed product" is an enzymatically digested soybean product derived from coarse soybeans and is largely chemically undetermined. Generally, soybean hydrolyzed products consist of aggregates of amino acids, proteins, carbohydrates, minerals, and vitamins. Soybean hydrolyzed products can be used, for example, in high-concentration solutions (e.g., HyClone). (商標) HyQ soy hydrolysis product solution) or powder (e.g., Sigma Aldrich® S1674 (Amisoy) (商標)Soy hydrolysates are plant-derived protein hydrolysates that are commercially available in the form of soy protein hydrolysates. As used herein, a “batch” or “lot” of soy hydrolysates refers to a production quantity of soy hydrolysates resulting from the hydrolysis of soybean grains. For example, each hydrolysis method may result in a unique “batch” or “lot” of soy hydrolysates having varying concentrations of components, such as vitamins, amino acids, peptides, and sugars. Soy hydrolysates are commonly used in conjunction with animal protein-free cell culture media for the growth of mammalian cell lines during the production of commercially available biotherapeutic agents, such as antibodies. More specifically, soy hydrolysates are added to the cell culture medium before or during cell inoculation. The cells are then cultured in the hydrolysate-containing medium until harvested. Due to the undetermined nature of soy hydrolysates, batches of soy hydrolysates can vary between batches (or lots), which can lead to variability in the commercial production of biotherapeutic agents.

[0048] This disclosure has confirmed that the concentration of certain components in a batch of soy hydrolysates affects the quality and composition of proteins produced in cell cultures using soy hydrolysates. This disclosure provides a method for screening batches of soy hydrolysates to select batches of certain soy hydrolysates containing desirable amounts of components, such as ornithine, putrescine, citrulline, arginine, or combinations thereof.

[0049] In certain embodiments, the screening method includes measuring the amount of ornithine or putrescine in at least a portion of a batch of soybean hydrolysate (i.e., a sample). In specific embodiments, the soybean hydrolysate sample is weighed and a portion thereof is dissolved to a desired concentration. In some embodiments, the soybean hydrolysate solution is then diluted in a solvent to a second desired concentration (e.g., 1 g / L to 25 g / L), and the composition of the resulting soybean hydrolysate solution is then determined.

[0050] In some embodiments, the measurement step uses an appropriate method for determining the molecular composition of the soybean hydrolysate sample, including, for example, colorimetric detection performed after the post-column ninhydrin reaction, or chromatography for the eluted ninhydrin-positive compound, such as HPLC or UPLC, and the unit used to express the measured amount of each component (e.g., ornithine or putrescine) may be any appropriate unit (e.g., micromoles / L, mg / L or g / L). In some embodiments, the measurement of the amount of ornithine or putrescine includes measuring the concentration of ornithine in the sample, or measuring the total amount of ornithine in the soybean hydrolysate sample. However, the amount of ornithine or putrescine is measured, and any unit is used to express the measured amount, and the concentration of ornithine or putrescine in a selected batch of soybean hydrolysate is 0.67 mg or less of ornithine or putrescine per gram of soybeans.

[0051] In one embodiment, a batch sample of soybean hydrolysate is obtained, and the ornithine or putrescine content of the sample is measured by amino acid chromatography using an ion-exchange column with post-columnar ninhydrin detection. More specifically, in a specific embodiment, the screening method includes acid hydrolysis of the soybean hydrolysate sample and reconstitution in a sample buffer. The hydrolyzed sample is then subjected to high-speed cation exchange separation using, for example, a sulfonated polystyrene resin (Dowex 50) column, followed by post-column derivatization, which enables highly sensitive detection of individual amino acids in the sample. For example, see Moore and Stein. J. Biol. Chem. (1954) Vol. 211 pp. 907-913, Nemkov, et al., Amino Acids 2015 Nov; 47(11): 2345-2357, and Wahl and Holzgrabe, “Amino acid analysis for pharmacopoeial purposes,” Talanta 154: 150-163, 1 July 2016. Following post-column color development with ninhydrin reagent, absorbance is measured in the purple range of ninhydrin, e.g., 570 nm. Data acquisition is achieved using chromatography software (e.g., EZChrom Elite version 3.1.5b chromatography software for Hitachi) to provide quantitative chromatograms showing micromoles / L, mg / L, or g / L per amino acid.

[0052] Those skilled in the art will recognize that other methods for identifying and measuring amino acids in a sample composition can be used according to the methods of this disclosure, for example, pre-column derivatization chromatography or reversed-phase liquid chromatography methods using liquid chromatography and mass spectroscopy.

[0053] In certain embodiments, liquid chromatography-mass spectrometry is used to screen soybean hydrolysis product samples. For example, a batch sample of soybean hydrolysis product can be obtained as described herein and subjected to a chromatographic run or a series of chromatographic runs using a high-performance liquid chromatography (HPLC) system, such as an Agilent 1100 or Agilent 1200SL. Mass spectrometry analysis can be performed to provide high-resolution quantitative data describing the composition of the soybean hydrolysis product sample being measured.

[0054] In some embodiments, the Disclosure provides a method comprising screening batches of soy hydrolysate for desired amounts of components, e.g., ornithine, putrescine, and / or citrulline, and selecting batches of soy hydrolysate having desired amounts of such components. For example, a sample containing a portion of a batch of soy hydrolysate powder can be screened as described above and compared to an amino acid standard profile prepared under the same conditions as the run of the sample. As shown in Figures 1A-1B, the resulting chromatogram(s) will provide the concentration of each amino acid component present in the soy hydrolysate sample (e.g., micromoles / L per amino acid, mg / L per amino acid, or g / L per amino acid). Analysis of the chromatogram facilitates the identification of batches (i.e., samples) of soy hydrolysate containing desired concentrations of components, e.g., ornithine, putrescine, and / or citrulline. Then, as described herein, batches of soy hydrolysate containing desired amounts of specific one or more components are selected for further use, e.g., in cell culture. Panel A of Figure 1 shows batches rejected after amino acid identification. Panel B of Figure 1 shows an example of a run of an acceptable batch of soy hydrolysate under identical conditions. The amino acid peak corresponding to ornithine is circled in both figures. The concentration of ornithine or putrescine can be determined by creating a calibration curve and interpolating the ornithine or putrescine concentration of the sample. Alternatively, the relative amount of ornithine or putrescine can be determined by determining the area under the curve for the ornithine or putrescine peak and dividing it by the sum of the areas under the peaks for all amino acids, or by comparing the peak area to a standard.

