Cell culture medium supplemented with taurine and method of use

Abstract

The present invention relates to a method for culturing eukaryotic cells expressing dupilumab, and a method for producing dupilumab. This specification describes a composition comprising a modified eukaryotic cell culture medium, which can be used to produce a protein of interest. Taurine can be added to a serum-free medium or a chemically defined medium to increase the yield of the protein of interest. Also included is a method for recombinantly expressing a high level of protein using the culture medium composition.

Description

[0001] This application is a divisional application of CN201680041992.X.

[0002] Field

[0003] The present invention relates to culturing cells and media and methods for producing recombinant proteins. In particular, the present invention relates to a taurine-supplemented medium and method for culturing recombinant eukaryotic cells for the production of protein biotherapeutics. Background

[0004] The organic acid taurine, commonly known as a β-amino acid, is highly concentrated in most tissues and is a derivative of the amino acid cysteine (Huxtable, RJ., 1992, Physiol Rev, 72: 101-163).

[0005]

[0006] Taurine is present in many tissues of humans and other mammalian species, such as the brain, retina, myocardium, bone and smooth muscle, platelets, and neutrophils. Taurine is known to help with osmotic regulation, membrane stabilization, and anti-inflammation, as well as regulate mitochondrial protein synthesis via protection against increased superoxide production in the electron transport chain (Jong et al., 2010, Journal of Biomedical Science 17(Suppl 1): S25; Jong et al., 2012, Amino Acids 42: 2223-2232sw). In primary neuron cultures, taurine has been characterized as a cytoprotectant due to its inhibition of glutamate-induced toxicity. Various media containing taurine for embryo culture have been developed.

[0007] Cell culture techniques involving amino acid feeding have a long history for the production of recombinant proteins from cultured cells. Amino acids are biosynthetic precursors, energy sources, osmolytes, etc., and their use in production cultures is strongly related to continuous cell growth and productivity.

[0008] However, there are numerous physiological events that contribute to productivity and high-yield protein expression, and competing metabolic activities and transport mechanisms make the design of culture strategies a challenge. The type and timing of amino acid supplementation can also have an impact on the quality of the protein produced in culture (Altamirano, et al., 2006, Electron. J. Biotechnol. 9: 61-67). The accumulation of by-products often becomes a problem in production cell cultures and is regarded as a consequence of nutrient imbalance in cell cultures, ultimately inhibiting cell growth (Fan, Y. et al. Biotechnol Bioeng. 2015 Mar; 112(3): 521-35). Taurine or its analogs or precursors have been proposed in cell culture in order to achieve the desired result of reducing the color concentration of a composition comprising a recombinantly produced polypeptide (WO2014145098A1, published September 18, 2014). A cell culture medium comprising taurine that promotes the maturation of immature retinal pigment epithelial cells into mature retinal pigment epithelial cells has been described (WO2013184809A1, published 12 December 2013). However, optimizing recombinant protein productivity in taurine-supplemented cultures has not been identified in the prior art. A cell culture method that increases the productivity of recombinantly expressed proteins while minimizing possible toxic cell metabolic by-products such as ammonia is highly desirable. Any constant gain in productivity could translate into a significantly higher supply of biotherapeutic products on a commercial scale.

[0009] Accordingly, there is a need in the art for culture media and methods for culturing mammalian cells, wherein the culture media allow for healthy and robust cell growth and maintenance, and high-titer production of biopharmaceutical substances. SUMMARY OF THE INVENTION

[0010] The inventors have surprisingly found that the inclusion of taurine in the culture medium increases the productivity per cell unit and results in fewer ammonia by-products in these cells. Various culture strategies incorporating taurine result in increased protein production titers. Additionally, the addition of taurine has no negative impact on culture performance or the quality of the antibodies produced.

[0011] The present invention provides a method for producing a therapeutic protein in high yield, which comprises culturing recombinant cells in a culture medium containing taurine, wherein the cells comprise a stably integrated nucleic acid encoding the therapeutic protein.

[0012] The present invention relates to a cell culture medium which is serum-free and contains from about 0.1 mM to about 10 mM taurine. The present invention relates to a cell culture medium which is serum-free and contains from about 0.1 mM to about 1 mM taurine, from about 0.2 to about 1 mM taurine, from about 0.3 to about 1 mM taurine, from about 0.4 to about 1 mM taurine, or from about 0.5 to about 1 mM taurine. The present invention relates to a cell culture medium which is serum-free and contains from about 1 mM to about 10 mM taurine. The present invention relates to a cell culture medium which is serum-free and contains from about 1 mM to about 5 mM taurine, from about 1 mM to about 6 mM taurine, from about 1 mM to about 7 mM taurine, from about 1 mM to about 8 mM taurine, or from about 1 mM to about 9 mM taurine.

[0013] In certain embodiments, the medium further comprises additional amino acids selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0014] In certain embodiments, the medium contains ≤16 g / L of hydrolysate. In certain embodiments, the medium does not contain any hydrolysate.

[0015] In one embodiment, the medium contains a chemically defined basal medium, such as a conventional formulation or a commercially available basal medium. In one embodiment, the complete medium is chemically defined, serum-free, and hydrolysate-free.

