Methods for improving protein titer in cell culture

Abstract

Methods for improving recombinant protein titers and cell titers in cell culture using cell culture media with reduced impurities are provided, as well as cell culture media with reduced impurities that can be used to produce recombinant proteins and cells with improved titers. The cell culture media with reduced impurities include a HEPES buffer, and the reduced impurities are HEPES-related impurities. In certain aspects, the methods and media improve protein titers, cell growth, and / or viable cell density.

Description

Technical Field

[0001] This application claims priority to U.S. Patent Application No. 63 / 139,494, filed on January 20, 2021, which is incorporated herein by reference.

[0002] The present invention relates to a method for culturing cells to improve titer and a method for producing a recombinant protein. The present invention relates to a method for culturing cells to improve titer using a medium having reduced impurities, a method for the production of protein biopharmaceuticals, and cells and cell cultures grown according to this method, and proteins produced by the cells and cell cultures.

Background Art

[0003] Biological agents, particularly proteins and polypeptides, are often developed as novel biopharmaceuticals. Engineered cells that produce high levels of a specific target protein have become extremely important for the successful commercial production of these biopharmaceuticals. The control and optimization of cell culture conditions are diverse and have a great impact on the level and quality of therapeutic proteins produced in cell culture.

[0004] It is customary to produce proteins via cell culture in a batch or fed-batch process. The initial stage of inoculation growth after vial thawing includes culturing the cells in a seed culture. Typically, the cells are grown at an exponential growth rate, such as in a seed train bioreactor, to gradually increase the size and / or volume of the cell population. After the cell mass is scaled up through several bioreactor stages, the cells are then transferred to a fed-batch production bioreactor while the cells are still in exponential growth (log phase) (Gambhir, A. et al., 2003, J Bioscience Bioeng 95(4):317-327).

[0005] After transferring to a fed-batch culture, the cells are cultured for a certain period while the culture medium composition is monitored and controlled to enable the production of the target protein or polypeptide. The produced protein or polypeptide is isolated after a certain yield is reached, or when it is determined that the culture should be terminated due to cell viability, waste accumulation, or nutrient depletion. Many significant advances have been made in the past decade with the aim of improving recombinant protein yields, and currently, titers of several grams per liter have been reached. Advances in protein production processes, as well as cell line engineering and the development of cell culture media and feeding, have contributed to the improvement of protein yields. For example, schemes for optimizing cell culture media and feeding include nutrient supply and the design of chemically defined serum-free media to support continuous cell proliferation and optimal product secretion.

[0006] However, there remains a need in the art for culture media and methods for culturing cells, and this medium enables healthy and robust cell proliferation and maintenance, as well as high-titer production of recombinant proteins. [Overview of the project]

[0007] In one embodiment, a method is provided for improving recombinant protein titer in recombinant protein production by culturing recombinant eukaryotic cells. In a particular embodiment, the method provides (a) a defined cell culture medium having reduced impurities, wherein the defined cell culture medium contains 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer and contains less than 4000 ppm of HEPES-related impurities with a molecular weight (MW) of 267.07 relative to the total amount of HEPES buffer in the medium (4000 μmol of HEPES impurities with a molecular weight (MW) of 267.07 per mole of total HEPES), and 221.06 ppm of HEPES-related impurities with a molecular weight (MW) relative to the total amount of HEPES buffer in the medium. The invention provides a cell culture medium having HEPES-related impurities of less than approximately 400 ppm (400 μmol of HEPES impurities with MW221.06 per mole of total HEPES), and includes (b) culturing the recombinant eukaryotic cells in the specified cell culture medium having reduced impurities, (c) expressing the recombinant protein of the target from the eukaryotic cells, and (d) producing a higher titer of the recombinant protein in the specified cell culture medium having reduced impurities compared to the titer of similar or identical cells cultured in a medium without reduced impurities.

[0008] In certain embodiments, the higher titer of the recombinant protein is increased by at least about 5% compared to the titer of similar or identical cells cultured in a non-impaired medium.

[0009] In certain embodiments, the eukaryotic cell may be a mammalian cell, an avian cell, an insect cell, or a yeast cell. In certain embodiments, the eukaryotic cell may be a CHO cell. In other embodiments, the recombinant protein may be any other recombinant protein, including Fc fusion proteins, receptor-Fc fusion proteins, trap proteins or mini-trap proteins, antibodies, antibody fragments, or ScFv-Fc fusion proteins, as disclosed herein.

[0010] In certain embodiments, the expression of the recombinant protein of interest may occur during the production phase, the proliferation phase, or both. In other embodiments, the culture of recombinant eukaryotic cells in a defined cell culture medium with reduced impurities occurs during the production phase, the proliferation phase, or both.

[0011] In further embodiments, the method improves cell culture performance, including improved cell proliferation, with cell proliferation of recombinant eukaryotic cells during culture being higher than that of similar or identical recombinant eukaryotic cells in a non-reduced impurity medium.

[0012] In another aspect of the present invention, a defined cell culture medium having reduced impurities is provided. In a particular embodiment, the medium comprises a defined cell culture medium having reduced impurities, the defined cell culture medium comprising 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer and having less than 800 ppm of HEPES-related impurities with a molecular weight (MW) of 267.07 (800 μmol of HEPES impurities per mole of total HEPES with MW 267.07) relative to the total amount of HEPES buffer in the medium, and less than 80 ppm of HEPES-related impurities with a molecular weight (MW) of 221.06 (80 μmol of HEPES impurities per mole of total HEPES with MW 221.06) relative to the total amount of HEPES buffer in the medium.

[0013] In yet another aspect of the present invention, a method is provided for selecting a prescribed cell culture medium for use in cell culture to improve cell culture performance. In a particular embodiment, the method generally includes (a) providing a prescribed cell culture medium comprising 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer; (b) analyzing the prescribed cell culture medium comprising HEPES buffer to determine the amount of HEPES-related impurities having a molecular weight (MW) of 267.07 and the amount of HEPES-related impurities having a molecular weight (MW) of 221.06 present in the prescribed cell culture medium; and (c) determining that the prescribed cell culture medium comprising HEPES buffer contains less than approximately 4000 ppm of HEPES-related impurities having a molecular weight (MW) of 267.07 (4000 μmol of HEPES impurities per mole of total HEPES) and less than approximately 400 ppm of HEPES-related impurities having a molecular weight (MW) of 221.06 relative to the total amount of HEPES buffer in the medium. The selection of a specified cell culture medium containing HEPES buffer for use in cell culture, provided that it is determined to have a certain amount of HEPES-related impurities (400 μmol of HEPES impurities with a molecular weight of 221.06 MW per mole of total HEPES), wherein the specified cell culture medium has less than approximately 4000 ppm of HEPES-related impurities with a molecular weight of 267.07 (4000 μmol of HEPES impurities with a molecular weight of 267.07 MW per mole of total HEPES) relative to the total amount of HEPES buffer in the medium, and less than approximately 400 ppm of HEPES-related impurities with a molecular weight of 221.06 (400 μmol of HEPES impurities with a molecular weight of 221.06 MW per mole of total HEPES) relative to the total amount of HEPES buffer in the medium, improves cell culture performance compared to cell culture performance in a medium without reduced HEPES-related impurities. In certain embodiments, the improved cell culture performance includes improved cell culture titer and / or cell proliferation.

[0014] In yet another aspect of the present invention, a method is provided for selecting a HEPES buffer for use in cell culture to improve cell culture performance. In a particular embodiment, the method generally comprises: (a) providing a 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer; (b) analyzing the HEPES buffer to determine the amount of HEPES-related impurities having a molecular weight (MW) of 267.07 and the amount of HEPES-related impurities having a molecular weight (MW) of 221.06 present in the HEPES buffer; and (c) determining that the HEPES buffer contains less than 4000 ppm of HEPES-related impurities having a molecular weight (MW) of 267.07 relative to the total amount of HEPES buffer used in conjunction with the cell culture (4000 μmol of HEPES impurities with a molecular weight of 267.07 per mole of total HEPES), and less than 400 ppm of HEPES-related impurities having a molecular weight (MW) of 221.06 relative to the total amount of HEPES buffer used in conjunction with the cell culture (total HEPES The selection of a HEPES buffer for use in cell culture includes, when it is determined that it has 400 μmol of HEPES-related impurities with a molecular weight (MW) of 267.07 per mole of total HEPES, and the use of a HEPES buffer having less than 4000 ppm of HEPES-related impurities with a molecular weight (MW) of 221.06 per mole of total HEPES, relative to the total amount of HEPES buffer used in association with the cell culture, improves cell culture performance compared to cell culture performance in the presence of a HEPES buffer with a higher amount of said impurities. In certain embodiments, the improved cell culture performance includes improved cell culture titer and / or cell proliferation.

[0015] Another aspect of the present invention provides a cell culture comprising (i) at least one recombinant eukaryotic cell capable of expressing a recombinant protein, and (ii) a cell culture medium, the cell culture being produced by a method comprising: (a) providing a defined cell culture medium having reduced impurities, wherein the defined cell culture medium has 4000 μmol of HEPES-related impurities having a molecular weight of 267.07 per mole of total HEPES and 400 μmol of HEPES-related impurities having a molecular weight of 221.06 per mole of total HEPES; (b) culturing the recombinant eukaryotic cell in the defined cell culture medium having reduced impurities; (c) expressing a recombinant protein of interest from the recombinant eukaryotic cell; and (d) producing a higher titer of the protein in the defined cell culture medium having reduced impurities compared to that of similar or identical cells cultured in a medium without reduced impurities.

[0016] Eukaryotic cells can be selected from the group consisting of mammalian cells, avian cells, insect cells, and yeast cells, and can be selected from the group consisting of CHO, COS, retinal cells, Vero, CV1, kidney, HeLa, HepG2, WI38, MRC5, Colo25, HB8065, HL-60, lymphocytes, A431, CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. For example, eukaryotic cells may be CHO cells.

[0017] The expression of the target recombinant protein may occur during the production phase, the proliferation phase, or both. Culture of recombinant eukaryotic cells in the specified cell culture medium with reduced impurities may occur during the production phase, the proliferation phase, or both. Cell proliferation of recombinant eukaryotic cells during such culture may be higher than that of similar or identical recombinant eukaryotic cells cultured in unreduced impurity medium. The higher titer of the recombinant protein may be increased by at least about 5% compared to the titer of similar or identical cells cultured in unreduced impurity medium.

[0018] Recombinant proteins may contain an Fc domain. Recombinant proteins may be antibodies, human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, antibody fragments, antigen-binding antibody fragments, single-chain antibodies, diabodies, triabodies or tetrabodies, Fab fragments or F(ab')2 fragments, IgD antibodies, IgE antibodies, IgM antibodies, IgG antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies. The recombinant proteins include anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL 2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 antibody, anti-CD3 / anti-CD20 bispecific antibody, anti-CD3 / anti-MUC16 bispecific antibody, and anti-CD3 / anti-PSMA bispecific antibody. For example, recombinant proteins can be selected from the group consisting of alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacirivimab, imudevimab, semiprimab, semiprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, facimmab, nesbacumab, trevoglumab, linucumab, and sarilumab.

[0019] Recombinant proteins can also be selected from the group consisting of Fc fusion proteins, receptor-Fc fusion proteins (TRAPs), mini-trap proteins, and ScFv-Fc fusion proteins, or any other recombinant proteins.

[0020] Another aspect of the present invention provides a recombinant protein produced in a cell culture comprising (i) at least one recombinant eukaryotic cell capable of expressing the recombinant protein, and (ii) a cell culture medium, wherein the recombinant protein is produced by a method comprising: (a) providing a defined cell culture medium having reduced impurities, wherein the defined cell culture medium has 4000 μmol of HEPES-related impurities having a molecular weight of 267.07 per mole of total HEPES and 400 μmol of HEPES-related impurities having a molecular weight of 221.06 per mole of total HEPES; (b) culturing the recombinant eukaryotic cell in the defined cell culture medium having reduced impurities; (c) expressing the recombinant protein of the target from the recombinant eukaryotic cell; and (d) producing a higher titer of the protein in the defined cell culture medium having reduced impurities compared to that of similar or identical cells cultured in a medium without reduced impurities.

[0021] Eukaryotic cells can be selected from the group consisting of mammalian cells, avian cells, insect cells, and yeast cells, and can be selected from the group consisting of CHO, COS, retinal cells, Vero, CV1, kidney, HeLa, HepG2, WI38, MRC5, Colo25, HB8065, HL-60, lymphocytes, A431, CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. For example, eukaryotic cells may be CHO cells.

[0022] The expression of the target recombinant protein may occur during the production phase, the proliferation phase, or both. Culture of recombinant eukaryotic cells in the specified cell culture medium with reduced impurities may occur during the production phase, the proliferation phase, or both. Cell proliferation of recombinant eukaryotic cells during such culture may be higher than that of similar or identical recombinant eukaryotic cells cultured in unreduced impurity medium. The higher titer of the recombinant protein may be increased by at least about 5% compared to the titer of similar or identical cells cultured in unreduced impurity medium.

[0023] Recombinant proteins may contain an Fc domain. Recombinant proteins may be antibodies, human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, antibody fragments, antigen-binding antibody fragments, single-chain antibodies, diabodies, triabodies or tetrabodies, Fab fragments or F(ab')2 fragments, IgD antibodies, IgE antibodies, IgM antibodies, IgG antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies. The recombinant proteins include anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL 2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 antibody, anti-CD3 / anti-CD20 bispecific antibody, anti-CD3 / anti-MUC16 bispecific antibody, and anti-CD3 / anti-PSMA bispecific antibody. For example, recombinant proteins can be selected from the group consisting of alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacirivimab, imudevimab, semiprimab, semiprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, facimmab, nesbacumab, trevoglumab, linucumab, and sarilumab.