[0055] In certain embodiments, the desired concentration of a component of the selected soy hydrolysate (e.g., ornithine or putrescine) is 5 mg / L or less. In one embodiment, the desired concentration of ornithine or putrescine in a batch of the selected soy hydrolysate ranges from 0.5 mg / L to 5.0 mg / L or from 0.5 mg / L to 2.0 mg / L. In other embodiments, the concentration of ornithine or putrescine in a selected batch of soy hydrolysate ranges from 0.5 mg / L to 4.5 mg / L, 0.5 mg / L to 4.0 mg / L, 0.5 mg / L to 3.5 mg / L, 0.5 mg / L to 3.0 mg / L, 0.5 mg / L to 2.5 mg / L, 0.5 mg / L to 2.0 mg / L, 0.5 mg / L to 1.5 mg / L or from 0.5 mg / L to 1.0 mg / L. In some embodiments, the concentration of ornithine or putrescine in a selected batch of soy hydrolysate ranges from 1.0 mg / L to 5.0 mg / L, 1.5 mg / L to 5.0 mg / L, 2.0 mg / L to 5.0 mg / L, 2.5 mg / L to 5.0 mg / L, 3.0 mg / L to 5.0 mg / L, 3.5 mg / L to 5.0 mg / L, 4.0 mg / L to 5.0 mg / L, or 4.5 mg / L to 5.0 mg / L.

[0056] In specific embodiments, the desired concentrations of ornithine or putrescine in a batch of soybean hydrolysate are at least 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, and 2.6 mg The concentrations are 1 / L, 2.7 mg / L, 2.8 mg / L, 2.9 mg / L, 3.0 mg / L, 3.1 mg / L, 3.2 mg / L, 3.3 mg / L, 3.4 mg / L, 3.5 mg / L, 3.6 mg / L, 3.7 mg / L, 3.8 mg / L, 3.9 mg / L, 4.0 mg / L, 4.1 mg / L, 4.2 mg / L, 4.3 mg / L, 4.4 mg / L, 4.5 mg / L, 4.6 mg / L, 4.7 mg / L, 4.8 mg / L, 4.9 mg / L, or 5.0 mg / L of ornithine or putrescine.

[0057] In other embodiments, the desired concentration of ornithine or putrescine in a batch of soybean hydrolysate is 0.67 mg or less of ornithine per gram of soybeans. In yet another embodiment, the desired concentration of ornithine or putrescine in a batch of soybean hydrolysate is 0.27 mg or less of ornithine per gram of soybeans. In yet another embodiment, the desired concentration of ornithine or putrescine in a batch of soybean hydrolysate is 0.24 mg or less of ornithine or putrescine per gram of soybeans. In some embodiments, the desired concentration of ornithine or putrescine in a batch of soybeans is 0.067 mg to 0.67 mg of ornithine or putrescine per gram of soybeans. In yet another embodiment, the desired concentration of ornithine or putrescine in a batch of soybean hydrolysate falls within the range of 0.067 mg to 0.27 mg of ornithine per gram of soybeans. In yet another embodiment, the desired concentration of ornithine or putrescine in a batch of soybean hydrolysate falls within the range of 0.067 mg to 0.24 mg of ornithine or putrescine per gram of soybeans.

[0058] In one embodiment, the relative amount of ornithine or putrescine in the selected soy hydrolysate product by mass (%w / w) (w / w = mass of ornithine or putrescine / total mass of hydrolysate product) is ≤0.067%, for example, 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, The percentages are 0.0008%, 0.0009%, 0.001%, 0.0015%, 0.002%, 0.0025%, 0.003%, 0.0035%, 0.004%, 0.0045%, 0.005%, 0.0055%, 0.006%, 0.0061%, 0.0062%, 0.0063%, 0.0064%, 0.0065%, and 0.0066% (all w / w).

[0059] In one embodiment, plant protein hydrolysis products are selected based on the production of glycoproteins having specific quality attributes. The quality of the glycoprotein can be determined by evaluating the level of one or more specific N-glycans on the glycoprotein, or by evaluating the level of one or more specific sugars or a combination of attributes on the glycoprotein. For example, a specific fucose level, e.g., a glycoprotein having 5 to 10 moles of fucose per mole of glycoprotein, may be a quality attribute criterion, or a specific sialic acid level, e.g., 5 to 15 moles of sialic acid per mole of glycoprotein, or a specific ratio of A1 N-glycans to the total amount of N-glycans, e.g., 10 to 17% (w / w), may be considered to have essential quality attributes. Plant protein hydrolysis products that enable the production of the glycoprotein may be considered selectable.

[0060] In one embodiment, plant protein hydrolysates are selected by producing glycoproteins in cells cultured in a medium containing a potentially selectable plant protein hydrolysate (e.g., soybean hydrolysate), purifying the glycoproteins, subjecting the glycoproteins to oligosaccharide fingerprinting, determining the relative amount of A1 N-glycans by calculating the area under the peak related to A1 N-glycans and dividing that value by the total area under the peak of all N-glycans, and selecting plant protein hydrolysates that enable the production of glycoproteins having a relative amount of A1 N-glycans of ≥10%, ≥10.5%, 10-17%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, or 18%.

[0061] cell culture This disclosure provides a method for culturing cells expressing a target protein in a cell culture medium using a selected batch of the above-described soy hydrolysate. This disclosure is the first to discover that the use of a selected batch of soy hydrolysate containing 5.0 mg / L or less ornithine in a cell culture medium reduces lot-to-lot variability and improves the quality of the protein product. This disclosure is the first to discover that the use of a selected batch of soy hydrolysate containing 5.0 mg / L or less putrescine in a cell culture medium reduces lot-to-lot variability and improves the quality of the protein product.

[0062] "Cell culture" or "culture" refers to the proliferation and expansion of cells outside of a multicellular organism or tissue. Appropriate culture conditions for mammalian cells are known in the art. See, for example, *Animal cell culture: A Practical Approach*, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or while attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shaking flasks, or agitated tank bioreactors, operated in batch, fed-batch, continuous, semi-continuous, or perfusion modes, with or without microcarriers, are available for mammalian cell culture. Cell culture medium or high-concentration feed medium can be added to the culture during culture, continuously or at intervals. For example, the culture may be fed once per day, every other day, every three days, or when the concentration of a particular medium component being monitored falls outside the desired range.

[0063] As used herein, the terms “cell culture medium,” “culture medium,” “cell culture medium,” or “culture medium” refer to any nutrient solution used to grow cells, for example, animal or mammalian cells, and generally providing at least one or more of the following: an energy source (usually in the form of carbohydrates such as glucose); one or more of all essential amino acids, and generally the 20 basic amino acids, plus cysteine; vitamins and / or other organic compounds, typically required in low concentrations; lipids or free fatty acids; and trace elements, typically required in very low concentrations, usually in the micromolar range, such as inorganic compounds or naturally occurring elements. In some embodiments, the cell culture medium is formed by combining soy or other plant protein hydrolysates with further components.