[0016] In certain embodiments, the entire process (including the basal medium and the feed) contains a total of at least 115 mM of a mixture of amino acids or amino acid salts. In one embodiment, the mixture of amino acids contains amino acids selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan, in amounts selected from Table 1.

[0017] In certain embodiments, the medium contains one or more fatty acids. In a specific embodiment, the medium contains a mixture of a fatty acid (or fatty acid derivative) and α-tocopherol. The fatty acid or fatty acid derivative is selected from: linoleic acid, linolenic acid, lipoic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, capric acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and caprylic acid.

[0018] In certain embodiments, the culture medium contains a mixture of nucleosides. In one embodiment, the culture medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0019] In certain embodiments, the culture medium contains a mixture of salts. The salts include divalent cations such as calcium and magnesium. In one embodiment, the culture medium contains calcium chloride and magnesium sulfate. Other salts may include phosphates.

[0020] In one embodiment, the culture medium (1) contains 0.1 ± 0.015 mM, 1 ± 0.015 mM, 3 ± 0.05 mM, 5 ± 0.10 mM, 7 ± 0.15 mM, or 10 ± 0.2 mM taurine, (2) contains ≤ 16 g / L of a hydrolysate, (3) is serum-free, (4) optionally further contains a mixture of amino acids, (5) contains a mixture of fatty acids, (6) contains a mixture of nucleosides including adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine, and (7) contains salts of calcium, magnesium, and phosphates.

[0021] The present invention provides a method for producing a protein of interest in high yield, which includes culturing a recombinant cell line in a cell culture medium containing at least about 0.1 mM to about 10 mM taurine, wherein the cell line contains a stably integrated nucleic acid encoding the protein. In other embodiments, the culture medium embodiments are any of the foregoing aspects of the present invention.

[0022] In a further aspect, the present invention provides a method for culturing eukaryotic cells to increase the yield of a recombinant protein, which includes the following steps: (a) proliferating or maintaining the cells in a chemically defined cell culture medium during the growth phase, (b) supplementing this basal cell culture medium with about 0.1 mM to about 10 mM L-taurine and expressing the recombinant protein of interest during the production phase, and (c) increasing the titer of the protein of interest by adding taurine. In certain embodiments, this taurine supplementation is provided at least once, or twice, three times, four times, or five times during the production phase, or is provided daily during the production phase. In other embodiments, this method further includes supplementing the culture medium with about 0.1 mM to about 10 mM L-taurine during the production phase. In certain embodiments, this method provides an increased yield of recombinant protein compared to eukaryotic cells lacking a taurine supplement or containing a taurine supplement of less than 0.1 mM or under otherwise identical conditions.

[0023] In another aspect, the present invention provides a method for culturing cells in a cell culture medium (such as an embodiment of the culture medium described in any of the foregoing aspects). In one embodiment, this method uses the step of proliferating or maintaining cells in a culture medium containing (1) taurine at a concentration of at least 0.1 mM ± 0.015 mM, (2) containing ≤ 16 g / L of hydrolysate or no hydrolysate, (3) being serum-free, and (4) and an optional mixture of amino acids selected from the amino acids in Table 1.

[0024] In one embodiment, this optional amino acid supplement mixture is selected from the group consisting of the amino acids in Table 1:

[0025] Table 1

[0026]

[0027] In certain embodiments, the cells are mammalian cells, avian cells, insect cells, yeast cells, or bacterial cells. In one embodiment, the cells are mammalian cells that can be used to produce recombinant proteins, such as CHO cells or derivatives thereof, CHO-K1. In certain embodiments, the cells express a protein of interest, such as a biotherapeutic protein. This biotherapeutic protein can be an antigen-binding protein, which may contain an Fc domain. In certain embodiments, the protein of interest is an Fc-fusion protein, such as a ScFv molecule or a trap molecule. Trap molecules include (but are not limited to) VEGF traps and IL-1 trap proteins. In certain embodiments, the protein of interest is an antibody, such as a human monoclonal antibody, a humanized monoclonal antibody, a bispecific antibody, or an antibody fragment.

[0028] If a positive effect on protein production is achieved by including taurine in various forms of serum-free media, then the cells cultured according to this method result in an increase in the average protein titer. In one embodiment, when compared to the protein titer of a medium not supplemented with taurine, the cells grown in the taurine-supplemented culture according to this method produce a protein titer that is at least 8% greater than the comparative control culture (i.e., the culture not supplemented with taurine). In one embodiment, the cells grown in the taurine-supplemented culture, when compared to the protein titer in a medium not supplemented with taurine, produce a titer that is 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%, or at least 29% greater than the comparative control culture.

[0029] Similarly, including taurine alone in a serum-free medium allows for lower ammonia by-products in cultured cells compared to not including taurine. In embodiments of a serum-free and hydrolysate-free medium supplemented with taurine, cell culture can result in at least a 4% reduction, up to a 32% reduction in the amount of ammonia by-products (mM NH 3 ) compared to similar cell cultures without the supplement (i.e., less than 0.1 mM taurine or no taurine supplement).