[0024] Recombinant proteins can also be selected from the group consisting of Fc fusion proteins, receptor-Fc fusion proteins (TRAPs), mini-trap proteins and ScFv-Fc fusion proteins, or any other recombinant proteins.

[0025] The present invention provides cells, cell cultures, recombinant proteins, and methods.

[0026] Although multiple embodiments are disclosed through this application, other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description that shows and describes exemplary embodiments of the present invention. As will be realized, the present invention is capable of modifications in various aspects without departing from the spirit and scope of the present invention. Therefore, the embodiments for carrying out the invention should be regarded essentially as illustrative and not as restrictive.

Brief Description of the Drawings

[0027] [Figure 1] Shows the relationship between HEPES-related impurities and protein titer according to one embodiment of the present invention. [Figure 2] Shows a negative correlation between HEPES-related impurities and protein titer according to one embodiment of the present invention. Figure 2A is based on the data from location 1. Figure 2B is based on the data from location 2. In Figure 2A, the data points overlap towards the right, which is also shown in Figure 1 for lot 1117000128 and lot 1117000130. [Figure 3] Shows the relationship between HEPES-related impurities and protein titer according to one embodiment of the present invention at locations 1 and 2. [Figure 4] Shows a negative correlation between HEPES-related impurities and protein titer according to one embodiment of the present invention. Figure 4A is based on the data from location 1. Figure 4B is based on the data from location 2. Figure 4A has an overlap of two data points. The first overlap is near the center, which is also shown in Figure 3 for lot 1117000129 and lot 1117000138. The second overlap is towards the right, which is also shown in Figure 3 for lot 1117000128 and lot 1117000130. [Figure 5A] Shows the HILIC separation of HEPES impurities. [Figure 5B] Shows the separation of HEPES impurities by mixed-mode column separation. [Figure 6A]This is an RP-LCMS plot of HEPES-[CH4] (also known as "221") derived from HEPES raw materials. [Figure 6B] This is a HILIC-LCMS plot of HEPES-[CH4] derived from HEPES raw materials. [Figure 7] This shows MS / MS fragmentation of HEPES-[CH4] (also known as "221") from HEPES raw materials.

[0028] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. [Modes for carrying out the invention]

[0029] According to aspects of the present invention, the use of cell culture media having reduced impurities has been unexpectedly found to improve cell culture performance, including improved cell proliferation and protein production by cells in the cell culture, compared to cell culture media without such reduced impurities.

[0030] More specifically, it has been unexpectedly found that impurities in cell culture media containing 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer affect cell culture performance. According to the present invention, HEPES-related impurities that have an effect, i.e., show a strong negative correlation with cell culture performance (e.g., protein titer), have been identified. In one embodiment, the HEPES-related impurities include HEPES-related impurities having a molecular weight (MW) of 267.07, HEPES-related impurities having a molecular weight of 221.06, and combinations thereof. In certain embodiments, it has been found that the use of cell culture media with reduced amounts of these HEPES-related impurities improves cell culture performance compared to cell culture media without such reduced amounts of HEPES-related impurities.

[0031] 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 art known in the art and, unless otherwise indicated, are carried out 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); 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).

[0032] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out the present invention, but specific methods and materials are described below.

[0033] In the context of numerical values ​​and ranges, the term "about" means a value or range that is close to, or nearly close to, the enumerated value or range, so that the invention can be performed as intended, having a desired rate, amount, degree, increase, decrease, or manifestation, concentration, or time range, as is evident from the teachings contained herein. Therefore, this term encompasses values ​​beyond those resulting merely from systematic errors.

[0034] The terms “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, sulfated material, 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. Examples of peptides, polypeptides, and proteins include, among others, antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins are produced by recombinant animal cell lines using cell culture methods.

[0035] As used herein, the term “heterogeneic polynucleotide sequence” refers to nucleic acid polymers that encode a protein of interest, such as a chimeric protein (e.g., a trap molecule), an antibody, or an antibody moiety (e.g., VH, VL, CDR3), produced as a biopharmaceutical drug substance. Heterogeneic polynucleotide sequences may be produced by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, intron-less sequences, etc.) and introduced into cells, where they may exist as episomes or be integrated into the cellular genome. Heterogeneic polynucleotide sequences may be naturally occurring sequences introduced into ectopic sites within the producing cellular genome. Heterogeneic polypeptide sequences may be naturally occurring sequences from another organism, such as sequences encoding human orthologues.

[0036] As used herein, the term “antibody” refers to an immunoglobulin molecule consisting of four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains. 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 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), which are dotted with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term “antibody” includes references to 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 to express antibodies. The term “antibody” also includes “bispecific antibodies,” which include heterotetrameric immunoglobulins capable of binding to more than one different epitopes. Bispecific antibodies are generally described in U.S. Patent Application Publication 2010 / 0331527.

[0037] The term "antigen-binding portion" (or "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a monovalent fragment consisting of a Fab fragment, VL, VH, CL, and CH1 domains; (ii) a bivalent fragment containing an F(ab')2 fragment, i.e., two Fab fragments linked by disulfide crosslinks at the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) a dAb fragment consisting of a VH domain (Ward et al. (1989) Nature 241:544-546); (vi) an isolated CDR; and (vii) scFv consisting of VL and VH, which are two domains of an Fv fragment linked by a synthetic linker so that the VL and VH regions pair up to form a single protein chain that forms a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also included in the term "antibody" (see, for example, Holliger et al. (1993) PNAS USA 90:6444-6448 and Poljak et al. (1994) Structure2:1121-1123).

[0038] Furthermore, an antibody or its antigen-binding moiety may be part of a larger immunoadhesion molecule formed by the covalent or non-covalent association of the antibody or antibody moiety with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include the use of a streptavidin core region to produce tetrameric scFv polypeptides (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to produce divalent biotinylated scFv molecules (Kipriyanov et al. (1994) Mol.Immunol.31:1047-1058). Antibody moieties such as Fab fragments and F(ab')2 fragments can be prepared from the whole antibody using conventional techniques, such as via papain digestion or pepsin digestion of the whole antibody. Furthermore, antibodies, antibody moieties, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques generally known in the field (see Sambrook et al., 1989).

[0039] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include, for example, amino acid residues in the CDR, particularly CDR3, that are 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). The term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, are grafted onto a human framework sequence.

[0040] 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 recombinant combinatorial human antibody libraries; antibodies isolated from animals transgenic for human immunoglobulin genes (e.g., mice) (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 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 transgenic animals for the human Ig sequence are used, in vivo somatic mutagenesis), and therefore 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 may not be naturally present in the human antibody germline repertoire in vivo.

[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, otherwise not found together in nature). The preparation of fusion proteins containing specific heterologous polypeptides fused to various portions of an antibody-derived polypeptide (including the Fc domain) is described, for example, by Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., “Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992. A "receptor Fc fusion protein" comprises one or more extracellular domains of a receptor ligated to the Fc portion, which in some embodiments include a hinge region, followed by the CH2 and CH3 domains of the immunoglobulin. In some embodiments, the Fc fusion protein contains two or more distinctly different receptor chains that bind to one or more ligands. For example, the Fc fusion protein is a trap containing, for example, an IL-1 trap (e.g., lilonacept containing an IL-1RAcP ligand-binding domain fused to an IL-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,044) or a VEGF trap (e.g., aflibercept containing 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 U.S. Patents No. 7,087,411 and 7,279,159).

[0042] In addition, trap proteins that use a polymerizing component (MC) instead of the Fc moiety include minitraps disclosed in U.S. Patent No. 7,279,159 and U.S. Patent No. 7,087,411.

[0043] The above derivatives, components, domains, chains, and fragments are also included.

[0044] All numerical limits and ranges shown herein include all numerical values ​​or values ​​that fall around or between the numerical values ​​of the range or limit. The ranges and limits described herein expressly represent and describe all integers, decimals, and fractional values ​​defined and encompassed by the range or limit. Accordingly, references to ranges of values ​​herein are intended to function simply as a simplified way of individually referring to each individual value included in the range unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually referred herein.

[0045] Cell culture medium In one embodiment, the present invention provides a cell culture medium having reduced impurities. In a particular embodiment, the cell culture medium comprises 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, and the reduced impurities are HEPES-related impurities. In a particular embodiment, the cell culture medium may be a chemically defined cell culture medium as discussed herein.

[0046] More specifically, according to the present invention, the cell culture medium contains less than 4000 ppm of HEPES-related impurities with a molecular weight (MW) of 267.07 relative to the amount of HEPES buffer present in the cell culture medium (4000 μmol of HEPES impurities with a molecular weight of 267.07 per mole of total HEPES), and less than 400 ppm of HEPES-related impurities with a molecular weight (MW) of 221.06 relative to the amount of HEPES buffer present in the cell culture medium (400 μmol of HEPES impurities with a molecular weight of 221.06 per mole of total HEPES).

[0047] At most biological pH ranges, HEPES is a biionic sulfonic acid buffer and is generally effective as a buffer at pH 6.8–8.2. HEPES is widely used in cell culture, partly due to its ability to maintain physiological pH despite changes in carbon dioxide concentration compared to bicarbonate buffers. The buffer strength for cell culture applications is typically in the range of 10–25 mM. HEPES buffers can be prepared by one of several methods. For example, HEPES free acid can be added to water and then titrated to the desired pH with approximately half a molar equivalent of sodium hydroxide or potassium hydroxide; a simple mixing table for preparing 0.05 M HEPES / NaOH has been published. Alternatively, equimolar concentrations of HEPES free acid and sodium HEPES can be mixed in approximately equal volumes and back-titrated to the appropriate pH with either solution. Other forms of HEPES include potassium HEPES and hemi-sodium HEPES. Any suitable HEPES buffer may be used in connection with the present invention such that the HEPES buffer has the reduced impurities discussed herein.

[0048] The terms "cell culture medium" and "culture medium" typically refer to a nutrient solution used for proliferating cells, such as eukaryotic cells, that supplies nutrients necessary to enhance cell proliferation, including carbohydrate energy sources, essential amino acids (e.g., phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine) and non-essential amino acids (e.g., alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine), trace elements, energy sources, lipids, and vitamins. Cell culture mediums may contain extracts that supply raw materials to support cell proliferation, such as serum or peptone (hydrolyzate). The medium may contain yeast-derived extracts or soybean extracts instead of animal-derived extracts. A chemically defined medium refers to a cell culture medium in which all chemical components are known (i.e., has a known chemical structure). A chemically defined medium is completely free of animal-derived components, such as serum or animal-derived peptone. In one embodiment, the culture medium is a chemically defined culture medium.

[0049] The culture medium may also contain components that promote growth and / or survival at a rate exceeding the minimum rate, including hormones and growth factors. The culture medium is preferably formulated to have an optimal pH and salt concentration for cell survival and growth.

[0050] In certain embodiments, the cell culture medium may be serum-free. "Serum-free" applies to cell culture media that do not contain animal serum, such as fetal bovine serum. Serum-free media may contain hydrolysates, such as soybean hydrolysate, at a concentration of ≤16 g / L. The present invention also provides a chemically defined medium having reduced impurities, which is not only serum-free but also hydrolysate-free. "Hydrolysate-free" applies to cell culture media that do not contain exogenous protein hydrolysates, such as animal or plant protein hydrolysates, such as peptone or tryptone.

[0051] "Basic medium" is the initial medium (e.g., present in the seed train and / or on day 0 of cell culture production) in which cells grow and which contains all the necessary nutrients, including the basic mixture of amino acids. Various recipes (i.e., formulations) of basic medium can be manufactured or purchased in commercial lots. Similarly, "basic supply medium" contains a mixture of supplemental nutrients that are commonly consumed during production culture and utilized in supply strategies (for so-called "fed-batch" culture). Various basic supply media are commercially available. "Supply" involves scheduled additions (one or more) to the medium at regular intervals, such as according to a protocol including a continuous supply culture system like a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov.-December; 17(6):1032-41) or according to a fed-batch culture process (YMHuang et al., Biotechnol Prog. 2010 Sept.-October; 26(5):1400-10). For example, the culture may be supplied once a day, once every two days, once every three days, or when the concentration of a specific culture medium component being monitored falls outside the desired range.

[0052] While not intended to be limiting, the present invention can be carried out in one or more of a variety of basic culture media or combinations thereof. Basic media are generally known in the art, particularly Eagle MEME (Minimum Essential Medium) (Eagle, Science, 1955, 112(3168):501-504), Ham F12 (Ham, Proc. Nat'l. Acad. Sci. USA, 1965, 53:288-293), F-12K medium, Dulbecco's medium, Dulbecco's modified Eagle medium (Proc. Natl. Acad. Sci. USA, August 1952; 38(8):747-752), DMEM / Ham F12 1:1, Trowell's T8 medium, A2 medium (Holmes and Wolf, Biophys. Biochem. Cytol., 1961, 10:389-401), Waymouth medium (Davidson and Waymouth, Biochem. J., 1945, 39(2):188-199, Williams E medium (William's et al., Exp. Cell Res., 1971, 69:105 et seq.), RPMI1640 (Moore et al., J. Amer. Med. Assoc., 1967, 199:519-524), MCDB104 / 110 medium (Bettger et al., Proc. Nat'l. Acad. Sci. USA, 1981, 78(9):5588-5592), Ventrex HL-1 medium, Albumin-globulin medium (Orr et al. al., Appl. Microbiol., 1973, 25(1):49-54), RPMI-1640 medium, RPMI-1641 medium, Iskov modified Dulbecco medium, McCoy 5A medium, Leibowitz L-15 medium, and serum-free media, e.g., EX-CELL(trademark) 300 series (JRH Biosciences, Lenexa, Kans.), protamine-zinc-insulin medium (Weiss et al.(1974, U.S. Patent No. 4,072,565), Biotin-Folic Acid Medium (Cartaya, 1978, U.S. Re30,985), Transferrin-Fatty Acid Medium (Baker, 1982, U.S. Patent No. 4,560,655), Transferrin-EGF Medium (Hasegawa, 1982, U.S. Patent No. 4,615,977; Chessebeu, 1984, U.S. Patent No. 4,786,599), and other medium substitutes (Inlow, U.S. Patent No. 6,048,728; Drapeau, U.S. Patent No. 7,294) Examples include: 484; Mather, 5,122,469; Furukawa, 5,976,833; Chen, 6,180,401; Chen, 5,856,179; Etcheverry, 5,705,364; Etcheverry, 7,666,416; Ryll, 6,528,286; Singh, 6,924,124; Luan, 7,429,491, etc.