[0064] As used herein, “further ingredients” includes, but is not limited to, water, energy sources, one or more of all essential amino acids, and generally 20 basic amino acids, plus cysteine; and one or more of the components of cell culture media, including vitamins and / or other organic compounds, lipids or free fatty acids, and trace elements, typically required in low concentrations.

[0065] In specific embodiments, the cell culture medium is supplemented with a certain amount of a selected batch of soy hydrolysate. In certain embodiments, the cell culture medium is supplemented with a selected batch of soy hydrolysate in amounts ranging from approximately 0.5 g / L to approximately 25 g / L. In some embodiments, the cell culture medium is supplemented with approximately 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L, 11.5 g / L, 12 g / L, 12.5 g / L, 13 g / L, Selected batches of soy hydrolysate in concentrations of 13.5g / L, 14g / L, 14.5g / L, 15g / L, 15.5g / L, 16g / L, 16.5g / L, 17g / L, 17.5g / L, 18g / L, 18.5g / L, 19g / L, 19.5g / L, 20g / L, 20.5g / L, 21g / L, 21.5g / L, 22g / L, 22.5g / L, 23g / L, 23.5g / L, 24g / L, 24.5g / L, or approximately 25g / L are replenished.

[0066] In one embodiment, the concentrations of ornithine or putrescine in the cell culture medium after the addition of plant protein hydrolysates are ≤5 mg / L, 0.6-3 mg / L, 0.01 mg / L, 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.05 mg / L, 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L, 0.010 mg / L, 0.015 mg / L, 0.02 mg / L, 0.025 mg / L, 0.03 mg / L, 0.035 mg / L, 0.04 mg / L, 0.045 mg / L, 0.05 mg / L, 0.055 mg / L, 0.06 mg / L, 0.065 mg / L, and 0.07 mg / L. g / L, 0.075mg / L, 0.08mg / L, 0.085mg / L, 0.09mg / L, 0.095mg / L, 0.1mg / L, 0.15mg / L , 0.2mg / L, 0.25mg / L, 0.3mg / L, 0.35mg / L, 0.4mg / L, 0.45mg / L, 0.5mg / L, 0.55mg / L , 0.6mg / L, 0.65mg / L, 0.7mg / L, 0.75mg / L, 0.8mg / L, 0.85mg / L, 0.9mg / L, 0.95mg / L , 1mg / L, 1.5mg / L, 2mg / L, 2.5mg / L, 3mg / L, 3.5mg / L, 4mg / L, 4.5mg / L or 5mg / L.

[0067] In one embodiment, the cultured cells are cells of a cell line capable of producing biotherapeutic proteins. Non-limiting examples of cell lines used to produce protein biotherapeutic agents include, in particular, primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, and chicken embryo cells. In one embodiment, the cell line is one or more CHO cell lines or several specific CHO cell variants optimized for large-scale protein production, such as CHO-K1 or CHO-K1-derived EESYR® (enhanced expression and stability regions) cells (U.S. Patent No. 7,771,997).

[0068] In one embodiment, the cultured cells expressing a heterologous glycoprotein are a population of cells obtained by clonal proliferation of cells expressing the glycoprotein (i.e., progenitor cells), where the glycoprotein is a complex multi-subunit protein such as an antibody. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100% of the constituent cells of the population of cells obtained from or derived from progenitor cells by clonal proliferation contain polynucleotides encoding the glycoprotein and express the glycoprotein.

[0069] Mammalian cells, such as CHO cells, can be cultured in small cell culture vessels, for example, a 125 ml vessel with about 25 ml of medium, a 250 ml vessel with about 50-100 ml of medium, or a 500 ml vessel with about 100-200 ml of medium. Alternatively, the culture may be on a larger scale, for example, a 1000 ml vessel with about 300-1000 ml of medium, a 3000 ml vessel with about 500-3000 ml of medium, an 8000 ml vessel with about 2000-8000 ml of medium, or a 15000 ml vessel with about 4000-15000 ml of medium. Cultures for production (i.e., generative cell cultures) may include 10,000 L or more of medium. Large-scale cell cultures or "generative cell cultures" for clinical production of protein therapeutics, etc., are typically maintained for several days or even weeks while the cells produce the desired protein(s). During this period, the culture can be supplemented with a high-concentration feed medium containing components consumed during the culturing process, such as nutrients and amino acids.

[0070] In certain embodiments, high-concentration feed media are used. High-concentration feed media can be based on any cell culture medium formulation. Such high-concentration feed media may contain many of the components of the cell culture mediums described herein, for example, in their usual useful amounts of about 5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×, 600×, 800×, or even about 1000×. High-concentration feed media are often used in fed-batch culture methods.

[0071] In some embodiments, cell culture media are supplemented with “point-of-use additives,” also known as additives, point-of-use components, or point-of-use chemicals, during the process of cell proliferation or proteinogenesis. Point-of-use additives include one or more of the following: growth factors or other proteins, buffers, energy sources, salts, amino acids, metals, and chelating agents. Other proteins include transferrin and albumin. Growth factors, including cytokines and chemokines, are generally known in the art and are known to stimulate cell proliferation, or in some cases, cell differentiation. Growth factors are typically proteins (e.g., insulin), small peptides, or steroid hormones, such as estrogen, DHEA, and testosterone. In some cases, growth factors may be non-natural chemicals that promote cell proliferation or proteinogenesis, such as tetrahydrofolate (THF) and methotrexate. Non-limiting examples of protein and peptide growth factors include angiopoietin, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth and differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatome-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), migration stimulants, and myostatin. Examples include tin (GDF-8), nerve growth factor (NGF) and other neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), WNT signaling pathway agonists, placental growth factor (PlGF), fetal bovine somatotropin (FBS), interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7. In one embodiment, the cell culture medium is supplemented with insulin, a point-of-use growth factor.In one embodiment, the concentration of insulin in the culture medium, i.e., the amount of insulin in the cell culture medium after addition, is approximately 0.1 μM to 10 μM. One or more point-of-use additives may also be included in the culture medium formulations of some embodiments.

[0072] Buffers are generally known in the art. The present invention is not limited to any specific buffer(s), and any person skilled in the art can select a suitable buffer(s) for use with a specific cell line that produces a specific protein. In one embodiment, the point-of-use buffer is a NaHCO3 / CO2 system. In one embodiment, the point-of-use buffer contains NaHCO3. In another embodiment, the buffer is HEPES.