[0030] In additional embodiments, the method includes the step of adding one or more point-of-use additives to the cell culture medium. In certain embodiments, the point-of-use additive is any one or more of NaHCO 3 , glutamine, insulin, glucose, CuSO 4 , ZnSO 4 , FeCl 3 , NiSO 4 , Na 4 EDTA, and sodium citrate. In one embodiment, the method uses the step of adding each of the following point-of-use chemicals to the cell culture medium: NaHCO 3 , glutamine, insulin, glucose, CuSO 4 , ZnSO 4 , FeCl 3 , NiSO 4 , Na 4 EDTA, and sodium citrate. In certain embodiments, the point-of-use additive can be included in the medium at the beginning.

[0031] In a specific embodiment, this aspect provides a method of culturing cells in a serum-free medium, the medium consisting essentially of (1) taurine at a concentration of at least 0.1 mM; (2) containing ≤16 g / L of hydrolysate, (3) being serum-free, and (4) optionally further containing at least about 20 mM, or at least about 25 mM, or at least about 30 mM, or at least about 40 mM, or at least about 50 mM, or at least about 60 mM, or at least about 70 mM of a mixture of total amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0032] In another aspect, the present invention provides a method for producing a protein of interest by using the following steps: (1) introducing a nucleic acid sequence encoding the protein of interest into a cell; (2) selecting one or more cells that express the protein of interest; (3) culturing the selected cells in an embodiment of the serum-free cell culture medium described in any of the foregoing aspects or in an embodiment of the method described herein; and (4) expressing the protein of interest in the cell, wherein the protein of interest is secreted into the culture medium. In certain embodiments, the cells used for producing the protein are mammalian cells capable of producing biotherapeutics, such as CHO, 293, and BHK cells, or any derivatives thereof. In one embodiment, the cells are CHO cells, such as CHO-K1 cells.

[0033] In certain embodiments, the protein of interest is an antigen-binding protein. In certain embodiments, the protein of interest is a protein having an Fc domain. In some cases, the two proteins of interest may overlap, such as in the case of a receptor-Fc-fusion protein, such as an antibody and a ScFv protein. Thus, in certain embodiments, the protein of interest is an antibody, such as a human antibody or a humanized antibody, an antibody fragment, such as Fab or F(ab’) 2 , a bispecific antibody, a capture trap molecule, such as a VEGF-trap or an IL-1-trap, a ScFv molecule, a soluble TCR-Fc fusion protein, and the like.

[0034] In one embodiment, the protein of interest can be produced at an average 14-, 15-, 16-, or 17-day titer that is at least 8% higher than the average 14-, 15-, 16-, or 17-day titer produced by similar cells in a serum-free cell culture medium containing less than 0.1 mM or not supplemented with taurine. In one embodiment, the protein of interest can be produced at an average 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, or 17-day titer that is 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%, or at least 29% higher than the average 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, or 17-day titer produced by similar cells in a serum-free cell culture medium containing less than 0.1 mM or not supplemented with taurine.

[0035] In additional embodiments, a protein of interest is produced by the steps of: (1) introducing into CHO cells a nucleic acid sequence encoding a protein of interest (e.g., an antibody or other antigen-binding protein); (2) selecting a cell that stably expresses the protein of interest; and (3) culturing the selected cell in a serum-free cell culture medium containing from about 0.1 mM to about 10 mM taurine. Brief Description of the Drawings

[0036] Figure 1 Shows the sample protein titers (yields) recovered daily from the production cultures of the cell cultures producing Ab3, where taurine-supplemented (solid squares connected by solid lines) is provided compared to no taurine supplementation (x connected by dashed lines). The benefit of taurine-supplemented culture on protein yield can be seen early, such as on day 6 of the production culture. Detailed Description of the Invention

[0037] It should be understood that the present invention is not limited to the specific methods and experimental conditions described, as the methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention is defined by the claims.

[0038] Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a" and "an" include plural referents. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein, and / or certain ones that will become apparent to those skilled in the art upon reading this disclosure.

[0039] Unless defined otherwise or stated otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the specific methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in their entirety.

[0040] The inventors have surprisingly found that, relative to cell culture media containing very little or no taurine, the addition of taurine to the cell culture medium increases the protein production of recombinant cells in the cell culture.

[0041] Before describing the cell culture and method, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as the methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0042] The paragraph headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Unless otherwise indicated, the methods and techniques described herein are generally carried out according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), and Julio E. Celis, Cell Biology: A Laboratory Handbook, 2 nd ed., Academic Press, New York, N.Y. (1998), and Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1995). All publications mentioned throughout this disclosure are incorporated herein by reference in their entirety.

[0043] Definition

[0044] "Taurine" is also known as 2-aminoethanesulfonic acid (IUPAC nomenclature; CAS Registry Number 107-35-7). "Taurine" and "L-taurine" are used interchangeably to refer to the same organic compound. Taurine is an organic acid containing an amino group, but is not considered an amino acid well-known in the prior art, while an amino acid contains both an amino group and a carboxyl group. The biosynthesis of taurine occurs when hypotaurine (a derivative of cysteine) is oxidized to taurine.

[0045] The terms "supplement" and like terms refer to the addition of ingredients, components, molecules, etc., which can be used in a culture medium for cell culture to maintain and / or promote cell growth and / or differentiation, extend or enhance the properties of the culture or the cells as a whole, or make up for deficiencies. For this purpose, supplementing with taurine involves adding a specific concentration of taurine solution to the culture medium.