[0053] In certain embodiments, the cell culture medium having reduced impurities according to the present invention comprises a basic medium containing all the nutrients necessary for viable cell culture and a HEPES buffer. The HEPES buffer may be a component of the basic medium or may be added to the cell culture medium. According to embodiments of the present invention, the cell culture medium having reduced impurities comprises less than 4000 ppm of HEPES-related impurities with a molecular weight (MW) of 267.07 (4000 μmol of HEPES impurities with MW 267.07 per mole of total HEPES) relative to the amount of HEPES buffer present in the cell culture medium, and less than 400 ppm of HEPES-related impurities with a molecular weight (MW) of 221.06 (400 μmol of HEPES impurities with MW 221.06 per mole of total HEPES) relative to the amount of HEPES buffer present in the cell culture medium.

[0054] For example, the amount of HEPES-related impurities relative to the amount of HEPES buffer present is generally related to the amount of impurities normalized to HEPES in the culture medium. For instance, the relative amount may be determined using standard analytical techniques such as HPLC or LC-MS, where relative amount (impurity, ppm) = peak area (impurity) / peak area (HEPES + HEPES dimer + HEPES adduct) × 1,000,000.

[0055] In certain embodiments, the culture medium contains HEPES-related impurities with a molecular weight (MW) of 267.07 in amounts less than 4000 ppm, less than 3500 ppm, less than 3200 ppm, less than 3000 ppm, less than 2900 ppm, less than 2500 ppm, less than 2200 ppm, less than 2000 ppm, less than 1800 ppm, less than 1500 ppm, less than 1200 ppm, less than 1000 ppm, and less than 800 ppm, relative to the amount of HEPES buffer present in the cell culture. In other words, these are HEPES impurities with a MW of 267.07 per mole of total HEPES, in amounts of less than approximately 4000 μmol, less than approximately 3500 μmol, less than approximately 3200 μmol, less than approximately 3000 μmol, less than approximately 2900 μmol, less than approximately 2500 μmol, less than approximately 2200 μmol, less than approximately 2000 μmol, less than approximately 1800 μmol, less than approximately 1500 μmol, less than approximately 1200 μmol, less than approximately 1000 μmol, and less than approximately 800 μmol. In certain embodiments, the culture medium contains HEPES-related impurities with a molecular weight (MW) of 221.06, in amounts such as less than 500 ppm, less than 450 ppm, less than 400 ppm, less than 390 ppm, less than 370 ppm, less than 350 ppm, less than 320 ppm, less than 300 ppm, less than 250 ppm, less than 200 ppm, less than 150 ppm, less than 100 ppm, less than 80 ppm, less than 75 ppm, and less than 70 ppm, relative to the amount of HEPES buffer present in the cell culture. In other words, these are HEPES impurities with a MW of 221.06 per mole of total HEPES, in amounts of less than approximately 500 μmol, less than approximately 450 μmol, less than approximately 400 μmol, less than approximately 390 μmol, less than approximately 370 μmol, less than approximately 350 μmol, less than approximately 320 μmol, less than approximately 300 μmol, less than approximately 250 μmol, less than approximately 200 μmol, less than approximately 150 μmol, less than approximately 100 μmol, less than approximately 80 μmol, less than approximately 75 μmol, and less than approximately 70 μmol.

[0056] In one particular embodiment, the culture medium contains less than 4000 ppm of HEPES-related impurities with a molecular weight (MW) of 267.07 relative to the amount of HEPES buffer present in the cell culture medium (4000 μmol of HEPES impurities with a molecular weight of 267.07 per mole of total HEPES), and less than 400 ppm of HEPES-related impurities with a molecular weight (MW) of 221.06 relative to the amount of HEPES buffer present in the cell culture medium (400 μmol of HEPES impurities with a molecular weight of 221.06 per mole of total HEPES). In another embodiment, the culture medium contains less than 3900 ppm of HEPES-related impurities with a molecular weight (MW) of 267.07 relative to the amount of HEPES buffer present in the cell culture medium, and less than 390 ppm of HEPES-related impurities with a molecular weight (MW) of 221.06 relative to the amount of HEPES buffer present in the cell culture medium. In further embodiments, the culture medium contains less than 800 ppm of HEPES-related impurities with a molecular weight (MW) of 267.07 and less than 80 ppm of HEPES-related impurities with a molecular weight (MW) of 221.06, relative to the amount of HEPES buffer present in the cell culture medium. As used herein, 1 ppm of HEPES impurities = 1 μmol of HEPES impurities / moles of total HEPES.

[0057] In other embodiments, the HEPES buffer itself has reduced HEPES-related impurities as described herein. For example, the HEPES buffer may contain HEPES-related impurities with a molecular weight (MW) of 267.07 in the total amount of HEPES buffer used in conjunction with a cell culture (e.g., in the culture medium) in amounts of less than 4000 ppm, less than 3500 ppm, less than 3200 ppm, less than 3000 ppm, less than 2900 ppm, less than 2500 ppm, less than 2200 ppm, less than 2000 ppm, less than 1800 ppm, less than 1500 ppm, less than 1200 ppm, less than 1000 ppm, or less than 800 ppm. In other words, these are HEPES impurities with a MW of 267.07 per mole of total HEPES, in amounts of less than approximately 4000 μmol, less than approximately 3500 μmol, less than approximately 3200 μmol, less than approximately 3000 μmol, less than approximately 2900 μmol, less than approximately 2500 μmol, less than approximately 2200 μmol, less than approximately 2000 μmol, less than approximately 1800 μmol, less than approximately 1500 μmol, less than approximately 1200 μmol, less than approximately 1000 μmol, and less than approximately 800 μmol. In certain embodiments, the culture medium contains HEPES-related impurities with a molecular weight (MW) of 221.06, such as less than 500 ppm, less than 450 ppm, less than 400 ppm, less than 390 ppm, less than 370 ppm, less than 350 ppm, less than 320 ppm, less than 300 ppm, less than 250 ppm, less than 200 ppm, less than 150 ppm, less than 100 ppm, less than 80 ppm, less than 75 ppm, and less than 70 ppm, relative to the total amount of HEPES buffer used in conjunction with the cell culture (e.g., in the medium). In other words, these are HEPES-related impurities with a MW of 221.06 per mole of total HEPES, in amounts of less than approximately 500 μmol, less than approximately 450 μmol, less than approximately 400 μmol, less than approximately 390 μmol, less than approximately 370 μmol, less than approximately 350 μmol, less than approximately 320 μmol, less than approximately 300 μmol, less than approximately 250 μmol, less than approximately 200 μmol, less than approximately 150 μmol, less than approximately 100 μmol, less than approximately 80 μmol, less than approximately 75 μmol, and less than approximately 70 μmol.

[0058] More specifically, according to an embodiment of the present invention, the HEPES-related impurity has the chemical formula and molecular weight (MW) shown in Table 1. [Table 1]

[0059] While not intended to be limited to theory, the following chemical structures have been proposed for HEPES-related impurities based on their chemical formulas and molecular weights (MW). However, the present invention is not limited to the presentation of these proposed chemical structures, and other chemical structures corresponding to the chemical formulas and molecular weights (MW) of HEPES-related impurities are assumed to be within the scope of the present invention. [ka]

[0060] The cell culture medium may also be supplied periodically (as in so-called "fed-batch" culture) depending on the requirements of the cells being cultured or the desired cell culture parameters, with or without increased concentrations of additional components such as polyamines or components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, and trace elements.

[0061] In certain embodiments, the cell culture medium may be depleted of amino acids over the course of recombinant protein production without additional amino acid supplementation, or the cell culture medium may be “non-depleted” with amino acid supplementation provided for the depleted amino acids (as described below).

[0062] In one embodiment, the culture medium also contains 100 μM ± 15 μM of ornithine, or 300 μM ± 45 μM of ornithine, or 600 μM ± 90 μM of ornithine, or 900 μM ± 135 μM of ornithine. In another embodiment, the culture medium contains at least about 5 mg / L ± 1 mg / L of ornithine.HCl, or at least about 10 mg / L ± 2 mg / L of ornithine.HCl, or at least about 15 mg / L ± 2.25 mg / L of ornithine.HCl, or at least about 50 mg / L ± 7.5 mg / L of ornithine.HCl, or at least about 100 mg / L ± 15 mg / L of ornithine.HCl, or at least about 150 mg / L ± 22.5 mg / L of ornithine.HCl.

[0063] Putrescine can be optionally added to supplemented culture media. Putrescine is included in very low concentrations as an ingredient in several cell culture medium formulations, for example, WO2005 / 028626; U.S. Patent No. 5,426,699 (0.08 mg / L); U.S. Patent No. RE30,985 (0.16 mg / L); U.S. Patent No. 5,811,299 (0.27 mg / L); U.S. Patent No. 5,122,469 (0.5635 mg / L); U.S. Patent No. 5,063,157 (1 mg / L); WO2008 / 154014 (approximately 100 mg / L). See U.S. Patent Application No. 2007 / 0212770 (0.5 to 30 mg / L polyamines; 2 mg / L putrescine + 2 mg / L ornithine; 2 mg / L putrescine + 10 mg / L ornithine).

[0064] In some embodiments, the cell culture medium is further supplemented with a combination of ornithine and putrescine, where the concentration of putrescine may be at least about 150–720 μM. In some embodiments, the medium is further supplemented with putrescine at a concentration of about 170–230 μM. In one embodiment, the medium contains ≥90 μM ± 15 μM of ornithine in addition to 200 μM ± 30 μM of putrescine. In one embodiment, the medium contains ≤15 mg / L ± 2.25 mg / L of ornithine in addition to ≤30 mg / L ± 4.5 mg / L of putrescine .2HCl. In another embodiment, the medium contains ≥15 mg / L ± 2.25 mg / L of ornithine .HCl in addition to ≥30 mg / L ± 4.5 mg / L of putrescine .2HCl. (See International Publication No. 2014 / 144198A1, published September 18, 2014.)

[0065] In yet another embodiment, ornithine is present in the culture medium at concentrations ranging from 0.09±0.014 mM to 0.9±0.14 mM, for example, 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine. In some embodiments, the culture medium also contains at least 0.20±0.03 mM of putrescine. In some embodiments, the additional putrescine is at concentrations ranging from 0.20±0.03 mM to 0.714±0.11 mM, for example, 0.20±0.03 mM, 0.35±0.06 mM, or 0.714±0.11 mM of putrescine.

[0066] In yet another embodiment, the culture medium may be supplemented with taurine at a concentration of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM (expressed as millimoles per liter).

[0067] Various other nutritional supplements may be added to the culture medium, and determining additional suitable conditions is within the scope of the art of the art. In some embodiments, to prevent depletion or when supplemental nutrients are needed, the culture medium is supplemented with a mixture of amino acids selected from the group consisting of aspartic acid, cysteine, glutamic acid, glycine, lysine, phenylalanine, proline, serine, threonine, valine, arginine, histidine, asparagine, glutamine, alanine, isoleucine, leucine, methionine, tyrosine, and tryptophan.

[0068] In one embodiment, the culture medium is further supplemented with approximately 170 μM to 175 μM of nucleoside. In one embodiment, the culture medium contains a purine derivative at a cumulative concentration of at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65 μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least 100 μM, or at least 105 μM. In one embodiment, the culture medium contains a purine derivative at a cumulative concentration of at least 100 μM to 110 μM. Examples of purine derivatives include hypoxanthine, nucleoside adenosine, and guanosine. In one embodiment, the culture medium contains a pyrimidine derivative at a cumulative concentration of at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, or at least 65 μM. In one embodiment, the culture medium contains approximately 65 μM to 75 μM of a pyrimidine derivative. Examples of pyrimidine derivatives include nucleoside thymidine, uridine, and cytidine. In one particular embodiment, the culture medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0069] In addition to including any of the above additives, in one embodiment the culture medium is further supplemented with micromolar amounts of fatty acids (or fatty acid derivatives) and tocopherol. In one embodiment the fatty acids include one or more of linoleic acid, linolenic acid, thiotic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. In one embodiment the culture medium contains tocopherol, linoleic acid, and thioctic acid.

[0070] In one embodiment, the culture medium may also be further supplemented with a mixture of vitamins containing other nutrients and essential nutrients at a cumulative concentration of at least about 700 μM or at least about 2 mM. In one embodiment, the vitamin mixture contains one or more of the following: D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, and vitamin B12. In one embodiment, the vitamin mixture contains all of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, and vitamin B12.

[0071] Various embodiments of the culture medium of the present invention having reduced impurities include any combination of the above embodiments, in addition to a chemically defined medium, HEPES buffer, and in particular (a) amino acids, (b) optionally nucleosides, (c) divalent cation salts, (d) fatty acids and tocopherols, and (e) vitamins.