[0073] Energy sources for use as point-of-use additives in cell culture are well known in the art. In one embodiment, but not limited to, the point-of-use additive energy source is glucose. Taking into account the specific and particular requirements of a particular cell line and the proteins to be produced, in one embodiment, glucose can be added to the culture medium to a concentration of about 1 to 20 mM.

[0074] Chelating agents are also well known in the fields of cell culture and protein synthesis. Tetrasodium EDTA anhydrous and citrate are two common chelating agents used in the art, but other chelating agents may be used in the implementation of the present invention. In one embodiment, the point-of-use chelating agent is tetrasodium EDTA dihydrate. In one embodiment, the point-of-use chelating agent is citrate, for example, Na3C6H5O7.

[0075] In one embodiment, the cell culture may be supplemented with one or more point-of-use amino acids, such as glutamine. Other point-of-use additives include one or more of various metal salts, such as salts of iron, nickel, zinc, and copper. In one embodiment, the cell culture medium is supplemented with one or more of copper sulfate, zinc sulfate, ferric chloride, and nickel sulfate.

[0076] In one embodiment, the culture medium is replenished at intervals between cell cultures according to a fed-batch method. Fed-batch culture is generally known in the art and is used for optimized protein production. See, for example, YMHuang et al., Biotechnol Prog. (2010) 26(5) pp. 1400-1410.

[0077] In another aspect of this disclosure, cells cultured in a medium containing a soy hydrolysate product with ornithine or putrescine at a desired concentration (i.e., 5.0 mg / L or less, e.g., 0.5 mg / L to 5.0 mg / L or 0.5 mg / L to 2.0 mg / L) produce a protein of the target with improved quality compared to cells cultured in a medium containing a soy hydrolysate product with ornithine or putrescine at a concentration greater than 5 mg / L. In certain embodiments, the improvement in protein quality is measured by the presence or absence of glycosylation at one or more amino acids in the protein of the target, the amount of glycan in the protein of the target, the presence of sialic acid at one or more glycosylation sites in the protein of the target, or a combination thereof. As used herein, “enhanced quality,” “improved quality,” or “high-quality” protein products may also refer to more consistent quality, such as post-translational modifications observed in biotherapeutic protein production lots. Consistent quality includes, for example, having a desired glycosylation profile that can be repeated after repeated production lines. Consistency, in terms of quality, refers to a certain degree of uniformity and standardization, but repeated production batches are inherently unchanging.

[0078] In certain embodiments, the protein product (the protein of interest) is an antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyspecific antibody, bispecific antibody, antigen-binding antibody fragment, single-chain antibody, diabody, triabody or tetrabody, Fab fragment or F(ab')2 fragment, IgD antibody, IgE antibody, IgM antibody, IgG antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody or IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0079] In some embodiments, the antibody is an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Publication No. US2015 / 0203579A1), an anti-programmed cell death ligand-1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Publication No. US2015 / 0203580A1), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., as described in U.S. Patent No. 9,018,356) Anti-AngPtl3 antibodies (such as those described in U.S. Patent No. 9,265,827), anti-PDGFR antibodies (for example, anti-Erb3 antibodies), anti-prolactin receptor antibodies (for example, anti-PRLR antibodies (for example, anti-PRLR antibodies (for example, anti-Prolactin Receptor Antibody R antibody (or anti-EGFRvIII antibody as described in U.S. Patent Publication No. US2015 / 0259423A1), anti-proprotein convertase subtilisin kexin-9 antibody (e.g., anti-PCSK9 antibody as described in U.S. Patent No. 8,062,640 or U.S. Patent Publication No. US2014 / 0044730A1), anti-growth differentiation factor-8 antibody (e.g., anti-GDF8 antibody, also known as anti-myostatin antibody, as described in U.S. Patent No. 8,871,209 or No. 9,260,515), anti-glucagon receptor Anti-GCGR antibodies (for example, as described in U.S. Patent Publication No. US2015 / 0337045A1 or No. US2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin-4 receptor antibodies (for example, anti-IL4R antibodies as described in U.S. Patent Publication No. US2014 / 0271681A1 or No. 8,735,095 or No. 8,945,559), anti-interleukin-6 receptor antibodies (for example, No. 7,582,298, No. 8,043,617 or No. 9,173,Anti-IL6R antibodies (as described in Patent No. 880), anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-interleukin 33 (e.g., anti-IL33 antibodies as described in U.S. Patent Publication No. US2014 / 0271658A1 or No. US2014 / 0271642A1), anti-respiratory rash virus antibodies (e.g., anti-RSV antibodies as described in U.S. Patent Publication No. US2014 / 0271653A1), anti-surface antigen classification 3 (e.g., U.S. Patent Publication No. U Anti-CD3 antibodies (as described in US Patent Publication No. S2014 / 0088295A1 and US20150266966A1 and US Patent Application No. 62 / 222,605), anti-surface antigen classification 20 (for example, anti-CD20 antibodies as described in US Patent Publication Nos. US2014 / 0088295A1 and US20150266966A1 and US Patent No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-surface antigen classification -48 (for example, anti-CD48 antibodies as described in US Patent No. 9,228,014), anti-Fel d1 antibody (e.g., described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibody described in U.S. Patent Publication No. US2015 / 0337029A1), anti-Ebola virus antibody (e.g., described in U.S. Patent Publication No. US2016 / 0215040), anti-Zika virus antibody, anti-lymphocyte activator gene 3 antibody (e.g., anti-LAG3 antibody or anti-CD223 antibody), anti-nerve growth factor antibody (e.g., U.S. Patent Publication No. US2016 / 0017029 and U.S. Patents No. 8,309,088 and 9,353,The anti-NGF antibody (as described in Patent No. 176) and anti-activin A antibody are selected from the group comprising these. In some embodiments, the bispecific antibody is selected from the group comprising anti-CD3 × anti-CD20 bispecific antibody (as described in U.S. Patent Publications US2014 / 0088295A1 and US20150266966A1), anti-CD3 × anti-mucin 16 bispecific antibody (e.g., anti-CD3 × anti-Muc16 bispecific antibody), and anti-CD3 × anti-prostate-specific membrane antigen bispecific antibody (e.g., anti-CD3 × anti-PSMA bispecific antibody). In some embodiments, the protein of interest is selected from the group comprising alirocumab, sarilumab, facinumab, nesbacumab, dupilumab, trevoglumab, evinacumab, and rinucumab. All publications referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0080] In other embodiments, the protein of interest is a recombinant protein (e.g., an Fc-fusion protein) comprising an Fc moiety and another domain. In some embodiments, the Fc-fusion protein is a receptor Fc-fusion protein comprising one or more extracellular domains of a receptor bound to the Fc moiety. In some embodiments, the Fc moiety comprises the hinge region of IgG, followed by CH2 and CH3 domains. In some embodiments, the receptor Fc-fusion protein comprises two or more different receptor chains bound to a single ligand or multiple ligands. For example, Fc-fusion proteins include trap proteins, such as IL-1 traps (e.g., lilonacept, which includes an IL-1RAcP ligand-binding domain fused to an IL-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated herein by reference), and VEGF traps (e.g., aflibercept or ziv-aflibercept, which includes an Ig domain 2 of VEGF receptor Flt1 fused to an Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1). See Bercept; U.S. Patents 7,087,411 and 7,279,159. Alternatively, see Convacept; U.S. Patent 8,216,575, which includes the Ig domain 2 of VEGF receptor Flt1 fused to the Ig domain 3 of VEGF receptor Flk1 fused to the Ig domain 4 of VEGF receptor Flk1 fused to the Fc of hIgG1, or TNF traps (e.g., etanercept; U.S. Patent 5,610,279, which includes a TNF receptor fused to the Fc of hIgG1). In other embodiments, the Fc-fusion protein is an ScFv-Fc-fusion protein which includes one or more antigen-binding domains of an antibody bound to the Fc portion, e.g., one or more variable heavy chain fragments and variable light chain fragments.