[0046] As used herein, the terms "peptide", "polypeptide" and "protein" are all used interchangeably and refer to molecules comprising 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 linkage, sulfation, γ-carboxylation of glutamate residues, alkylation, hydroxylation and ADP-ribosylation. Peptides, polypeptides and proteins may have scientific or commercial interests, including protein drugs. In particular, peptides, polypeptides and proteins include antibodies and chimeric or fusion proteins. Peptides, polypeptides and proteins can be produced by using cell culture methods in recombinant animal cell lines.

[0047] As used herein, the term "heterologous polynucleotide sequence" refers to a nucleic acid polymer encoding a protein of interest, such as a chimeric protein (e.g., a capture trap molecule), an antibody or an antibody portion (e.g., VH, VL, CDR3) produced as a biopharmaceutical substance. Heterologous polynucleotide sequences can be produced by genetic engineering techniques (e.g., such as a sequence encoding a nucleoprotein, or a codon-optimized sequence, an intronless sequence, etc.) and introduced into cells, where it can reside as an episome or can integrate into the genome of the cells. The heterologous polynucleotide sequence can be a naturally occurring sequence introduced at an ectopic location within the genome of a production cell. This heterologous polynucleotide sequence can be a naturally occurring sequence from another organism, such as a sequence encoding a human xenologue.

[0048] "Antibody" refers to an immunoglobulin molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains) linked by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or V H) and the 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 hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes any isotype or subclass of glycosylated and non-glycosylated immunoglobulins mentioned. The term "antibody" includes antibody molecules prepared, expressed, created, or isolated by recombinant methods, such as antibodies isolated from transfected host cells expressing such antibodies. The term antibody also includes bispecific antibodies, which include heterotetrameric immunoglobulins that can bind to more than one different epitope. Bispecific antibodies are generally described in U.S. Patent Application Publication No. 2010 / 0331527, which is incorporated herein by reference.

[0049] The "antigen-binding portion" (or "antibody fragment") of an antibody refers to one or more antibody fragments 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) Fab fragments, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments, consisting of VH and CH1 regions; (iv) Fv fragments, consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al. (1989) Nature 241: 544-546), which consist of VH domains, (vi) isolated CDRs, and (vii) scFv, which consists of the two regions VL and VH of an Fv fragment, joined by a synthetic linker to form a single polypeptide chain, wherein the VL and VH domains pair to form a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also encompassed by the term "antibody" (see, e.g., Holliger et al. (1993) PNAS USA 90: 6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).

[0050] Further, the antibody or antigen-binding portion thereof can be part of a larger immunoadhesion molecule formed by covalent or non-covalent attachment of the antibody or antibody portion to one or more other proteins or polypeptides. Examples of immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine residues, a tag protein, and C-terminal polyhistidine tags to generate divalent and biotinylated scFv molecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab’)2 fragments, can be prepared from intact antibodies using well-known techniques, such as by papain or pepsin digestion of the intact antibody. In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques well known in the art (see Sambrook et al., 1989).

[0051] 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 invention may also include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), such as in the CDRs and in particular CDR3. However, as used herein, the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0052] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody libraries, antibodies from transgenic animals (e.g., mice) that have the human immunoglobulin genes isolated therefrom (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20: 6287-6295), or antibodies prepared, expressed, produced or isolated by any other method that involves splicing human immunoglobulin gene sequences to other DNA sequences. Recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, the recombinant human antibodies are mutated in vitro (or in vivo somatic mutations when using transgenic animals with human Ig sequences) and thus the amino acid sequences of the VH and VL regions of the recombinant antibody, when derived from or related to human germline VH and VL sequences, may be sequences that are not found in the human antibody germline repertoire naturally occurring in vivo.

[0053] "Fc fusion protein" includes part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, which are not found together in nature. The preparation of fusion proteins comprising a fusion of a specific heterologous polypeptide with different portions of an antibody-derived polypeptide (including the Fc domain) has been described, e.g., Ashkenazi et al., Proc. Natl. Acad. Sci. 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. "Receptor Fc-fusion protein" includes one or more extracellular regions of a receptor that bind to the Fc portion, which in certain embodiments includes a hinge region followed by the CH2 and CH3 regions of an immunoglobulin. In certain embodiments, this Fc-fusion protein contains two or more different receptor chains that bind to one or more ligands. For example, this Fc-fusion protein is a trap, e.g., an IL-1 trap (e.g., rilonacept, which contains the ligand-binding region of IL-1RAcP fused to the extracellular region of IL-1R1, and the extracellular region of IL-1R1 is fused to the Fc domain of hIgG1; see U.S. Patent No. 6,927,004), or a VEGF trap (e.g., aflibercept, which contains the Ig domain 2 of VEGF receptor Flt1 fused to the Ig3 region of VEGF receptor Flk1, and the Ig domain 3 of VEGF receptor Flk1 is fused to the Fc domain of hIgG1; see U.S. Patents Nos. 7,087,411 and 7,279,159).