[0072] In certain embodiments, the cell culture medium having reduced impurities may be chemically defined and may include HEPES buffer, an amino acid mixture as considered herein, CaCl22H2O;KCl;MgSO4;NaCl;Na2HPO4 or other phosphates;pyruvic acid;D-biotin;choline chloride;folic acid;myo-inositol;niacinamide;pyridoxine HCl;D-pantothenic acid;riboflavin;thiamine HCl;vitamin B12;ρ-aminobenzoic acid;ethanolamine HCl;poloxamer 188;DL-α-tocopherol phosphate;linolenic acid;Na2SeO3;thiotic acid;and glucose;and optionally adenosine;guosan;cytidine;uridine;thymidine;and hypoxanthine disodium.

[0073] In one embodiment, the initial osmotic molar concentration of the culture medium of the present invention is 200-500, 250-400, 275-350, or about 300 mOsm. While growing cells in the culture medium of the present invention, in particular after any feed according to a fed-batch culture protocol, the osmotic molar concentration of the culture can increase to a maximum of about 350, 400, 450, 500, or a maximum of about 500 mOsm.

[0074] In some embodiments where the osmotic molar concentration of the culture medium is less than about 300, the osmotic molar concentration can be adjusted to about 300 by adding one or more salts in amounts greater than specified. In one embodiment, the osmotic molar concentration is increased to a desired level by adding one or more osmoregulators selected from sodium chloride, potassium chloride, magnesium salts, calcium salts, amino acid salts, fatty acid salts, sodium bicarbonate, sodium carbonate, potassium carbonate, chelating agents which are salts, sugars (e.g., galactose, glucose, sucrose, fructose, fucose, etc.), and combinations thereof. In one embodiment, the osmoregulator is added in a concentration greater than or equal to that of the components already present in the specified culture medium (e.g., sugars are added in a concentration greater than or equal to that of the sugar components).

[0075] Any embodiment of the culture medium described herein, as well as any other culture medium containing reduced amounts of HEPES-related impurities as described herein, shall be referred to as a culture medium with reduced impurities or a culture medium with reduced HEPES-related impurities. Conversely, a culture medium containing amounts of HEPES-related impurities exceeding those levels considered herein shall hereafter be referred to as a non-reduced impurity culture medium or a non-reduced HEPES-related impurity culture medium. In some embodiments, a non-reduced impurity culture medium contains the same basic medium and nutritional supplements as a culture medium with reduced impurities, except for the presence of the impurities considered herein.

[0076] cell culture One aspect of the present invention provides a cell culture comprising a cell line expressing a recombinant protein of interest in a medium having reduced impurities, as described herein. Examples of cell lines routinely used to produce recombinant proteins include, among others, 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, 3T3 cells, 293 cells, Per.C6 cells, and chicken embryo cells. In one embodiment, the cell line is a CHO cell line, or one or more of several specific CHO cell variants optimized for large-scale protein production, for example, CHO-K1.

[0077] Another aspect of the present invention relates to a method for culturing cells using a medium having reduced impurities as described herein, the use of such a medium, compared to culturing such cells in a medium without reduced impurities, particularly by its use in production culture and / or seed train culture, improves the titer of one or more recombinant proteins of interest by such cells and promotes the proliferation of recombinant eukaryotic cells while maintaining cell viability.

[0078] In some embodiments, recombinant protein titer is improved compared to cells grown in unreduced impurity medium. In some embodiments, the protein titer obtained from cell cultures in the medium having reduced impurities of the present invention is at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 35%, at least about 40%, or at least about 50% greater than the protein titer (yield) obtained from cells cultured in unreduced impurity medium. In some embodiments, the protein titer of cell cultures formed in the medium containing reduced impurities of the present invention is greater than that of similar or identical cells cultured in a medium without reduced impurities.

[0079] In some embodiments, cell proliferation (e.g., doubled acceleration), viable cell density, cell viability, and combinations thereof are improved compared to cells grown in a medium without reduced impurities.

[0080] In some embodiments, the doubling acceleration of viable cells in a medium having reduced impurities according to the present invention is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 3 times greater than the doubling acceleration of cells cultured in a medium without reduced impurities. In some embodiments, the doubling acceleration of viable cells in the culture medium having reduced impurities according to the present invention is about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% greater than the doubling acceleration of viable cells in the un-impured culture medium.

[0081] In some embodiments, the doubling time for actively cycling mammalian cells is less than 30 hours, less than 29 hours, less than 28 hours, less than 27 hours, less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, or less than 18 hours in a medium with reduced impurities. In some embodiments, the doubling time for actively growing mammalian cells is less than 28 hours in a medium with reduced impurities. In some embodiments, the doubling time for mammalian cells is about 27±1 hours, about 26±1 hours, about 25±1 hours, about 24±1 hours, about 23±1 hours, about 22±1 hours, or about 21±1 hours in a medium with reduced impurities. In some embodiments, the doubling time for actively cycling mammalian cells is about 24±1 hours in a medium with reduced impurities. In some embodiments, the doubling time of actively dividing cells cultured in a medium with reduced impurities is at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, or at least 25% shorter than the doubling time of actively cycling cells cultured in a medium without reduced impurities.

[0082] With respect to cell viability, cells grown in a medium with reduced impurities exhibit a viability at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, or at least three times higher than cells grown in a medium with reduced impurities.

[0083] In a production culture vessel or bioreactor, the basic culture medium and cells are supplied to the culture vessel after seed culture or growth phase. In certain embodiments, the cell supernatant or cell lysate is collected after production culture. In other embodiments, the polypeptide or protein of interest is recovered from the culture medium or cell lysate, or in any case, depending on the location of the protein of interest, using techniques well known in the art.

[0084] A "cell line" refers to one or more cells derived from a specific lineage through the continuous passage or subculturing of cells. The term "cell" is used interchangeably with "cell population."

[0085] The term "cell" includes any cell suitable for the expression of recombinant nucleic acid sequences. Cells include non-human animal cells, mammalian cells, human cells, avian cells, insect cells, yeast cells, or eukaryotic cells such as cell fusions, such as hybridomas or quadromas. In certain embodiments, cells are human, monkey, ape, hamster, rat, or mouse cells. In certain embodiments, the cells are selected from the following cells: CHO (e.g., CHOK1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC5, Colo25, HB8065, HL-60, lymphocytes (e.g., Jurkat (T lymphocyte) or Daudi (B lymphocyte)), A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cells include one or more viral genes, for example, retinal cells expressing a viral gene (e.g., PER.C6® cells). In some embodiments, the cells are CHO cells. In other embodiments, the cells are CHO K1 cells.

[0086] In recombinant protein production, “fed-batch cell culture” or “fed-batch culture” refers to a fed-batch culture in which animal cells and culture medium are initially supplied to the culture tube, and additional culture nutrients are slowly supplied to the culture in continuous or individual increments during the culture, with or without periodic collection of cells and / or products before the end of the culture. Fed-batch culture includes “semi-continuous fed-batch culture,” in which the entire culture (which may include cells and medium) is periodically removed and replaced with fresh medium. Fed-batch culture is distinguished from simple “batch culture,” in which all components for cell culture (including animal cells and all culture nutrients) are supplied to the culture vessel at the start of the culture process in batch culture. Fed-batch culture can be further distinguished from perfusion culture insofar as the supernatant is removed from the culture vessel during the process, whereas in perfusion culture, cells are left in the culture, for example by filtration, and the culture medium is continuously or intermittently introduced and removed from the culture vessel. However, the removal of samples from the fed-batch cell culture for validation purposes is intended. The fed-batch culture process is continued until it is determined that the maximum working volume and / or maximum protein production has been reached.

[0087] As used herein, the term “continuous cell culture” refers to a technique typically used to continuously grow cells in a particular growth phase. For example, if a constant supply of cells is required, or if the production of a specific polypeptide or protein of interest is required, cell culture may require maintenance in a particular growth phase. Therefore, conditions must be continuously monitored and adjusted as needed to maintain the cells in that particular phase.

[0088] One aspect of the present invention relates to a seed culture in which a cell population is expanded before protein production and harvesting in a production culture. In certain embodiments, a cell culture medium having reduced impurities may be used with the seed cell culture, as further described herein.

[0089] Another aspect of the present invention relates to a production culture from which proteins are produced and harvested. Prior to the production phase, there is typically a growth phase (also known as a seed train or seed culture) in which all components for cell culture are supplied to the culture vessel at the start of the culture process, and the cell population is then expanded until it is ready for production scale. Thus, the culture vessel is inoculated with cells at a seeding density suitable for the initial cell growth phase, depending on the starting cell line. In some embodiments, a cell culture medium with reduced impurities may be used with the seed cell culture to further improve or enhance the productivity of the cells in the subsequent production phase. In other embodiments, the cell culture medium with reduced impurities may be used with the production cell culture as further described herein.

[0090] Examples of culture vessels include, but are not limited to, well plates, T-flasks, vibrating flasks, stirring vessels, spinner flasks, hollow fiber vessels, and air-lift bioreactors. A preferred cell culture vessel is a bioreactor. A bioreactor refers to a culture vessel manufactured or operated to manipulate or control environmental conditions. Such culture vessels are well known in the art.

[0091] Bioreactor processes and systems have been developed to optimize gas exchange, supply sufficient oxygen to maintain cell growth and productivity, and remove CO2. Maintaining the efficiency of gas exchange is a critical criterion for ensuring the successful scale-up of cell culture and protein production. Such systems are well known to those skilled in the art.

[0092] In one embodiment, the culture medium is replenished at intervals during cell culture according to a fed-batch culture process. Fed-batch culture is generally known in the art and is used to optimize protein production (see YMHuang et al., Biotechnol Prog. 2010 Sep-October;26(5):1400-10). The fed-batch culture process is typically used during the production phase.

[0093] Supplementation may be performed daily or every 2-3 days during the production culture period, containing additional nutrients as described herein, such as vitamins, amino acids, and other nutrients. Supplementation may be performed at least twice, or at least eight times, throughout the duration of the production culture for cultures lasting two weeks or more (by adding supplement medium containing nutrients). In another embodiment, supplementation may be performed daily during the culture period. Alternative culture supply schedules are also envisioned.

[0094] Non-depleted media can also be provided by supplementing with additional amino acids, the depleted amino acids being known in the art and determined according to the methods described herein. When using this regime, additional amino acids are preferably supplemented or added daily or every 2-3 days during the period of production culture, depending on the determination of amino acid depletion. In one embodiment, a mixture of additional amino acids to maintain non-depleted cell culture medium is added around day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, and day 14 for cultures of two weeks or more. Alternative culture supply schedules are also envisioned.

[0095] Eukaryotic cells such as CHO cells can be cultured in small-scale cultures, such as 125 mL containers with approximately 25 mL of medium, 250 mL containers with approximately 50–100 mL of medium, and 500 mL containers with approximately 100–200 mL of medium. Alternatively, cultures can be large-scale, for example, 1000 mL containers with approximately 300–1000 mL of medium, 3000 mL containers with approximately 500–3000 mL of medium, 8000 mL containers with approximately 2000–8000 mL of medium, and 15000 mL containers with approximately 4000–15000 mL of medium. Cultures for production can contain 10,000 L or more of medium. Large-scale cell cultures, such as those for the clinical production of protein therapeutics, are typically maintained for several days or weeks, during which time the cells produce the desired protein. During this time, the culture can be replenished with concentrated supply medium containing nutrients and components such as amino acids consumed during the culture process. Concentrated feed media can be based on any cell culture medium formulation. Such concentrated feed media can contain most of the components of the cell culture medium in typical useful amounts, for example, about 5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×, 600×, 800×, or about 1000×. Concentrated feed media are often used in fed-batch culture processes.

[0096] In some embodiments, cell culture may be further 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 protein production. 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 usually 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 protein production, such as tetrahydrofolate (THF) and methotrexate. Non-exclusive 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 line-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), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), transition-stimulating factor, myoglobulin Examples include statins (GDF-8), nerve growth factors (NGF) and other neurotrophic factors, 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 (PIGF), fetal bovine growth hormone (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 growth factor insulin added at the time of use.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.

[0097] Buffers are generally known in the art. The present invention is not limited to any particular one or more buffers, and those skilled in the art can select a suitable buffer or buffer system for use with a particular cell line that produces a particular protein. In one embodiment, the point-of-use buffer is NaHCO3. In another embodiment, the buffer is HEPES. In yet another embodiment, the point-of-use buffer contains both NaHCO3 and HEPES. In embodiments in which the buffer contains HEPES, the HEPES buffer contains reduced amounts of HEPES-related impurities as described herein.

[0098] Energy sources for use as point-of-use additives in cell culture are also well known in the art. In one embodiment, but not limited to, the point-of-use energy source is glucose. Taking into account the specific and particular requirements of a particular cell line and the protein to be produced, in one embodiment glucose can be added to the culture medium to a concentration of about 1 to 20 mM. In some cases, glucose can be added at high levels of 20 g / L or more.

[0099] Chelating agents are also well known in cell culture and protein production techniques. Tetrasodium EDTA dihydrate 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 chelating agent added at the time of use is tetrasodium EDTA dihydrate. In one embodiment, the chelating agent added at the time of use is a citrate such as Na3C6H5O7.

[0100] In one embodiment, the cell culture medium may be supplemented with one or more time-of-use added amino acids as an energy source, such as glutamine. In one embodiment, the cell culture medium is supplemented with time-of-use added glutamine at a final concentration of approximately 1 mM to 13 mM.

[0101] Other point-of-use additives include one or more of various metal salts, such as iron, nickel, zinc, and copper salts. In one embodiment, the cell culture medium is supplemented with any one or more of copper sulfate, zinc sulfate, ferrous chloride, and nickel sulfate.

[0102] Protein production In addition to culture media having reduced impurities and methods for culturing cells in such media, the present invention provides methods for improving cell culture performance, including improving recombinant protein titer in recombinant protein production by culturing recombinant eukaryotic cells. In some embodiments, the recombinant eukaryotic cells contain stably incorporated nucleic acids encoding recombinant proteins. In other embodiments, the methods of the present invention provide improved cell proliferation (e.g., doubled acceleration), viable cell density, cell viability, and combinations thereof.