[0081] Protein synthesis The target protein can be expressed by host cells using methods known to those skilled in the art. Generally, any target protein suitable for expression in mammalian cells can be generated by this method, but glycoproteins will particularly benefit from this method. For example, in specific embodiments, the target protein may be an antibody or its antigen-binding fragment, a bispecific antibody or its fragment, a chimeric antibody or its fragment, an ScFv or its fragment, an Fc-tagged protein (e.g., a trap protein) or its fragment, a growth factor or its fragment, a cytokine or its fragment, or the extracellular domain or fragment of a cell surface receptor.

[0082] Glycoproteins containing asparagine-linked (N-linked) glycans are ubiquitous in eukaryotic cells. The biosynthesis of these glycans and their translocation to polypeptides occurs in the endoplasmic reticulum (ER). The structure of N-glycans is further modified in the ER and Golgi complex by several glycosidases and glycosyltransferases. The protein synthesis used in this method aims to improve the consistency of the desired N-glycan structure in order to eliminate immunogenic epitopes ("glycotopes"). Detailed structural analysis of glycan-linked proteins can be correlated with the functional characteristics of the protein. Such analyses characterizing protein glycosylation typically involve several steps: i) enzymatic or chemical liberation of the bound glycan, ii) derivatization of the liberated glycan by reductive amination or complete methylation using aromatic or aliphatic amines, and iii) analysis of the glycan. Many variations for analyzing glycosylation patterns are known to those skilled in the art. Glycoproteins may possess several types of glycoforms occupying different sites at specific concentrations, and therefore, their complexity can make reproducibility difficult in certain synthesis methods. Consistency in glycoform type and quantity is measurable and corresponds to desirable outcomes for therapeutic protein synthesis.

[0083] This disclosure shows that producing multiple batches of the target protein in batch or fed-batch culture by culturing cells expressing the target protein in a medium containing a soy hydrolysate product having a specific concentration of ornithine or putrescine enhances the quality of the produced protein and improves consistency between batches. Accordingly, another aspect of this disclosure provides multiple protein preparations produced by culturing cells in a medium containing separate batches of soy hydrolysate product containing a predetermined amount of ornithine or putrescine. In certain embodiments, each batch of soy hydrolysate product selected for use in cell culture has a concentration of 0.67 mg ornithine or putrescine or less per gram of soy, in particular, 0.0067 mg to 0.67 mg of ornithine or putrescine per gram of soy, or 0.0067 to 0.27 mg of ornithine or putrescine per gram of soy.

[0084] In other embodiments, the concentration of ornithine or putrescine in the soybean hydrolysate-containing cell culture medium ranges from 0.5 mg / L to 4.5 mg / L, 0.5 mg / L to 4.0 mg / L, 0.5 mg / L to 3.5 mg / L, 0.5 mg / L to 3.0 mg / L, 0.5 mg / L to 2.5 mg / L, 0.5 mg / L to 2.0 mg / L, 0.5 mg / L to 1.5 mg / L, or 0.5 mg / L to 1.0 mg / L. In some embodiments, the concentration of ornithine or putrescine in the soy hydrolysate-containing cell culture medium ranges from 1.0 mg / L to 5.0 mg / L, 1.5 mg / L to 5.0 mg / L, 2.0 mg / L to 5.0 mg / L, 2.5 mg / L to 5.0 mg / L, 3.0 mg / L to 5.0 mg / L, 3.5 mg / L to 5.0 mg / L, 4.0 mg / L to 5.0 mg / L, or 4.5 mg / L to 5.0 mg / L.

[0085] In specific embodiments, the cell culture medium containing soybean hydrolysate is available in concentrations of 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, 2.6 mg / L, and 2.7 mg / L. Contains ornithine or putrescine in amounts of L, 2.8 mg / L, 2.9 mg / L, 3.0 mg / L, 3.1 mg / L, 3.2 mg / L, 3.3 mg / L, 3.4 mg / L, 3.5 mg / L, 3.6 mg / L, 3.7 mg / L, 3.8 mg / L, 3.9 mg / L, 4.0 mg / L, 4.1 mg / L, 4.2 mg / L, 4.3 mg / L, 4.4 mg / L, 4.5 mg / L, 4.6 mg / L, 4.7 mg / L, 4.8 mg / L, 4.9 mg / L, or 5.0 mg / L.

[0086] In other embodiments, the desired concentration of ornithine or putrescine in a batch of culture medium containing soybean hydrolysate is 5.0 mg / L or less. In yet another embodiment, the desired concentration of ornithine or putrescine in a batch of culture medium containing soybean hydrolysate is 2.0 mg / L or less. In yet another embodiment, the desired concentration of ornithine or putrescine in a batch of culture medium containing soybean hydrolysate is 1.8 mg / L or less. In some embodiments, the desired concentration of ornithine or putrescine in a batch of culture medium containing soybeans is between 0.5 mg / L and 5.0 mg / L. In yet another embodiment, the desired concentration of ornithine or putrescine in a batch of culture medium containing soybean hydrolysate falls within the range of 0.5 mg / L to 2.0 mg / L. In yet another embodiment, the desired concentration of ornithine or putrescine in a batch of culture medium containing soybean hydrolysate falls within the range of 0.5 mg / L to 1.8 mg / L.