[0054] Cell culture

[0055] The terms "cell culture medium" and "culture medium" refer to a nutrient solution for growing mammalian cells, which typically provides essential nutrients to promote cell growth, such as 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, vitamins, etc. The cell culture medium may contain extracts, such as serum or peptone (hydrolysate), which supply raw materials to support cell growth. The culture medium may contain yeast-derived or soy extracts, rather than animal-derived extracts. A chemically defined medium is a cell culture medium in which all chemical components are known (i.e., having 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 medium is a chemically defined medium.

[0056] This solution may also contain components that promote growth and / or above minimum viability, including hormones and growth factors. This solution is preferably formulated to a pH and salt concentration suitable for cell survival and proliferation.

[0057] "Cell line" refers to one or more cells derived from a specific strain via serial passage or subculture of cells. The term "cell" may be used interchangeably with "cell population".

[0058] The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include eukaryotic cells, such as non-human animal cells, mammalian cells, human cells, avian cells, insect cells, yeast cells, or cell fusions, such as hybridomas or quadromas. In certain embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In certain embodiments, the cell is a cell selected from the following cells: CHO (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, lymphocytes, such as Jurkat (T lymphocytes) or Daudi (B lymphocytes), 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 foregoing cells. In some embodiments, the cell includes one or more viral genes, such as retinal cells expressing viral genes (e.g., PER. cells). In certain embodiments, the cells are CHO cells. In other embodiments, the cells are CHO K1 cells.

[0059] One aspect of the present invention relates to seeding culture, in which a cell population is expanded prior to protein production and harvested in production culture. According to the present invention as described herein, taurine can be added to the basal medium as a seeding culture formulation.

[0060] Another aspect of the present invention relates to production culture, in which a protein is produced and harvested. Prior to the production phase, there is typically a growth phase (also referred to as seeding training or seeding culture), in which all components for cell culture are supplied to the culture vessel at the start of the culture procedure, and then the cell population is expanded until ready for production scale. In this regard, depending on the starting cell line, the culture vessel is inoculated with cells at a suitable seeding density and the initial cell growth phase is carried out. In certain aspects, according to the present invention as described herein, taurine can be added to the basal medium as a seeding culture formulation to further increase or enhance the productivity of the cells in the subsequent production phase.

[0061] One aspect of the present invention relates to production culture, in which cell culture conditions are modified to increase the growth of recombinant prokaryotic cells, while increasing the production of one or more recombinant proteins of interest in the cells and maintaining cell viability, particularly by adding taurine to the production cell medium and / or seeding culture. In the production culture vessel or bioreactor, the basal medium and cell line are supplied to the culture vessel after the seeding culture or growth phase. In certain embodiments, the cell supernatant or cell lysate is harvested after production culture. In other embodiments, the polypeptide or protein of interest is recovered from the cell medium or cell lysate, or in any case, depending on the location of the protein of interest, using techniques well known in the art.

[0062] Culture vessels include (but are not limited to) multi-well plates, T-flasks, shake flasks, stirred vessels, spinner flasks, hollow fibers, airlift bioreactors, and the like. A suitable cell culture vessel is a bioreactor. A bioreactor refers to any culture vessel that has been produced or modified to manipulate or control environmental conditions. Such culture vessels are well known in the prior art.

[0063] Bioreactor procedures and systems have been developed to optimize gas exchange, to supply sufficient oxygen to maintain cell growth and productivity, and to remove CO 2 . Maintaining the efficacy of gas exchange is an important criterion for ensuring successful scale-up of cell culture and protein production. Such systems are well known to those skilled in the prior art.

[0064] In the polypeptide production phase, "fed-batch cell culture" or "fed-batch culture" refers to a process in which animal cells and a culture medium are supplied to a culture vessel at the start, and additional culture nutrients are slowly injected into the culture during the culture period either continuously or in discrete increments, regardless of whether there is or is not a periodic harvest of cells and / or product before the end of the culture. Fed-batch culture includes "semicontinuous 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 differs from simple "batch culture", in which all components of the cell culture (including animal cells and all culture nutrients) are supplied to the culture vessel at the start of the culture procedure. Fed-batch culture can be further distinguished from perfusion culture as long as supernatant is not removed from the culture vessel during the process, whereas in perfusion culture, cells are retained in the culture, e.g., by filtration, and the medium is continuously or intermittently introduced and removed from the culture vessel. However, samples may be removed during fed-batch culture for assay purposes. This fed-batch procedure continues until it is determined that the maximum working volume and / or protein production has been reached.

[0065] The term "continuous cell culture" as used herein refers to techniques for culturing cells continuously, typically at a particular growth stage. For example, if a constant supply of cells is required, or if a particular polypeptide or protein of interest needs to be produced, the cell culture may need to be maintained at a particular growth stage. Therefore, conditions must be continuously monitored and adjusted accordingly in order to maintain the cells at a specific stage.