[0103] In some embodiments, the method of the present invention comprises providing a cell culture medium having reduced impurities of the present invention; culturing recombinant eukaryotic cells in the medium; expressing a target recombinant protein from the recombinant eukaryotic cells; and producing a higher titer of the recombinant protein from recombinant eukaryotic cells cultured in the medium having reduced impurities compared to similar or identical recombinant eukaryotic cells cultured in a medium without reduced impurities.

[0104] In some embodiments, the protein production rate or titer, which can be expressed in grams of protein product per liter of culture medium, from cells cultured in a medium containing reduced impurities, is at least 100 mg / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, at least 2.5 g / L, at least 3 g / L, at least 3.5 g / L, at least 4 g / L, at least 4.5 g / L, at least 5 g / L, at least 5.5 g / L, at least 6 g / L, at least 6.5 g / L, at least 7 g / L, at least 7.5 g / L, at least 8 g / L, at least 8.5 g / L, at least 9 g / L, at least 9.5 g / L, at least 10 g / L, at least 15 g / L, or at least 20 g / L.

[0105] In some embodiments, the protein titer obtained from cells cultured in a medium with reduced impurities is at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, or at least about 29% greater than the protein titer (yield) from cells cultured in a medium without reduced impurities or similar cells.

[0106] In some embodiments, the protein titer (yield) of mammalian cells cultured in a medium containing the reduced impurities described herein is at least 100 mg / L, at least 0.5 g / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, and at least 2.5 g / L greater than the protein titer of similar or identical cells cultured in a medium without reduced impurities.

[0107] The methods of the present invention are useful for improving protein production through cell culture processes. The cell lines used in the present invention can be genetically engineered to express recombinant proteins of commercial or scientific interest. Genetically engineering cell lines involves transfection, transformation, or transduction of cells with recombinant polynucleotide molecules (e.g., by homologous recombination and gene activation, or by fusion of recombinant and non-recombinant cells) to cause host cells to express a desired recombinant polypeptide. Methods and vectors for genetically engineered cells or cell lines to express the polypeptide of interest are well known to those skilled in the art, and various techniques are exemplified, for example, in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69. A diverse range of cell lines suitable for growing cultures can be obtained from the American Type Culture Collection (Manassas, Va.) and commercial vendors.

[0108] In some embodiments, the protein product (target protein) is an antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, multispecific 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.

[0109] In some embodiments, the antibody may be an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Publication No. 2015 / 0203579A1), an anti-programmed cell death ligand-1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Publication No. 2015 / 0203580A1), an anti-DII4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), or an anti-angiopoietin-like 3 antibody (e.g., as described in U.S. Patent No. 9,018,356). Anti-AngPtl3 antibodies (such as), anti-platelet-derived growth factor receptor antibodies (e.g., anti-PDGFR antibodies as described in U.S. Patent No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies as described in U.S. Patent No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies as described in U.S. Patent Publication No. 2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies as described in U.S. Patent No. 9,132,192), Or anti-EGFRvIII antibodies as described in U.S. Patent Application Publication No. 2015 / 0259423A1), anti-precursor protein-converting enzyme subtilisin kexin-9 (e.g., anti-PCSK9 antibodies as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. 2014 / 0044730A1), anti-proliferation and differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies as described in U.S. Patent No. 8,871,209 or U.S. Patent No. 9,260,515), anti-glucagon receptor ( For example, anti-GCGR antibodies as described in U.S. Patent Publication No. 2015 / 0337045A1 or No. 2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin-4 receptor antibodies (for example, anti-IL4R antibodies as described in U.S. Patent Publication No. 2014 / 0271681 or U.S. Patent No. 8,735,095 or No. 8,945,559), anti-interleukin-6 receptor antibodies (for example, U.S. Patent No. 7,582,298, No. 8,043,617 or No. 9,173,Anti-IL6R antibodies (such as those 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 (for example, anti-IL33 antibodies as described in U.S. Patent Publication No. 2014 / 0271658A1 or 2014 / 0271642A1), anti-respiratory syncytial virus antibodies (for example, U.S. Patent Publication No. 2014 / 0271653A Anti-RSV antibodies as described in item 1), anti-differentiation antigen group 3 (for example, anti-CD3 antibodies as described in U.S. Patent Application Publication No. 2014 / 0088295A1 and No. 2015026666A1, and U.S. Patent Application No. 62 / 222,605), anti-differentiation antigen group 20 (for example, as described in U.S. Patent Application Publication No. 2014 / 0088295A1 and No. 2015266966A1, and U.S. Patent No. 7,879,984) Anti-CD20 antibodies, anti-CD19 antibodies, anti-CD28 antibodies, anti-differentiation antigen group 48 (e.g., anti-CD48 antibody as described in U.S. Patent No. 9,228,014), anti-Feld1 antibodies (e.g., as described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome virus (MMS antibody as described in U.S. Patent Publication No. 2015 / 0337029A1), anti-Ebola virus antibodies (e.g., U.S. Patent Publication No. 9,079,948) The antibodies are selected from the group consisting of anti-Zika virus antibodies (such as those described in Patent Application Publication No. 2016 / 0215040), anti-lymphocyte activator gene 3 antibodies (e.g., anti-LAG3 antibody or anti-CD223 antibody), anti-neuronal growth factor antibodies (e.g., anti-NGF antibodies as described in U.S. Patent Application Publication No. 2016 / 0017029, and U.S. Patents No. 8,309,088 and No. 9,353,176), and anti-activin A antibodies. In some embodiments, the bispecific antibody is selected from the group consisting of anti-CD3 × anti-CD20 bispecific antibodies (as described in U.S. Patent Publications 2014 / 0088295A1 and 20150266966A1), anti-CD3 × anti-mucin 16 bispecific antibodies (e.g., anti-CD3 × anti-Muc16 bispecific antibody), and anti-CD3 × anti-prostate-specific membrane antigen bispecific antibodies (e.g., anti-CD3 × anti-PSMA bispecific antibody).

[0110] In some embodiments, the protein of interest is selected from the group consisting of alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacilibimab, imdevimab, semiprimab, semiprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, facimmab, nesbacumab, trevoglumab, linucumab, and sarilumab.

[0111] In some embodiments, the protein of interest is a recombinant protein containing an Fc moiety and another domain (e.g., an Fc fusion protein). In some embodiments, the Fc fusion protein is a receptor Fc fusion protein containing one or more extracellular domains of a receptor linked to the Fc moiety. In some embodiments, the Fc moiety includes a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor Fc fusion protein contains two or more different receptor chains that bind to a single ligand or to a plurality of ligands. For example, the Fc fusion protein is a TRAP protein, such as an IL-1 trap (e.g., lilonacept containing an IL-1RAcP ligand-binding domain fused to the II-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,044), or a VEGF trap (e.g., aflibercept or ziv-aflibercept containing the Ig domain 2 of VEGF receptor Flt1 fused to the Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patents No. 7,087,411 and 7,279,159). In other embodiments, the Fc fusion protein is an ScFv-Fc fusion protein containing one or more antigen-binding domains, such as variable heavy chain fragments and variable light chain fragments of an antibody bound to the Fc portion.

[0112] In addition, trap proteins that use a polymerizing component (MC) instead of the Fc moiety include minitraps disclosed in U.S. Patent No. 7,279,159 and U.S. Patent No. 7,087,411.

[0113] The above derivatives, components, domains, chains, and fragments are also included.

[0114] The present invention is not limited to any particular type of cell for recombinant protein production. Examples of cell types suitable for recombinant protein production include mammalian cells, insect cells, avian cells, bacterial cells, and yeast cells. The cells may be stem cells or recombinant cells transformed with a vector for recombinant gene expression, or cells transfected with a virus for the production of a viral product. The cells may contain a recombinant heteroseed polynucleotide construct encoding the protein of interest. The construct may be an episome, or it may be an element that is physically integrated into the cell's genome. The cells may also produce the protein of interest without having the protein encoded on a heteroseed polypeptide construct. In other words, the cells may naturally encode the protein of interest, such as antibody-producing B cells. The cells may also be primary cells, such as chicken embryo cells or primary cell lines.

[0115] Examples of useful cells include CHO, COS, retinal cells, Vero, CV1, kidney cells, HeLa, HepG2, WI38, MRC5, Colo25, HB8065, HL-60, lymphocytes, A431, CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. In various embodiments, the cell line is a CHO cell derivative, such as CHO-K1, CHO DUX B-11, CHO DG-44, Veggie-CHO, GS-CHO, S-CHO, or CHO Iec mutant.

[0116] The production phase can be carried out in cultures of any scale, from shaking flasks or wave bags to 1-liter bioreactors and large-scale industrial bioreactors. Similarly, the seed train expansion phase can be carried out in cultures of any scale, from shaking flasks or wave bags to bioreactors of 1 liter or more. Large-scale processes can be carried out in volumes of approximately 100 liters to 20,000 liters or more. One or more of several means may be used to control protein production, such as temperature shifts or chemical induction. The growth phase may occur at a higher temperature than the production phase. For example, the growth phase may occur at a first temperature of approximately 35°C to 38°C, and the production phase may occur at a second temperature of approximately 29°C to 37°C, and optionally at approximately 30°C to 36°C or approximately 30°C to 34°C. In addition, chemical inducers of protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene bisacetoamide (HMBA), may be added simultaneously with, before, or after the temperature shift. If inducers are added after the temperature shift, they can be added 1 to 5 days after the temperature shift, such as 1 to 2 days after the shift. The producing cell cultures may be operated as a continuous feed culture system according to a chemostat (see C. Altamirano et al., 2001 (above)) or a fed-batch culture process (Huang, 2010 (above)).

[0117] Selection of culture medium and buffer solution A further aspect of the present invention relates to a method for screening cell culture media or HEPES buffers for selection for use in cell culture to thereby improve cell culture performance, such as improved protein titer, improved cell proliferation, improved viable cell density, etc. Such a method may be used to select a medium having reduced impurities according to the present invention for use in cell culture, or to select a HEPES buffer having reduced impurities for use in cell culture.

[0118] In some embodiments, a method is provided for selecting a cell culture medium for use in cell culture to improve cell culture performance. The method may generally include providing a cell culture medium containing HEPES buffer; analyzing the cell culture medium containing HEPES buffer to determine the amount of HEPES-related impurities having a molecular weight of 267.07 (MW) and the amount of HEPES-related impurities having a molecular weight of 221.06 (MW) present in the cell culture medium; and selecting the cell culture medium containing HEPES buffer for use in cell culture when it is determined that the cell culture medium containing HEPES buffer has reduced HEPES-related impurities as discussed herein. According to the present invention, the use of a cell culture medium selected according to such a method improves cell culture performance compared to cell culture performance in cell culture medium without reduced HEPES impurities.

[0119] In other embodiments, a method is provided for selecting a HEPES buffer for use in cell culture to improve cell culture performance. The method may generally include providing a HEPES buffer, analyzing the HEPES buffer to determine the amount of HEPES-related impurities having a molecular weight (MW) of 267.07 and the amount of HEPES-related impurities having a molecular weight (MW) of 221.06 present in the cell culture medium, and selecting a HEPES buffer for use in cell culture if it is determined that the HEPES buffer has reduced HEPES-related impurities as discussed herein. According to the present invention, the use of a HEPES buffer selected according to such a method improves cell culture performance compared to cell culture performance using a non-reduced impurity HEPES buffer.

[0120] Any suitable method for analyzing a culture medium or HEPES buffer to quantitatively determine the presence of HEPES-related impurities may be used in connection with the methods disclosed herein. Analytical methodologies for use according to the present invention include HPLC, LC-MS, and other methodologies, including all analytical, separation, and purification methodologies disclosed herein.

[0121] The present invention is not limited to the scope of the specific embodiments described herein, which are intended as examples of individual aspects or embodiments of the present invention. Functionally equivalent methods and components are within the scope of the present invention. In addition to those described herein, various modifications of the present invention will be obvious to those skilled in the art from the foregoing specification and the accompanying drawings. Such modifications are within the scope of the present invention. [Examples]

[0122] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0123] Example 1 - Identification of HEPES-related impurities ANOVA analysis of lot lineages of 53 different components of chemically defined culture media was performed between lots used in "high-titer" and "low-titer" cell culture operations. HEPES acid and salt were identified as "high-risk" components and showed the strongest correlation with the final titer (the correlation was stronger than the correlation between titer and the culture medium as a whole). HEPES was also identified as a "high-risk component" in an independent risk-based analysis that considered the weight fractionation of components in the culture medium formulation, the COA purity of the components, and their manufacturing methods.

[0124] As a follow-up, FTIR and Raman spectroscopy were performed on the culture medium lots. A strong correlation was found between absorption in specific regions of the FTIR spectrum of the medium and the final titer, which were assigned to HEPES (based on known features of the HEPES spectrum) and later confirmed by comparison with the FTIR spectrum from HEPES retainers. The spectral difference between "low titer" and "high titer" lots was consistent with the observed titer results. Data were obtained from additional culture medium lots and used to build a predictive model of the titer performance of incoming culture medium lots.

[0125] Several bands in the Raman spectrum of CDM1B were also found to have a strong correlation with the final titer. Similar to FTIR analysis, these bands were assigned to HEPES by matching the Raman spectrum of the culture medium with the spectrum of the HEPES-retaining sample.

[0126] After identifying HEPES as a "high-risk" component with a strong correlation to the final titer, chemical composition analysis of HEPES buffer lots was performed, including LC-MS and titer correlation assessment. Based on these studies, two HEPES-related impurities were identified, which showed a negative correlation to the titer for all production runs analyzed.