[0087] In certain embodiments, the quality of the target protein produced or the amount of certain glycans in each of a group of protein preparations is improved compared to protein preparations produced by a method comprising culturing cells in a medium supplemented with soy hydrolysate containing ornithine or putrescine at concentrations greater than 5 mg / L. In certain embodiments, the improvement in protein quality exhibited by each protein preparation is measured by the presence or absence of glycosylation of one or more amino acids in the target protein, the amount of glycans in the target protein, the presence of sialic acid at one or more glycosylation sites of the target protein, or a combination thereof. In one embodiment, protein quality corresponds to the glycosylation status of individual members of the population of proteins produced in culture. In certain embodiments, quality is improved by regulating glycosylation substitutions present on individual glycoproteins in the population of proteins produced in culture by culturing cells in a medium supplemented with soy hydrolysates having concentrations of 5.0 mg / L or less of ornithine or putrescine, 0.5 mg / L to 5.0 mg / L of ornithine or putrescine, or 0.5 mg / L to 2.0 mg / L of ornithine or putrescine.

[0088] In one embodiment, protein quality is determined by comparing the abundance of at least one glycan molecule in each batch of protein derived from multiple protein preparations with the abundance of the same glycan molecule(s) in another batch of protein. As used herein, the term “abundance” refers to the percentage of protein containing a particular glycan molecule in a particular batch, or the amount of protein containing a particular glycan molecule relative to the total amount of all types of glycan molecules in the batch. In some embodiments, the glycan molecule is selected from the group consisting of A1, A1F, A2, A2F, Man5, NA2, NA2F, NA2G1, NA2G1F, NGA2, and NGA2FI. In a specific embodiment, the glycan molecule is A1 (e.g., peak 11 in Figure 2).

[0089] The target protein produced by the cell culture method of this disclosure exhibits desirable quality characteristics. Protein quality can be measured, for example, using methods well known to those skilled in the art, such as weak cation exchange chromatography, capillary isoelectric focusing, size exclusion chromatography, high-performance liquid chromatography (HPLC), ELISA, and / or Western blot analysis. In some embodiments, protein quality is measured by mass spectrometry, for example, capillary electrophoresis mass spectrometry (CE-MS). In specific embodiments, protein quality is determined by comparing mass spectrometric readouts of each batch of protein from multiple protein preparations.

[0090] In this specification, as illustrated in Tables 2-4, high-performance liquid chromatography (HPLC) with fluorescence detection of exemplary production lots shows that the target protein (glycoprotein) produced by cells cultured in a medium containing soy hydrolysate at concentrations of 0.5 mg / L to 5.0 mg / L of ornithine or putrescine has a more consistent glycan expression and glycosylation pattern.

[0091] Oligosaccharide profiling The degree and distribution of specific N-linked glycans on glycoproteins can be determined by oligosaccharide profiling. In one embodiment, the glycoprotein is deglycosylated by peptide:N-glycosidase F (PNGase F) to cleave and remove N-linked oligosaccharides from the asparagine side chains. The oligosaccharides are then derivatized with a fluorescent reagent, such as anthranilic acid. The glycans are then separated by normal-phase anion-exchange HPLC, detected with a fluorescence detector, and an HPLC chromatogram is generated.

[0092] In another embodiment, as part of an overall carbohydrate characterization analysis, individual glycopolypeptides are isolated following trypsin digestion of a reduced and alkylated glycoprotein. The individual trypsinic glycopolypeptides are separated by reverse-phase HPLC, with a subsequent C18 column for enhanced resolution, if necessary. Oligosaccharides are released from each of the separated glycopolypeptides by PNGase F digestion, derivatized with anthranilic acid, and analyzed by fluorescence HPLC to obtain site-specific oligosaccharide profiles of the glycoprotein. In one embodiment, where the glycoprotein is lilonacept (SEQ ID NO: 1), asparagine residues N37, N87, N91, N98, and optionally N176, N189, N279, N418, N511, N551, N567, N581, N615, and N730 are glycosylated. In one embodiment, one or more residues N37, N98, N418, and N511 of lilonacept (the positions of the residues correspond to SEQ ID NO: 1) contain an A1 oligosaccharide. In one embodiment, where the glycoprotein is aflibercept (SEQ ID NO: 2), the asparagine residues N36, N68, N123, N196, and N282 are glycosylated. In one embodiment, one or both residues N123 and N196 of aflibercept (the positions of the residues correspond to SEQ ID NO: 2) contain an A1 oligosaccharide.

[0093] In another embodiment, an oligosaccharide pool is generated from glycoproteins by deglycosylation of the protein with PNGase F, followed by anthranilic acid derivatization, and then solid-phase extraction (SPE). The mass of the oligosaccharides is then measured using MALDI-TOF in negative linear mode with 2,4,6-trihydroxyacetophenone (THAP) as the matrix.

[0094] Each observed mass is assigned to a unique oligosaccharide structure based on the mass of N-linked glycans commonly observed in recombinant proteins. The expected mass assignments for all peaks are outlined in Table 1. The expected masses are average masses calculated based on the proposed N-linked glycan structure, including the mass of anthranilic acid residues. Monosaccharide compositions are also listed based on the proposed N-linked glycan structure.

[0095] In another embodiment, a quantitative oligosaccharide fingerprint assay using capillary electrophoresis is used to characterize the N-glycan (oligosaccharide) structure of a glycoprotein of interest. The glycoprotein is denatured and then deglycosylated by treatment with PNGase F. The liberated oligosaccharides are then isolated by precipitation after protein removal. The isolated oligosaccharide pool is labeled with fluorophore 8-aminopyrene 1,3,6-trisulfonate (APTS). The labeled oligosaccharides are then separated by capillary electrophoresis and monitored with a laser-induced fluorescence detector using an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0096] For the aflibercept glycoprotein, as shown in Figure 2, all quantifiable peaks are numbered (21 peaks in total in this example), and an electrophoresis map is generated. The complete integrated peak area (total peak area) for the oligosaccharide fingerprint is determined. The relative amount of each oligosaccharide can be determined by dividing the peak area for that particular oligosaccharide (e.g., the A1 peak area) by the total peak area.

[0097] In some embodiments, the quality of the glycoprotein in question is assessed by determining the level of sialylation (amount of sialic acid residues per glycoprotein) or fucosylation (amount of fucose residues per glycoprotein). In one embodiment, the total number of sialic acids on the glycoprotein is determined using a quantitative HPLC assay. In this assay, sialic acids are released from the glycoprotein using mild acid hydrolysis, then derivatized with o-phenylenediamine, separated by HPLC, and detected by either a UV or fluorescence detector. The quantification of sialic acids can be assessed by comparison with a calibration curve using, for example, sialyl lactose. The sialic acid content is calculated from the moles of sialic acid released and the moles of glycoprotein used in the reaction.