[0066] Culture medium

[0067] The present invention provides a cell culture medium containing from about 0.1 mM to 10 mM taurine, which is serum-free. "Serum-free" applies to cell culture media that do not contain animal sera, such as fetal bovine serum. Serum-free media may contain ≤16 g / L of hydrolysates, such as soy hydrolysates. The present invention also provides a chemically defined medium that is not only serum-free but also hydrolysate-free. "Hydrolysate-free" applies to cell culture media that do not contain exogenous proteolytic hydrolysates, such as animal or plant proteolytic hydrolysates, such as peptone, tryptone, and the like. A "basal medium" is the initial medium (present at inoculation and / or in cell culture production on day 0) in which cells proliferate and contains all the necessary nutrients, which includes a basal mixture of amino acids. Various formulations (i.e., preparations) for basal media can be produced or purchased in commercial quantities. Similarly, a "basal feed medium" contains a mixture of supplementary nutrients that are normally consumed during production culture and is utilized in a feeding strategy (for so-called "fed-batch" culture). A variety of basal media are commercially available. "Feeding" includes the periodic addition or addition at regular intervals to the culture medium, for example, according to a schedule, including continuous feeding culture systems, such as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec; 17(6): 1032-41), or according to a fed-batch procedure (Y.M. Huang et al., Biotechnol Prog. 2010 Sep-Oct; 26(5): 1400-10). For example, the culture can be fed daily, every other day, every three days, or can be fed under monitoring when the concentration of a specific culture medium component falls outside the desired range.

[0068] The elimination of serum and the reduction or elimination of hydrolysates from cell culture media, while reducing batch-to-batch variability and increasing downstream processing steps, unfortunately reduces cell growth, viability, and protein expression. Therefore, chemically defined serum-free and low- to hydrolysate-free media require additional components to increase cell growth and protein production.

[0069] Accordingly, the cell culture medium of the present invention includes a basal medium that contains all the necessary nutrients for viable cell culture. According to the present invention as described herein, taurine can be added to the basal medium as an inoculation culture preparation. In addition, taurine can be added to the basal medium as a production culture preparation, which can then be periodically fed (such as in a so-called "fed-batch" culture) with or without additional components (such as polyamines) or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, and the like, depending on the needs of the cells being cultured or the desired cell culture parameters.

[0070] The present invention provides that during protein production culture in a cell culture medium supplemented with taurine, amino acids may be depleted without providing additional amino acid supplementation, or this cell culture medium supplemented with taurine may be "non-depleting", where amino acid supplementation provides for the depleted amino acids (as described below). The inventors have observed that cultures supplemented with taurine during the production phase increase recombinant protein production under various culture conditions as described above.

[0071] The present invention provides a medium supplemented with taurine, which contains 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 in millimoles per liter).

[0072] In one embodiment, the medium further contains 100 μM ± 15 μM ornithine, or 300 μM ± 45 μM ornithine, or 600 μM ± 90 μM ornithine, or even 900 μM ± 135 μM ornithine. In another embodiment, the medium contains at least about 5 mg / L ± 1 mg / L ornithine·HCl, or at least about 10 mg / L ± 2 mg / L ornithine·HCl, 15 mg / L ± 2.25 mg / L ornithine·HCl, or at least about 50 mg / L ± 7.5 mg / L ornithine·HCl, or at least about 100 mg / L ± 15 mg / L ornithine·HCl, or at least about 150 mg / L ± 22.5 mg / L ornithine·HCl.

[0073] Putrescine may optionally be added to the supplemented medium. In certain cell culture formulations, very low concentrations of putrescine are included as a component; see, for example, WO 2005 / 028626, which describes 0.02 - 0.08 mg / L putrescine; 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); WO 2008 / 154014 (~100 μM - ~1000 μM); U.S. Patent Application No. 2007 / 0212770 (0.5 - 30 mg / L polyamine; 2 mg / L putrescine; 2 mg / L putrescine + 2 mg / L ornithine; 2 mg / L putrescine + 10 mg / L ornithine).

[0074] In certain embodiments, the culture medium is further supplemented with a composition of ornithine and putrescine, wherein the putrescine can be at a concentration of at least about 150 to 720 μM. In certain embodiments, the culture medium is further supplemented with putrescine at a concentration of at least about 170 to 230 μM. In one embodiment, the culture medium contains 200 μM ± 30 μM putrescine in addition to ≥ 90 μM ± 15 μM ornithine. In one embodiment, the culture medium contains ≤ 30 mg / L ± 4.5 mg / L putrescine·2HCl in addition to ≤ 15 mg / L ± 2.25 mg / L ornithine. In additional embodiments, the culture medium contains ≥ 30 mg / L ± 4.5 mg / L putrescine·2HCl in addition to ≥ 15 mg / L ± 2.25 mg / L ornithine·HCl (see International Publication No. WO2014 / 144198A1, published on September 14, 2014, which is incorporated herein by reference in its entirety).

[0075] In other embodiments, ornithine is present in the culture medium at a concentration ranging from 0.09 ± 0.014 mM to 0.9 ± 0.14 mM, such as 0.09 ± 0.014 mM, 0.3 ± 0.05 mM, 0.6 ± 0.09 mM, or 0.9 ± 0.14 mM ornithine. In certain embodiments, the culture medium also contains at least 0.20 ± 0.03 mM putrescine. In certain embodiments, this additional putrescine concentration ranges from 0.20 ± 0.03 mM to 0.714 ± 0.11 mM, such as 0.20 ± 0.03 mM, 0.35 ± 0.06, or 0.714 ± 0.11 mM putrescine.

[0076] A variety of other supplements can be added to the culture medium and are within the skill of the art to determine additional suitable conditions. In certain embodiments, 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 for the purpose of facilitating non-depleted or when additional nutrients are needed.