[0127] The two identified HEPES-related impurities were determined to have the chemical formulas and molecular weights (MW) shown in Table 2 below. [Table 2]

[0128] Table 3 below shows all identified impurities, including HEPES+[O2]-[H2] and HEPES-[CH4]. [Table 3]

[0129] Surprisingly, the impurities identified below in Table 4 (compounds in Table 4) did not adversely affect cell titer, even though many were present in greater quantities than HEPES+[O2]-[H2] and HEPES-[CH4]. One or more of the compounds in Table 4 may be present in the culture medium without causing excessive adverse effects on cells. [Table 4]

[0130] While not intended to be limited to theory, the following chemical structures have been proposed for HEPES-related impurities based on their chemical formulas and molecular weights (MW). However, the present invention is not limited to the presentation of these proposed chemical structures, and other chemical structures corresponding to the chemical formulas and molecular weights of HEPES-related impurities are assumed to be within the scope of the present invention. [ka]

[0131] Example 2 - HEPES-related impurities negatively correlated with titer To produce dupilumab, mass production operations were carried out at multiple locations. According to the present invention, as discussed herein, it was found that the amount of HEPES-related impurities affects the protein titer. Based on the findings and in accordance with the present invention, improved protein titers can be obtained by using a culture medium having reduced HEPES-related impurities in accordance with the present invention.

[0132] Figure 1 shows the relationship between the relative amount of HEPES+[O2]-[H2] and protein titer. Figures 2A and 2B show a negative correlation between titer and HEPES+[O2]-[H2] for multiple production runs of dupilumab at two different locations (Figure 2A: 20 production runs at location 1; Figure 2B: 7 production runs at location 2). A summary of the fitted data is shown below each graph in Figures 2A and 2B.

[0133] Figure 3 shows the relationship between the relative amount of HEPES-[CH4] and protein titer. Figures 4A and 4B show a negative correlation between titer and HEPES-[CH4] for multiple production runs of dupilumab at two different locations (Figure 4A, 20 production runs at location 1; Figure 4B, 7 production runs at location 2). A summary of the fitted data is shown below each graph in Figures 4A and 4B.

[0134] As shown in the figure, at both location 1 and location 2, HEPES+[O2]-[H2] and HEPES-[CH4] showed higher abundances in lower potency production operations and exhibited a negative correlation with potency. Similar results observed at different production sites increase the confidence in the conclusion that higher abundances of HEPES+[O2]-[H2] and HEPES-[CH4] are negatively correlated with potency.

[0135] Example 3 - Elucidation of the structure of impurities in HEPES buffer HEPES+[O2]-[H2] and HEPES-[CH4] were separated from HEPES using a hydrophilic interaction liquid chromatography (HILIC) column (Figure 5A shows the target). A mixed-mode column was used to further separate HEPES+[O2]-[H2] and HEPES-[CH4] from other HEPES impurities collected in the fraction from the HILIC separation (Figure 5B shows the target). The combination of both columns can be used for further purification of HEPES+[O2]-[H2] and HEPES-[CH4].

[0136] The structures of HEPES+[O2]-[H2] and HEPES-[CH4] were confirmed using reversed-phase liquid chromatography-mass spectrometry (RP-LCMS), hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LCMS), and MS / MS fragmentation. Figure 6A is the RP-LCMS plot of HEPES-[CH4] (also referred to as "221") supplied from HEPES raw materials. Figure 6B is the HILIC-LCMS plot of HEPES-[CH4] supplied from HEPES raw materials. Figure 7 shows the MS / MS fragmentation of HEPES-[CH4] derived from HEPES raw materials.

[0137] Example 4 - Protein The present invention can be employed in the production of biological products and pharmaceuticals and is suitable for the proliferation of cells containing genes encoding the protein of interest. Each embodiment and example disclosed herein may be used in conjunction with the following in the production of biological products and pharmaceuticals: such proteins may include, but are not limited to, antibodies, receptors, fusion proteins, antagonists, inhibitors, enzymes (such as those used in enzyme replacement therapy), factors and cofactors, cytokines, chemokines, inhibitors, activators, ligands, reporter proteins, selection proteins, protein hormones, protein toxins, structural proteins, storage proteins, transport proteins, neurotransmitters, and contractile proteins. Specific types of proteins that can be produced according to the present invention are discussed in more detail below.

[0138] Antibodies (also called "immunoglobulins") are examples of proteins with multiple polypeptide chains and extensive post-translational modifications. A typical immunoglobulin protein (e.g., IgG) contains four polypeptide chains—two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cysteine ​​disulfide bond, and the two heavy chains are linked to each other via two cysteine ​​disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at various residues (e.g., asparagine residues) by various polysaccharides, which can vary by species and may affect the antigenicity of therapeutic antibodies. Butler and Spearman, “The choice of mammalian cell host and possibilities for glycosylation engineering”, Curr. Opin. Biotech. 30:107-112 (2014).

[0139] The antibody heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are dotted with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody is defined as having at least 10 when measured by surface plasmon resonance, e.g., BIACORE® or solution affinity ELISA. -9 M, at least 10 -10 M, at least 10 -11 M, or at least 10 -12 This refers to antibodies that have binding affinity to those targets of M.

[0140] The antibody light chain comprises an immunoglobulin light chain constant region sequence from any organism, and unless otherwise specified, includes human kappa and lambda light chains. The light chain variable (VL) domain typically includes three light chain CDRs and four framework (FR) regions unless otherwise specified. Generally, a full-length light chain includes a VL domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus, and a light chain constant domain. Examples of light chains that can be used in these inventions include light chains that do not selectively bind to either a first or second antigen selectively bound by an antigen-binding protein. Preferred light chains can be identified by screening for the light chains most commonly used in existing antibody libraries (wet libraries or in silico), and the light chain does not substantially interfere with the affinity and / or selectivity of the antigen-binding domain of the antigen-binding protein. Preferred light chains can bind to one or both epitopes bound by the antigen-binding domain of the antigen-binding protein.

[0141] The antibody variable domain includes an amino acid sequence (optionally modified) of an immunoglobulin light or heavy chain containing the following amino acid regions (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the N-terminus to the C-terminus. The "variable domain" includes an amino acid sequence that can fold into a standard domain (VH or VL) having a double beta-sheet structure, where the beta sheets are connected by disulfide bonds between the residues of the first beta-sheet and the residues of the second beta-sheet.

[0142] An antibody complementarity-determining region ("CDR") typically (i.e., in wild-type animals) comprises an amino acid sequence encoded by a nucleic acid sequence of an immunoglobulin gene of an organism, appearing between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor). A CDR may be encoded by, for example, a germline sequence, or by, for example, a sequence that is rearranged or not rearranged by a naive or mature B cell or T cell. In some situations (e.g., in the case of CDR3), a CDR may be encoded by two or more sequences (e.g., germline sequences) that are not adjacent (e.g., in an unrearranged nucleic acid sequence) but are adjacent in a B cell nucleic acid sequence, for example, as a result of sequence splicing or linking (e.g., VDJ recombination forming a heavy chain CDR3). Each of the above components of an antibody can be produced according to the present invention.

[0143] A bispecific antibody contains an antibody that can selectively bind to two or more epitopes. Generally, a bispecific antibody contains two distinct heavy chains, each specifically binding to a different epitope on either two different molecules (e.g., antigens) or the same molecule (e.g., the same antigen). If a bispecific antibody can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain to the first epitope is generally at least one to two, three, or four orders of magnitude lower than the affinity of the first heavy chain to the second epitope, and vice versa. The epitopes recognized by a bispecific antibody can be on the same or different targets (e.g., the same or different proteins). A bispecific antibody can be constructed, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen may be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences may be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each having three heavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that does not confer antigen-binding specificity but can associate with each heavy chain, or can associate with each heavy chain and can associate with one or more epitopes to which the heavy chain antigen-binding region binds, or can associate with each heavy chain and can bind one or both of the heavy chains to one or both of the epitopes, and can be produced according to the present invention.

[0144] For example, in antibody embodiments, the present invention is modifiable for research and production use for diagnosis and treatment based on all major antibody classes, namely IgG, IgA, IgM, IgD, and IgE. Preferred classes of IgG include IgG1 (including IgG1λ and IgG1κ), IgG2, and IgG4. Exemplary antibodies produced according to the present invention include alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacirivimab, imudevimab, semiprimab, semiprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, facimmab, nesbacumab, trevoglumab, linucumab, and sarilumab. Further embodiments of the antibody include human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, antigen-binding antibody fragments, single-chain antibodies, diabodies, triabodies or tetrabodies, Fab fragments or F(ab')2 fragments, IgD antibodies, IgE antibodies, IgM antibodies, IgG antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies. 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.

[0145] In additional embodiments, the antibodies include anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Publication No. 2015 / 0203579A1), anti-programmed cell death ligand-1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Publication No. 2015 / 0203580A1), anti-DII4 antibody, anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), and anti-angiopoietin-like 3 antibody (e.g., as described in U.S. Patent No. 9,018,356). Anti-AngPtl3 antibody, anti-platelet-derived growth factor receptor antibody (e.g., anti-PDGFR antibody as described in U.S. Patent No. 9,265,827), anti-Erb3 antibody, anti-prolactin receptor antibody (e.g., anti-PRLR antibody as described in U.S. Patent No. 9,302,015), anti-complement 5 antibody (e.g., anti-C5 antibody as described in U.S. Patent Application Publication No. 2015 / 0313194A1), anti-TNF antibody, anti-epidermal growth factor receptor antibody (e.g., anti-EGFR antibody as described in U.S. Patent No. 9,132,192), (For example, anti-EGFRvIII antibodies as described in U.S. Patent Application Publication No. 2015 / 0259423A1), anti-precursor protein-converting enzyme subtilisin kexin-9 (e.g., anti-PCSK9 antibodies as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. 2014 / 0044730A1), anti-proliferation and differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies also known as anti-myostatin antibodies as described in U.S. Patent No. 8,871,209 or U.S. Patent No. 9,260,515), anti-glucagon receptors (e.g., For example, anti-GCGR antibodies as described in U.S. Patent Publication No. 2015 / 0337045A1 or No. 2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin-4 receptor antibodies (for example, anti-IL4R antibodies as described in U.S. Patent Publication No. 2014 / 0271681 or U.S. Patent No. 8,735,095 or No. 8,945,559), anti-interleukin-6 receptor antibodies (for example, U.S. Patent No. 7,582,298, No. 8,043,617 or No. 9,173,Anti-IL6R antibodies (such as those 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 (for example, anti-IL33 antibodies as described in U.S. Patent Publication No. 2014 / 0271658A1 or 2014 / 0271642A1), anti-respiratory syncytial virus antibodies (for example, anti-RSV antibodies as described in U.S. Patent Publication No. 2014 / 0271653A1) ), anti-differentiation antigen group 3 (e.g., anti-CD3 antibodies as described in U.S. Patent Application Publication Nos. 2014 / 0088295A1 and 2015026666A1, and U.S. Patent Application No. 62 / 222,605), anti-differentiation antigen group 20 (e.g., anti-CD20 antibodies as described in U.S. Patent Application Publication Nos. 2014 / 0088295A1 and 2015266966A1, and U.S. Patent No. 7,879,984), anti-CD19 antibody, anti-CD28 antibody, anti-differentiation antigen group 4 8 (e.g., anti-CD48 antibody as described in U.S. Patent No. 9,228,014), anti-Feld1 antibody (e.g., as described in U.S. Patent No. 9,079,948), SARS-CoV-2 treatment (REGN-COV™ including casirivimab and imudevimab), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibody as described in U.S. Patent Application Publication No. 2015 / 0337029A1), antibody cocktail against Ebola (atorutivimab, mafici REGN-EB3 (INMAZEB®), including bimab and odesibimab-ebgn, anti-Ebola virus antibodies (e.g., as described in U.S. Patent Application Publication 2016 / 0215040), anti-Zika virus antibodies, anti-lymphocyte activator gene 3 antibodies (e.g., anti-LAG3 antibody or anti-CD223 antibody), anti-neuronal growth factor antibodies (e.g., U.S. Patent Application Publication 2016 / 0017029, and U.S. Patents No. 8,309,088 and No. 9,353,The bispecific antibody is selected from the group consisting of anti-NGF antibodies (such as those described in Patent Publication No. 176) and anti-activin A antibodies. In some embodiments, the bispecific antibody is selected from the group consisting of anti-CD3 × anti-CD20 bispecific antibodies (such as those described in U.S. Patent Publication Nos. 2014 / 0088295A1 and 20150266966A1), anti-CD3 × anti-mucin 16 bispecific antibodies (e.g., anti-CD3 × anti-Muc16 bispecific antibody), and anti-CD3 × anti-prostate-specific membrane antigen bispecific antibodies (e.g., anti-CD3 × anti-PSMA bispecific antibody). See also U.S. Patent Publication No. 2019 / 0285580A1.

[0146] Antibody derivatives and fragments are modifiable for production according to the present invention and include, but are not limited to, antibody fragments (e.g., ScFv-Fc, dAB-Fc, semi-antibodies), multispecificity (e.g., IgG-ScFv, IgG-dab, ScFV-ScFV, triplicate), and Fc fusion proteins (e.g., Fc fusion (N-terminus), Fc fusion (C-terminus), mono-Fc fusion, bispecificity Fc fusion). The phrase "Fc-containing protein" includes antibodies, bispecificity antibodies, antibody derivatives containing Fc, Fc, antibody fragments containing Fc, Fc fusion proteins, immunoadhesins, and other binding proteins containing at least functional portions of the immunoglobulin CH2 and CH3 regions. The "functional portion" refers to the CH2 and CH3 regions that can bind to Fc receptors (e.g., FcyR or FcRn (neonatal Fc receptor)) and / or be involved in complement activation. If the CH2 and CH3 regions include deletions, substitutions, and / or insertions or other modifications that prevent them from binding to any FC receptor and from activating complement, then the CH2 and CH3 regions are not functional.