[0098] In one embodiment, the sialic acid content of lilonacept glycoprotein is approximately 30-70 molar sialic acid (mol / mol) per mole of glycoprotein, approximately 35-65 mol / mol, 30 mol / mol, 31 mol / mol, 32 mol / mol, 33 mol / mol, 34 mol / mol, 35 mol / mol, 36 mol / mol, 37 mol / mol, 38 mol / mol, 39 mol / mol, 40 mol / mol, 41 mol / mol, 42 mol / mol, 43 mol / mol, 44 mol / mol, 45 mol / mol, 46 mol / mol l, 47mol / mol, 48mol / mol, 49mol / mol, 50mol / mol, 51mol / mol, 52mol / mol, 53mol / mol, 54mol / mol, 55mol / mol, 56mol / mol, 57mol / mol, 58mol / mol, 5 9mol / mol, 60mol / mol, 61mol / mol, 62mol / mol, 63mol / mol, 64mol / mol, 65mol / mol, 66mol / mol, 67mol / mol, 68mol / mol, 69mol / mol or 70mol / mol.

[0099] In one embodiment, the sialic acid content of aflibercept glycoprotein is approximately 5-15 moles (mol / mol), approximately 8-12 mol / mol, 4 mol / mol, 5 mol / mol, 6 mol / mol, 7 mol / mol, 8 mol / mol, 9 mol / mol, 10 mol / mol, 11 mol / mol, 12 mol / mol, 13 mol / mol, 14 mol / mol, 15 mol / mol, 16 mol / mol, 17 mol / mol, 18 mol / mol, 19 mol / mol, or 20 mol / mol per mole of sialic acid in glycoprotein.

[0100] In one embodiment, oligosaccharide profiling is used to determine the degree and distribution of sialylation of N-linked glycans on glycoproteins. The glycoprotein is deglycosylated with PNGase F and then derivatized with a fluorescent reagent, namely anthranilic acid. The oligosaccharides are then separated by normal-phase anion-exchange HPLC and detected with a fluorescence detector to generate an HPLC chromatogram of the oligosaccharide profile. The Z number for the glycoprotein (which evaluates the average degree of sialylation) is calculated using the following formula:

[0101] (OS A*O)+(ISA*111-(2SA*2)+(3SA*3)+...(nSA*n)1 / (OSA+15A+2SA+35A+...n5A)

[0102] To determine the Z number, the area of ​​each peak from the oligosaccharide profile is aggregated. Total sialic acid is calculated as the sum of the areas of the 0 sialic acid / chain peak (multiplied by 0), the 1 sialic acid / chain peak (multiplied by 1), the 2 sialic acid / chain peak (multiplied by 2), and the 3 sialic acid / chain peak (multiplied by 3). The total number of glycans is generated as the sum of the areas of all peaks. The Z number is the total sialic acid area divided by the total glycan area.

[0103] In one embodiment, the Z number of sialic acid in lilonacept glycoprotein is approximately 1.3-1.6, 1.4-1.5, 1.41-1.48, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, or 1.60.

[0104] In one embodiment, the Z number of sialic acid in aflibercept glycoprotein is approximately 0.5-2, 1-1.5, 1-1.2, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0 These are 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, or 1.3. [Examples]

[0105] The following examples are provided to those skilled in the art to the methods and methods for preparing and using compositions as described herein, and are not intended to limit the scope of what the inventors consider to be their invention. While efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), some experimental error and deviation should be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0106] Example 1: Screening of soy hydrolysate products to determine amino acid concentration A sample of soybean hydrolysate was weighed, and 20 grams of it was dissolved in 1 L of water to obtain a starting concentration of 20 g / L. The resulting soybean hydrolysate solution was then further diluted with water to the desired concentration for use in cell culture, and the molecular composition of the resulting soybean hydrolysate solution was determined by chromatography.

[0107] The amino acid concentrations in soybean hydrolysis product samples were measured by ion-exchange column chromatography, along with post-column ninhydrin detection. See, for example, Moore and Stein. J. Biol. Chem. (1954) Vol. 211 pp. 907-913. The soybean hydrolysis product samples were diluted to enable highly sensitive separation and resolution of individual peaks (amino acids) when eluted from the HPLC column and compared to the standard. The concentration of each eluate was determined by comparing the peak area of ​​each chromatogram shown in Figures 1A and 1B with the standard.

[0108] To determine whether a batch of soy hydrolysate powder contains less than 0.67 milligrams of ornithine or putrescine per gram of soy, the chromatogram of each representative sample is compared to a standard. For example, Figure 1A shows a batch of soy hydrolysate with an eluate containing ornithine at a retention time of 89.02, which reveals a peak area equivalent to 1.57 mg of ornithine per gram of soy when compared to the standard. Figure 1B illustrates a batch of soy hydrolysate with an ornithine concentration of less than 0.67 mg of ornithine per gram of soy. To produce biotherapeutic proteins with more consistent protein glycosylation between lots, batches of soy hydrolysate containing 0.067 to 0.67 mg of ornithine per gram of soy were selected for use in cell culture methods. However, to determine the effect of ornithine concentration in soy hydrolysates on protein synthesis, further experiments were conducted using batches of soy hydrolysates containing ornithine concentrations exceeding less than 0.67 mg per gram of soy, as described below.

[0109] Example 2: Expression and glycosylation profiles of the target protein To determine which amino acid components affect the quality of the protein produced, CHO cells expressing a trap protein (receptor-Fc fusion protein, VEGF-trap) were cultured in a proprietary medium containing soy hydrolysates with varying amounts of ornithine, putrescine, and citrulline, or combinations thereof. Table 2 shows that, regardless of citrulline concentration, the level of ornithine in the hydrolysates negatively correlates with the quality of the protein production lot for protein lots produced as a result of culturing CHO cells in a medium supplemented with soy hydrolysates containing ornithine at concentrations of less than 5.0 mg / L. This is indicated by an increase in the area under the curve relative to key N-glycans.