[0077] In one embodiment, the culture medium is further supplemented with from about 170 μM to 175 μM nucleosides. In one embodiment, the culture medium contains 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 cumulative concentration of purine derivatives. In one embodiment, the culture medium contains from about 100 μM to 110 μM of purine derivatives. Purine derivatives include hypoxanthine and the nucleosides adenosine and guanosine. In one embodiment, the culture medium contains 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 cumulative concentration of pyrimidine derivatives. In one embodiment, the culture medium contains from about 65 μM to 75 μM of pyrimidine derivatives. Pyrimidine derivatives include the nucleosides thymidine, uridine, and cytidine. In a specific embodiment, the culture medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0078] 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 tocopherols. In one embodiment, such fatty acids include any one or more of linoleic acid, linolenic acid, lipoic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, capric acid, lauric acid, caproic acid, pelargonic acid, myristic acid, and caprylic acid. In one embodiment, the culture medium contains tocopherol, linoleic acid, and lipoic acid.

[0079] In one embodiment, the culture medium may also be further supplemented with a mixture of vitamins, which includes at least about 700 μM or at least about 2 mM cumulative concentration of other nutrients and essential nutrients. In one embodiment, the mixture of vitamins contains one or more of D-biotin, choline chloride, folic acid, inositol, nicotinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, vitamin B12, and the like. In one embodiment, the mixture of vitamins includes all of D-biotin, choline chloride, folic acid, inositol, nicotinamide, pyridoxine HCl, D-pantothenic acid (hemiCa), riboflavin, thiamine HCl, and vitamin B12.

[0080] Various embodiments of the culture medium of the present invention include combinations of any of the above embodiments, including chemically defined, hydrolysate-free, serum-free culture media, which contain a specified amount of taurine, plus (in particular) (a) amino acids; (b) optional nucleosides; (c) salts of divalent cations; (d) fatty acids and tocopherols; and (e) vitamins. In certain embodiments, all minor amounts of hydrolysates may be added to the culture medium supplemented with taurine.

[0081] The applicant contemplates that in the practice of the present invention, one or more various basal media or combinations thereof may use taurine. Basal media are generally known in the prior art and include (inter alia) Eagle's MEM (Minimum Essential Medium) (Eagle, Science, 1955, 112(3168): 501-504), Ham's 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., 1952 August; 38(8): 747-752), DMEM / Ham's F12 1:1, Trowell's T8, 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.), RPMI 1640 (Moore et al., J. Amer. Med. Assoc., 1967, 199: 519-524), MCDB 104 / 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., Appl. Microbiol., 1973, 25(1): 49-54), RPMI-1640 medium, RPMI-1641 medium, Iscove's Modified Dulbecco Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as EX-CELL TM300 Series (JRH Biosciences, Lenexa, Kansas), protamine-zinc-insulin medium (Weiss et al., 1974, US 4,072,565), biotin-folic acid medium (Cartaya, 1978, US Re30,985), transferrin-fatty acid medium (Baker, 1982, US 4,560,655), transporter protein-EGF medium (Hasegawa, 1982, US 4,615,977; Chessebeuf, 1984, US 4,786,599), and other medium replacements (see Inlow, US 6,048,728; Drapeau, US 7,294,484; Mather, US 5,122,469; Furukawa, US 5,976,833; Chen, US 6,180,401; Chen, US 5,856,179; Etcheverry, US 5,705,364; Etcheverry, US 7,666,416; Ryll, US 6,528,286; Singh, US 6,924,124; Luan, US 7,429,491; etc.).

[0082] In a specific embodiment, the medium is chemically defined and contains, in addition to taurine: a mixture of amino acids as defined herein, CaCl 2 2H 2 O; HEPES buffer, KCl; MgSO 4 ; NaCl; Na 2 HPO 4 or other phosphates; pyruvic acid; D-biotin; choline chloride; folic acid; inositol; nicotinamide; pyridoxine HCl; D-pantothenic acid; riboflavin; thiamine HCl; vitamin B12; ρ-aminobenzoic acid; ethanolamine HCl; poloxamer 188; DL-α-tocopherol phosphate; linoleic acid; Na 2 SeO 3 ; lipoic acid; and glucose; and optionally adenosine; guanosine; cytidine; uridine; thymidine and hypoxanthine 2Na.

[0083] In one embodiment, the starting osmotic pressure of the medium of the present invention is 200 - 500, 250 - 400, 275 - 350 or about 300 mOsm. During the growth phase of the cells in the medium of the present invention, and especially after any feeding according to the fed-batch method, the osmotic pressure of the culture can increase to a maximum of about 350, 400, 450, 500 or up to about 550 mOsm.

[0084] In certain embodiments, the osmolality of the defined-component medium is less than about 300, and this osmolality is adjusted to about 300 by adding one or more specified salts in excess. In one embodiment, the osmolality is increased to the desired level by adding one or more osmolytes selected from the group consisting of sodium chloride, potassium chloride, magnesium salts, calcium salts, amino acid salts, fatty acid salts, sodium bicarbonate, sodium carbonate, potassium carbonate, chelators of salts, sugars (such as galactose, glucose, sucrose, fructose, fucose, etc.), and combinations thereof. In one embodiment, the osmolytes are added in addition to the concentrations of the components already present in the defined-component medium (e.g., sugars are added in addition to the specified sugar components).