[0147] Antigen-binding molecules (ABMs) and ABM conjugates having unnatural forms such as Fab domains with unnatural configurations can be expressed according to the present invention and are disclosed in WO2021 / 026409A1. Multispecificity-binding molecules (MBMs) and MBM conjugates can be produced according to the present invention and are disclosed in WO2021 / 091953A1 and WO2021 / 030680A1.

[0148] Fc-containing proteins may include modifications to the immunoglobulin domain, including modifications that affect one or more effector functions of the binding protein (e.g., modifications that affect FcyR binding, FcRn binding, and therefore half-life, and / or CDC activity). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to the EU numbering of the immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, ​​383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

[0149] For example, but not limited to, the binding protein is an Fc-containing protein exhibiting an enhanced serum half-life (compared to the same Fc-containing protein without the described modifications), and having modifications at position 250 (e.g., E or Q), position 250 and position 428 (e.g., L or F), position 252 (e.g., L / Y / F / W, or T), position 254 (e.g., S or T), and position 256 (e.g., S or T), or at position 428 and / or 433 (e.g., L / R / SI / P / Q, or K) and / or position 434 (e.g., H / F or Y), or at position 250 and / or 428, or at position 307 or 308 (e.g., 308F, V308F) and position 434. In another example, modifications could include modifications 428L (e.g., M428L) and 434S (e.g., N434S), modifications 428L, 2591 (e.g., V259I), and 308F (e.g., V308F), modifications 433K (e.g., H433K) and 434 (e.g., 434Y), modifications 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications 250Q and 428L (e.g., T250Q and M428L), and modifications 307 and / or 308 (e.g., 308F or 308P).

[0150] As described above, the present invention is also suitable for the production of other molecules, including fusion proteins. These proteins may contain part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, which is not fused in its native state. Examples of Fc fusion proteins include Fc fusion (N-terminus), Fc fusion (C-terminus), mono-Fc fusion, and bispecific Fc fusion. The preparation of fusion proteins containing specific heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) is described, for example, in Ashkenazi et al., Proc. Natl. Acad. Sci USA 88:10535-39 (1991); Byrn et al., Nature 344:677-70, 1990; and Hollenbaugh et al., “Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11 (1992). Receptor Fc-containing proteins are also described in C. Huang, “Receptor-Fc fusion therapeutics, traps, and MFMETIBODY technology,” 20(6) Curr. Opin. Biotechnol. 692-9 (2009).

[0151] A receptor Fc fusion protein comprises one or more extracellular domains of a receptor bound to the Fc portion, and in some embodiments, includes a hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc fusion protein contains two or more distinctly different receptor chains that bind to one or more ligands. Some receptor Fc fusion proteins may contain ligand-binding domains of multiple different receptors.

[0152] In some embodiments, the Fc fusion protein is a receptor Fc fusion protein containing one or more extracellular domains of a receptor linked to the Fc portion. In some embodiments, the Fc portion includes a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor Fc fusion protein contains two or more different receptor chains that bind to a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, e.g., an IL-1 trap (e.g., lilonacept containing an IL-1RAcP ligand-binding region fused to an Il-1R1 extracellular region fused to the Fc of hIgG1; see U.S. Patent No. 6,927,044), or a VEGF trap (e.g., aflibercept or ziv-aflibercept containing 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 U.S. Patents No. 7,087,411 and 7,279,159). In other embodiments, the Fc fusion protein is an ScFv-Fc fusion protein containing one or more antigen-binding domains, such as a variable heavy chain fragment and a variable light chain fragment of an antibody bound to the Fc portion.

[0153] Mini-trap proteins are trap proteins that use a polymerizing component (MC) instead of an Fc moiety, and are disclosed in U.S. Patent Nos. 7,279,159 and 7,087,411, and can be produced according to the present invention.

[0154] (Note) [Note 1] A method for improving recombinant protein titer in recombinant protein production by culturing recombinant eukaryotic cells, (a) To provide a defined cell culture medium having reduced impurities, wherein the defined cell culture medium contains less than 4000 μmol of HEPES-related impurities having a molecular weight of 267.07 per mole of total HEPES, and less than 400 μmol of HEPES-related impurities having a molecular weight of 221.06 per mole of total HEPES. (b) The recombinant eukaryotic cells are cultured in the specified cell culture medium having reduced impurities, (c) Expressing the target recombinant protein from the recombinant eukaryotic cells, (d) A method comprising producing a higher titer of the recombinant protein in the specified cell culture medium having reduced impurities compared to the titer of similar or identical cells cultured in a medium without reduced impurities. [Note 2] The method according to Appendix 1, wherein the eukaryotic cells are selected from the group consisting of mammalian cells, avian cells, insect cells, and yeast cells. [Note 3] The method according to Appendix 1, wherein the eukaryotic cells are selected from the group consisting of CHO, COS, retinal cells, Vero, CV1, kidney, HeLa, HepG2, WI38, MRC5, Colo25, HB8065, HL-60, lymphocytes, A431, CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. [Note 4] The method according to Appendix 3, wherein the eukaryotic cell is a CHO cell. [Note 5] The method according to Appendix 1, wherein the expression of the target recombinant protein occurs during the production phase, the proliferation phase, or both. [Note 6] The method according to Appendix 1, wherein the culture of the recombinant eukaryotic cells in the specified cell culture medium having reduced impurities occurs during the production phase, the proliferation phase, or both. [Note 7] The method according to Appendix 1, wherein the cell proliferation of the recombinant eukaryotic cells during culture is higher than that of similar or identical recombinant eukaryotic cells in a non-reduced impurity medium. [Note 8] The method according to Appendix 1, wherein the higher titer of the recombinant protein is increased by at least about 5% compared to the titer of similar or identical cells cultured in a non-impaired medium. [Note 9] The method according to Appendix 1, wherein the recombinant protein is an antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, multispecific 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. [Note 10] The method according to Appendix 1, wherein the recombinant protein contains an Fc domain. [Note 11] The method according to Appendix 10, wherein the recombinant protein is selected from the group consisting of Fc fusion proteins, receptor-Fc fusion proteins (TRAP), antibodies, antibody fragments, and ScFv-Fc fusion proteins. [Note 12] The recombinant protein may be anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody, anti- IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD4 antibody 8 antibody, an anti-CD3 / anti-CD20 bispecific antibody, an anti-CD3 / anti-MUC16 bispecific antibody, and an anti-CD3 / anti-PSMA bispecific antibody. [Note 13] The method according to Appendix 11, wherein the recombinant protein is selected from the group consisting of alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacilibimab, imudevimab, semiprimab, semiprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, facimmab, nesbacumab, trevoglumab, linucumab, and sarilumab. [Note 14] The method according to Appendix 13, wherein the recombinant protein is dupilumab. [Note 15] A cell culture medium having reduced impurities, wherein the medium comprises a defined cell culture medium having reduced impurities, and the defined cell culture medium comprises a 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer and contains less than 800 μmol of HEPES-related impurities having a molecular weight of 267.07 per mole of total HEPES, and less than 80 μmol of HEPES-related impurities having a molecular weight of 221.06 per mole of total HEPES. [Note 16] The cell culture medium described in Appendix 15, wherein the culture medium does not contain hydrolysates. [Note 17] The culture medium is a cell culture medium as defined in Appendix 15, which is chemically defined. [Note 18] A cell culture medium as described in Appendix 15, further containing insulin. [Note 19] The cell culture medium described in Appendix 15 further comprises ≥0.09 mM ± 0.014 mM ornithine, ≥0.20 ± 0.03 mM putrescine, or a combination thereof. [Note 20] The cell culture medium described in Appendix 15 further comprises a mixture of amino acids or salts thereof in an area of ​​≥40±6 mM. [Note 21] The cell culture medium described in Appendix 20, wherein the mixture of amino acids comprises alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. [Note 22] The cell culture medium described in Appendix 15, further comprising one or more fatty acids and tocopherols. [Note 23] The cell culture medium according to Appendix 22, wherein the one or more fatty acids are selected from the group consisting of linoleic acid, linolenic acid, thiotic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. [Note 24] The cell culture medium described in Appendix 15, further comprising a mixture of nucleosides. [Note 25] The cell culture medium according to Appendix 24, wherein the mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine. [Note 26] A cell culture medium as described in Appendix 25, comprising adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine. [Note 27] The cell culture medium described in Appendix 15, further comprising one or more salts of divalent cations. [Note 28] The cell culture medium according to Appendix 27, wherein the divalent cation is magnesium, calcium, or both. [Note 29] Ca 2+ and Mg 2+ Cell culture media as described in Appendix 28, including the above. [Note 30] The cell culture medium described in Appendix 15 is a chemically defined medium comprising HEPES buffer, a mixture of amino acids, optionally a mixture of nucleosides, one or more fatty acids and tocopherols, one or more salts of divalent cations, and one or more vitamins. [Note 31] A cell culture medium as described in Appendix 30, further containing insulin. [Note 32] A method for selecting a prescribed cell culture medium for use in cell culture in order to improve cell culture performance, (a) To provide a defined cell culture medium containing 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer, (b) Analyze the specified cell culture medium containing the HEPES buffer to determine the amount of HEPES-related impurities having a molecular weight of 267.07 and the amount of HEPES-related impurities having a molecular weight of 221.06 present in the specified cell culture medium, (c) Selecting the specified cell culture medium containing the HEPES buffer for use in cell culture if the specified cell culture medium containing the HEPES buffer contains less than 4000 μmol of HEPES-related impurities having a molecular weight of 267.07 per mole of total HEPES, and less than 400 μmol of HEPES-related impurities having a molecular weight of 221.06 per mole of total HEPES. A method for improving cell culture performance by using the specified cell culture medium, comprising a HEPES buffer containing less than 4000 μmol of HEPES-related impurities having a molecular weight of 267.07 per mole of total HEPES, and less than 400 μmol of HEPES-related impurities having a molecular weight of 221.06 per mole of total HEPES, compared to cell culture performance in a medium without reduced HEPES-related impurities. [Note 33] The method according to Appendix 32, wherein the improved cell culture performance includes improved cell culture titer and / or cell proliferation. [Note 34] A method for selecting HEPES buffer for use in cell culture in order to improve cell culture performance, (a) To provide 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer, (b) Analyze the HEPES buffer to determine the amount of HEPES-related impurities having a molecular weight of 267.07 and the amount of HEPES-related impurities having a molecular weight of 221.06 present in the HEPES buffer, (c) Selecting the HEPES buffer for use in cell culture when it is determined that the HEPES buffer contains less than 4000 μmol of HEPES-related impurities with a molecular weight of 267.07 per mole of total HEPES, and less than 400 μmol of HEPES-related impurities with a molecular weight of 221.06 per mole of total HEPES. A method for improving cell culture performance by using a HEPES buffer containing less than 4000 μmol of HEPES-related impurities with a molecular weight of 267.07 per mole of total HEPES used in association with a cell culture, and less than 400 μmol of HEPES-related impurities with a molecular weight of 221.06 per mole of total HEPES, compared to cell culture performance in the presence of a HEPES buffer containing a higher amount of the impurities. [Note 35] The method according to Appendix 34, wherein the improved cell culture performance includes improved cell culture titer and / or cell proliferation. [Note 36] A cell culture comprising cells and cell culture medium as described in any one of the appendices 15 to 31. [Note 37] The cell culture described in Appendix 36, wherein the cells are eukaryotic cells. [Note 38] The cell culture described in Appendix 37, wherein the eukaryotic cells are selected from the group consisting of mammalian cells, avian cells, insect cells, and yeast cells. [Note 39] The cell culture described in Appendix 36, wherein the cells can express an antibody selected from the group consisting of alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacilibimab, imdevimab, semiprimab, semiprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, facimmab, nesbacumab, trevoglumab, linucumab, and sarilumab. [Note 40] The cell culture described in Appendix 39, wherein the cells are capable of expressing dupilumab. [Note 41] The method according to Appendix 8, wherein the higher titer of the recombinant protein is increased by at least about 10% compared to the titer of similar or identical cells cultured in a non-impaired medium. [Note 42] The method according to Appendix 8, wherein the higher titer of the recombinant protein is increased by at least about 15% compared to the titer of similar or identical cells cultured in a non-impaired medium. [Note 43] The method according to Appendix 8, wherein the higher titer of the recombinant protein is increased by at least about 25% compared to the titer of similar or identical cells cultured in a non-impaired medium. [Note 44] The method according to Appendix 1, wherein the doubling acceleration of viable cells in a culture medium with reduced impurities is at least 5% higher than the doubling acceleration of cells cultured in a culture medium without reduced impurities. [Note 45] The method according to Appendix 44, wherein the doubling acceleration of viable cells in a culture medium with reduced impurities is at least 10% higher than the doubling acceleration of cells cultured in a culture medium without reduced impurities. [Note 46] The method according to Appendix 44, wherein the doubling acceleration of viable cells in a culture medium with reduced impurities is at least 15% higher than the doubling acceleration of cells cultured in a culture medium without reduced impurities. [Note 47] The method according to Appendix 44, wherein the doubling acceleration of viable cells in a culture medium with reduced impurities is at least 25% higher than the doubling acceleration of cells cultured in a culture medium without reduced impurities. [Note 48] The cell culture medium according to Appendix 15, wherein the cell culture medium contains at least a compound selected from the group consisting of vinyl sulfonic acid, HEPES+[O]-[H2], acetamidomethanesulfonic acid, HEPES+[O], 2,2-dihydroxyethanesulfonic acid, and HEPES-[C2H6]+[O] [SO3-containing] species. [Note 49] (i) at least one recombinant eukaryotic cell capable of expressing a recombinant protein, and (ii) a cell culture medium, wherein the cell culture comprises (a) A step of providing a defined cell culture medium having reduced impurities, wherein the defined cell culture medium has less than 4000 μmol of HEPES-related impurities having a molecular weight of 267.07 per mole of total HEPES, and less than 400 μmol of HEPES-related impurities having a molecular weight of 221.06 per mole of total HEPES, (b) A step of culturing the recombinant eukaryotic cells in the specified cell culture medium having reduced impurities, (c) A step of expressing the target recombinant protein from the recombinant eukaryotic cells, (d) A cell culture produced by a method comprising the step of producing a cell culture medium having reduced impurities, wherein the higher titer of the recombinant protein is compared to the titer of similar or identical cells cultured in a medium without reduced impurities. [Note 50] The cell culture described in Appendix 49, wherein the eukaryotic cells are selected from the group consisting of mammalian cells, avian cells, insect cells, and yeast cells. [Note 51] The cell culture described in Appendix 50, wherein the eukaryotic cells are selected from the group consisting of CHO, COS, retinal cells, Vero, CV1, kidney, HeLa, HepG2, WI38, MRC5, Colo25, HB8065, HL-60, lymphocytes, A431, CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. [Note 52] The cell culture described in Appendix 51, wherein the eukaryotic cells are CHO cells. [Note 53] The cell culture described in Appendix 49, wherein the expression of the target recombinant protein occurs during the production phase, the proliferation phase, or both. [Note 54] The cell culture according to Appendix 49, wherein the culture of the recombinant eukaryotic cells in the specified cell culture medium having reduced impurities occurs during the production phase, the proliferation phase, or both. [Note 55] The cell culture according to Appendix 49, wherein the cell proliferation of the recombinant eukaryotic cells during culture is higher than that of similar or identical recombinant eukaryotic cells in a non-reduced impurity medium. [Note 56] The cell culture according to Appendix 49, wherein the higher titer of the recombinant protein is increased by at least about 5% compared to the titer of similar or identical cells cultured in a non-impaired medium. [Note 57] The cell culture described in Appendix 49, wherein the recombinant protein is an antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, multispecific 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. [Note 58] The cell culture described in Appendix 49, wherein the recombinant protein contains an Fc domain. [Note 59] The cell culture described in Appendix 58, wherein the recombinant protein is selected from the group consisting of Fc fusion protein, receptor-Fc fusion protein (TRAP), antibody, antibody fragment, and ScFv-Fc fusion protein. [Note 60] The recombinant protein is anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF Antibodies, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody, anti-I L3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 antibody 59. The cell culture according to claim 59, wherein the cell culture is selected from the group consisting of anti-CD3 / anti-CD20 bispecific antibody, anti-CD3 / anti-MUC16 bispecific antibody, and anti-CD3 / anti-PSMA bispecific antibody. [Note 61] The cell culture described in Appendix 59, wherein the recombinant protein is selected from the group consisting of alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacilibimab, imudevimab, semiprimab, semiprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, facimmab, nesbacumab, trevoglumab, linucumab, and sarilumab. [Note 62] The cell culture described in Appendix 61, wherein the recombinant protein is dupilumab. [Note 63] (i) a recombinant protein produced in a cell culture comprising at least one recombinant eukaryotic cell capable of expressing a recombinant protein, and (ii) a cell culture medium, wherein the recombinant protein is (a) A step of providing a defined cell culture medium having reduced impurities, wherein the defined cell culture medium has less than 4000 μmol of HEPES-related impurities having a molecular weight of 267.07 per mole of total HEPES, and less than 400 μmol of HEPES-related impurities having a molecular weight of 221.06 per mole of total HEPES, (b) A step of culturing the recombinant eukaryotic cells in the specified cell culture medium having reduced impurities, (c) A step of expressing the target recombinant protein from the recombinant eukaryotic cells, (d) Recombinant proteins produced by a method comprising the step of producing the recombinant protein in a specified cell culture medium having reduced impurities, with a higher titer of the recombinant protein compared to that of similar or identical cells cultured in a medium without reduced impurities. [Note 64] The recombinant protein described in Appendix 63, wherein the eukaryotic cell is selected from the group consisting of mammalian cells, avian cells, insect cells, and yeast cells. [Note 65] The aforementioned eukaryotic cells are selected from the group consisting of CHO, COS, retinal cells, Vero, CV1, kidney, HeLa, HepG2, WI38, MRC5, Colo25, HB8065, HL-60, lymphocytes, A431, CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells, as described in Appendix 64, which is a recombinant protein. [Note 66] The aforementioned eukaryotic cell is a CHO cell, and the recombinant protein is as described in Appendix 65. [Note 67] The recombinant protein described in Appendix 63, wherein the expression of the target recombinant protein occurs during the production phase, the proliferation phase, or both. [Note 68] The recombinant eukaryotic cells are cultured in the specified cell culture medium having reduced impurities, resulting in the recombinant protein described in Appendix 63, which occurs during the production phase, the proliferation phase, or both. [Note 69] The recombinant protein according to Appendix 63, wherein the cell proliferation of the recombinant eukaryotic cells during culture is higher than that of similar or identical recombinant eukaryotic cells in a non-reduced impurity medium. [Note 70] The recombinant protein according to Appendix 63, wherein the higher titer of the recombinant protein is increased by at least about 5% compared to the titer of similar or identical cells cultured in a non-impaired medium. [Note 71] The recombinant protein described in Appendix 63 is an antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, multispecific 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. [Note 72] The recombinant protein described above is the recombinant protein described in Appendix 63, wherein the recombinant protein includes an Fc domain. [Note 73] The recombinant protein described in Appendix 72 is selected from the group consisting of Fc fusion proteins, receptor-Fc fusion proteins (TRAP), antibodies, antibody fragments, and ScFv-Fc fusion proteins. [Note 74] The recombinant protein described in Appendix 73 is selected from the group consisting of anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 antibody, anti-CD3 / anti-CD20 bispecific antibody, anti-CD3 / anti-MUC16 bispecific antibody, and anti-CD3 / anti-PSMA bispecific antibody. [Note 75] The recombinant protein described in Appendix 73 is selected from the group consisting of alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacilibimab, imudevimab, semiprimab, semiprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, facimumab, nesbacumab, trevoglumab, linucumab, and sarilumab. [Note 76] The recombinant protein described in Appendix 75, wherein the recombinant protein is dupilumab. [Note 77] Cells described in any one of the appendices 1 to 76. [Note 78] A cell culture described in any one of the appendices 1 to 77. [Note 79] The method described in any one of the appendices 1 to 78. [Note 80] Recombinant protein as described in any one of the appendices 1 to 79. The present invention has been described with respect to exemplary embodiments, but those skilled in the art will understand that various changes can be made without departing from the scope of the present invention and equivalents can be substituted for its elements. In addition, many modifications can be made to adapt a particular situation or material to the teachings without departing from the essential scope of the present invention. Therefore, the present invention is not limited to the particular embodiments disclosed as the best mode contemplated for carrying out the present invention, but the present invention is intended to include all embodiments falling within the scope of the appended claims.