[0110] As shown in Table 2 and illustrated in Figure 3, lots of VEGF-trap protein products produced by cells cultured in a medium containing soy hydrolysate with ornithine concentrations of 2.0 mg / L or less produce higher quality protein products compared to cells cultured in a medium containing ornithine, citrulline, or putrescine at concentrations exceeding 5.0 mg / L. [Table 2]

[0111] To determine whether ornithine affected the protein glycosylation profile, detailed glycan analysis was performed for each lot of glycoprotein using HPLC and chromatography based on a well-known method for fluorescent anthranilic acid (AA) tagging (Anumula, and Dhume, Glycobiology (1998) 8(7) pp.685-694). As shown in Table 3, cell culture in media containing soy hydrolysates with ornithine concentrations of 0.67 mg or less per g of soy resulted in more consistent protein production across lots. More specifically, approximately 90% of the production lots cultured in media containing selected soy hydrolysates met the FDA production criteria. In contrast, only 57% of the production lots cultured in media containing soy hydrolysates with ornithine concentrations exceeding 5 mg / L met the FDA production criteria (area under the curve for specific N-glycan peaks). As shown in Table 3, lots of VEGF-trap protein products produced by cells cultured in a medium containing soy hydrolysates with an ornithine concentration of 0.67 mg or less per gram of soybeans exhibit improved product quality and more consistent quality across lots. [Table 3]

[0112] Each generated lot was also compared (with respect to the glycan profile) to a reference standard corresponding to a therapeutically acceptable batch of the exemplary VEGF-trap protein. Table 4 shows representative glycan analyses for protein lots generated from cells cultured in medium supplemented with soy hydrolysates resulting in final ornithine concentrations between 0.5 mg / L and 2.0 mg / L. Compared to the reference, each generated trap protein (75% of the analyzed lots) contained a consistent glycan profile with peaks within the acceptable range. In contrast, each lot generated by cells cultured in medium supplemented with soy hydrolysates containing ornithine above 5.0 mg / L failed to meet the FDA acceptance criteria. As shown in Table 4, protein generation lots generated by cells cultured in medium containing soy hydrolysates with ornithine concentrations between 0.5 mg / L and 2.0 mg / L produced higher quality lots than those cultured in medium containing more soy hydrolysates with ornithine concentrations above 5.0 mg / L, as indicated by the A1 N-glycan levels below the product acceptance criteria. [Table 4]

[0113] Figure 4 shows a strong negative correlation between the level of ornithine in soy hydrolysates and the quality of glycoprotein (aflibercept), as indicated by the A1 N-glycan level.

[0114] Example 3: Glycoprotein synthesis titer Sixteen lots of soy hydrolysate were tested for their ability to influence the metabolomics of lilonacept CHO cell generation. Approximately 426 soy hydrolysate analytes were measured and compared to final glycoprotein titers and lactate metabolism. Figure 5 shows a loading plot of the correlation between soy hydrolysate analytes and maximum lactate and final glycoprotein titers. The determination of lactate and glycoprotein titers shows a negative correlation with ornithine in soy hydrolysate.

[0115] Example 4: Marker confirmation by spiking study Figures 6A and 6B show CHO cell cultures under control medium and feed conditions, respectively, with either ornithine or putrescine spiked to demonstrate the effects of ornithine and putrescine on cell proliferation and glycosylation. Table 6B highlights the effects, particularly the significant peak 11.

[0116] While embodiments of the present invention have been described with reference to the attached drawings, it should be understood that the present invention is not limited to specific embodiments, and that those skilled in the art can make various modifications and alterations within the scope or spirit of the invention as defined in the attached claims without departing from it.

Claims

1. A method for producing an Fc fusion protein in a cell culture medium containing soy hydrolysate, the method being as follows: (a) Measure the amount of ornithine in the hydrolyzed soybean product; (b) Select a soy hydrolysate containing 0.003% to 0.027% (by weight) ornithine; and (c) A step of culturing a population of cells expressing the Fc fusion protein in a cell culture medium containing the soy hydrolysate selected in step (b), where the Fc fusion protein is produced by the cells. The method, including the method described above.

2. The method according to claim 1, wherein the cell population is obtained by clonal proliferation of cells expressing an Fc fusion protein.

3. The method according to claim 1 or 2, wherein the culture medium contains ornithine or putrescine in a concentration of 5 mg or less.

4. The method according to any one of claims 1 to 3, wherein the culture medium comprises 0.6 to 3 mg / L of ornithine or putrescine.

5. The method according to any one of claims 1 to 4, wherein the Fc fusion protein includes a trap molecule.

6. The method according to claim 5, wherein the trap molecule comprises an IL-1 trap molecule, a VEGF trap molecule, or a TNF trap molecule.

7. The method according to any one of claims 1 to 6, wherein the Fc fusion protein comprises A1 N-glycan and at least one other N-glycan species, and the relative amount of A1 N-glycan is 10% (by weight) or more of the total amount of all N-glycan species in the Fc fusion protein.

8. A selection method including soy hydrolysate, a. A step of culturing cells expressing an Fc fusion protein in a cell culture medium containing soybean hydrolysate, wherein the cells produce an Fc fusion protein; b. The process of purifying the Fc fusion protein; c. The process of subjecting the purified Fc fusion protein to oligosaccharide fingerprint analysis; d. A step of measuring the relative amount of A1 N-glycan to the total amount of N-glycan species in the Fc fusion protein; and e. The method comprising the step of selecting a soy hydrolysate that provides at least 10% (by weight) of A1 N-glycans relative to the total amount of N-glycan species of the Fc fusion protein, wherein the soy hydrolysate contains 0.003% to 0.027% (by weight) of ornithine.

9. The method according to claim 8, wherein the culture medium comprises 0.6 to 3 mg / L of ornithine or putrescine.

10. The method according to claim 8 or 9, wherein the Fc fusion protein is a trap molecule.

11. The method according to claim 10, wherein the trap molecule comprises an IL-1 trap molecule, a VEGF trap molecule, or a TNF trap molecule.

12. A method for producing a cell culture medium for producing Fc fusion proteins, the method being as follows: a. A step to measure the amount of ornithine in soy hydrolysate; b. A step of selecting a soy hydrolysate containing 0.003% to 0.027% (w / w) ornithine; and c. The method comprising the step of combining a selected soy hydrolysate with a cell culture medium that does not contain animal protein to form a cell culture medium containing soy hydrolysate, wherein the cell culture medium containing soy hydrolysate contains ≤5 mg / L of ornithine.

13. The method according to claim 12, wherein the cell culture medium comprises 0.6 to 3 mg / L of ornithine or putrescine.

14. The method according to claim 12 or 13, wherein the Fc fusion protein is a trap molecule.

15. The method according to claim 14, wherein the trap molecule comprises an IL-1 trap molecule, a VEGF trap molecule, or a TNF trap molecule.

16. The method according to any one of claims 12 to 15, wherein the Fc fusion protein comprises A1 N-glycan and at least one other N-glycan species, and the relative amount of A1 N-glycan is 10% (by weight) or more of the total amount of all N-glycan species in the Fc fusion protein.

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