Claims

1. A method for improving recombinant protein titer in recombinant protein production by culturing recombinant eukaryotic cells, (a) To provide a defined cell culture medium having reduced impurities, wherein the defined cell culture medium comprises a 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer and contains less than 4000 μmol of HEPES+[O2]-[H2] and less than 400 μmol of HEPES-[CH4] per mole of total HEPES, (b) The recombinant eukaryotic cells are cultured in the specified cell culture medium having reduced impurities, (c) Expressing the target recombinant protein from the recombinant eukaryotic cells, (d) A method comprising producing a higher titer of the recombinant protein in the specified cell culture medium having reduced impurities, compared to the titer of similar or identical cells cultured in a medium without reduced impurities.

2. The method according to claim 1, wherein the eukaryotic cells are selected from the group consisting of mammalian cells, avian cells, insect cells, and yeast cells.

3. The method according to claim 1, wherein the eukaryotic cells are selected from the group consisting of CHO, COS, retinal cells, Vero, CV1, kidney cells, HeLa, HepG2, WI38, MRC5, Colo25, HB8065, HL-60, lymphocytes, A431, CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells.

4. The method according to claim 3, wherein the eukaryotic cell is a CHO cell.

5. The method according to claim 1, wherein the expression of the target recombinant protein occurs during the production phase, the proliferation phase, or both.

6. The method according to claim 1, wherein the culture of the recombinant eukaryotic cells in the specified cell culture medium having reduced impurities occurs during the production phase, the proliferation phase, or both.

7. The method according to claim 1, wherein the higher titer of the recombinant protein is increased by at least about 5% compared to the titer of similar or identical cells cultured in a non-impaired medium.

8. The method according to claim 1, wherein the recombinant protein is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody or tetrabody, a Fab fragment or F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

9. The method according to claim 1, wherein the recombinant protein comprises an Fc domain.

10. The method according to claim 9, wherein the recombinant protein is selected from the group consisting of Fc fusion protein, receptor-Fc fusion protein (TRAP), antibody, antibody fragment, and ScFv-Fc fusion protein.

11. The recombinant protein is anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TN F antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody, anti- IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 11. The method of claim 10, wherein the method is selected from the group consisting of antibodies, anti-CD3 / anti-CD20 bispecific antibodies, anti-CD3 / anti-MUC16 bispecific antibodies, and anti-CD3 / anti-PSMA bispecific antibodies.

12. The method according to claim 10, wherein the recombinant protein is selected from the group consisting of alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacilibimab, imudevimab, semiprimab, semiprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, fasimumab, nesbacumab, trevoglumab, linucumab, and sarilumab.

13. The method according to claim 12, wherein the recombinant protein is dupilumab.

14. A cell culture medium having reduced impurities, wherein the medium comprises a defined cell culture medium having reduced impurities, and the defined cell culture medium comprises a 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer and has less than 4000 μmol of HEPES+[O2]-[H2] and less than 400 μmol of HEPES-[CH4] per mole of total HEPES.

15. The cell culture medium according to claim 14, wherein the culture medium does not contain hydrolysates.

16. The cell culture medium according to claim 14, wherein the culture medium is chemically defined.

17. The cell culture medium according to claim 14, further comprising insulin.

18. The cell culture medium according to claim 14, further comprising ≥0.09 mM ± 0.014 mM ornithine, ≥0.20 ± 0.03 mM putrescine, or a combination thereof.

19. The cell culture medium according to claim 14, further comprising a mixture of amino acids or salts thereof in an area of ​​≥40±6 mM.

20. The cell culture medium according to claim 19, wherein the mixture of amino acids comprises alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

21. The cell culture medium according to claim 14, further comprising one or more fatty acids and tocopherols.

22. The cell culture medium according to claim 21, wherein the one or more fatty acids are selected from the group consisting of linoleic acid, linolenic acid, thiotic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid.

23. The cell culture medium according to claim 14, further comprising a mixture of nucleosides.

24. The cell culture medium according to claim 23, wherein the mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

25. The cell culture medium according to claim 24, comprising adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

26. The cell culture medium according to claim 14, further comprising one or more salts of a divalent cation.

27. The cell culture medium according to claim 26, wherein the divalent cation is magnesium, calcium, or both.

28. Ca ２＋ and Mg ２＋ The cell culture medium according to claim 27, comprising:

29. The cell culture medium according to claim 14, wherein the culture medium is a chemically defined culture medium comprising HEPES buffer, a mixture of amino acids, optionally a mixture of nucleosides, one or more fatty acids and tocopherols, one or more salts of divalent cations, and one or more vitamins.

30. The cell culture medium according to claim 29, further comprising insulin.

31. A cell culture comprising cells and a cell culture medium according to any one of claims 14 to 30.

32. The cell culture according to claim 31, wherein the cells are eukaryotic cells.

33. The cell culture according to claim 32, wherein the eukaryotic cells are selected from the group consisting of mammalian cells, avian cells, insect cells, and yeast cells.

34. The cell culture according to claim 31, wherein the cells can express an antibody selected from the group consisting of alirocumab, atorutivimab, maftivimab, odesibimab, odesibimab-ebgn, cacilibimab, imudevimab, semiprimab, semprimab-rwlc, dupilumab, evinacumab, evinacumab-dgnb, facimmab, nesbacumab, trevoglumab, linucumab, and sarilumab.

35. The cell culture according to claim 34, wherein the cells are capable of expressing dupilumab.

36. The method according to claim 7, wherein the higher titer of the recombinant protein is increased by at least about 10% compared to the titer of similar or identical cells cultured in a non-impaired medium.

37. The method according to claim 7, wherein the higher titer of the recombinant protein is increased by at least about 15% compared to the titer of similar or identical cells cultured in a non-impaired medium.

38. The method according to claim 7, wherein the higher titer of the recombinant protein is increased by at least about 25% compared to the titer of similar or identical cells cultured in a non-impaired medium.

39. The cell culture medium according to claim 14, wherein the cell culture medium contains at least a compound selected from the group consisting of vinyl sulfonic acid, HEPES+[O]-[H2], acetamidomethanesulfonic acid, HEPES+[O], 2,2-dihydroxyethanesulfonic acid, and HEPES-[C2H6]+[O][SO3-containing] species.

40. The method according to claim 1, wherein the specified cell culture medium has less than 800 μmol of HEPES+[O2]-[H2] and less than 80 μmol of HEPES-[CH4] per mole of total HEPES.

41. The cell culture medium according to claim 14, comprising a defined cell culture medium having less than 800 μmol of HEPES+[O2]-[H2] and less than 80 μmol of HEPES-[CH4] per mole of total HEPES.

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