[0085] Each of the above-described embodiments of the medium, and any other serum-free medium containing at least about 0.1 mM taurine, is referred to as a taurine-supplemented medium. Conversely, a medium that does not contain taurine, or contains less than 0.1 mM taurine, is hereinafter referred to as a non-taurine-supplemented medium or a non-supplemented medium.

[0086] Fed-batch culture

[0087] The feeding strategy for cell culture is aimed at ensuring optimal growth and proliferation of cells outside a multicellular organism or tissue. Culture conditions suitable for mammalian cells are known in the art. See, for example, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, spinner flasks, shake flasks, or stirred tank bioreactors (with or without microcarriers, and operating in batch, fed-batch, continuous, semi-continuous, or perfusion modes) can be used for mammalian cell culture. The cell culture medium or concentrated feed medium can be added continuously or at intervals during the culture. For example, the culture can be fed daily, every other day, every three days, or can be fed under monitoring when the concentration of a particular medium component falls outside the desired range.

[0088] In addition to including taurine, in one embodiment, the culture medium may be further supplemented with at least 20 mM cumulative concentration of amino acids. In one embodiment, the initial amino acid concentration in the starting cell culture medium does not include this supplemented cumulative (total) concentration of amino acids. In an embodiment of a cell culture medium, or in a method of culturing cells or a method of producing a protein of interest, the culture medium may be supplemented with an amount greater than about 20 mM, greater than about 25 mM, greater than about 30 mM, greater than about 40 mM, greater than about 50 mM, or greater than about 60 mM, greater than about 70 mM, greater than about 100 mM, greater than about 200 mM, greater than about 300 mM, greater than about 400 mM, or greater than about 500 mM. See also Table 1 in the text. In one embodiment, the amount of amino acids added to the culture medium is about 30 mM ± 10 mM or more.

[0089] The feeding supplementation protocol can be optimized by those skilled in the art to support cell growth during the production phase, minimize cell stress, or provide a "non-depleted medium".

[0090] A "non-depleted medium" includes a cell culture medium that has been assayed to have nutrients, particularly the amino acids required for producing a recombinant protein of interest. Amino acid feeding typically supplements the amino acids required for the building blocks of producing a recombinant protein in cell culture. However, depending on the requirements of the specific protein produced by the cells in culture, some amino acids may be consumed faster than others. In a non-depleted medium, it has been determined that the feeding protocol must therefore replenish essential amino acids as they are consumed. Thus, the depletion and subsequent optimal consumption rate (pg / cell-day) can be determined by the following steps: culturing eukaryotic cells expressing a protein of interest in a cell culture medium; measuring the concentration of each amino acid in the culture medium at time points to establish the consumption amount; identifying the consumption time points at which the amino acid concentration falls below the consumption amount; calculating the consumption rate of each amino acid; and determining the optimal consumption rate as the consumption rate at the time point just before the depletion time point. The cell culture is then supplemented with specific amino acids at appropriate concentrations as needed to maintain the optimal consumption rate for use as a non-depleted medium.

[0091] It should be understood that the present invention provides a taurine-supplemented cell culture medium that increases protein titer in both depleted and non-depleted cultures.

[0092] The present invention provides for cell culture in a cell medium supplemented with taurine as described above, which comprises a cell line expressing a protein of interest. Examples of cell lines conventionally used for the production of protein biotherapeutics include (inter alia) 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 specific CHO cell variants optimized for large-scale protein production, such as CHO-K1.

[0093] In one embodiment, the cell culture supplemented with taurine contains insulin, which can be added to the medium as a point-of-use ingredient or can be included in the medium formulation. In one embodiment, the cells comprise cells capable of producing a biotherapeutic protein.

[0094] In one embodiment, the medium is supplemented at intervals during the culture according to the fed-batch method. Fed-batch culture has generally been known in the prior art and applied to optimize protein production (see Y.M. Huang et al., Biotechnol Prog. 2010 Sep-Oct; 26(5): 1400-10).

[0095] The cell growth phase or inoculation culture (i.e., the initial cell culture) in which no medium exchange is provided is typically followed by a different second culture, called the polypeptide production phase. During the production phase, the fed-batch method is generally used.

[0096] The present invention provides a cell medium comprising from about 0.1 mM to about 10 mM taurine at the start of production cell culture (day 0). Alternatively, a cell medium comprising from about 0.1 mM to about 10 mM taurine can be supplemented on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, and / or day 10 of the production cell culture. The cell medium added to the production culture on multiple days comprises a total amount of from about 0.1 mM to about 10 mM taurine. The cell medium comprising a total amount of from about 0.1 mM to about 10 mM taurine can be added in any order.

[0097] Taurine can also be supplemented to the basal medium during the inoculation expansion phase.

[0098] During production culture, feeding supplements can be carried out at intervals, daily or every 2 - 3 days, to include additional nutrients such as vitamins, amino acids, and other nutrients as described above. During the production culture for a total of 2 weeks or longer, feeding supplements (addition of a supplemented medium containing nutrients) can be carried out at least 2 times, or at least 8 times. In an alternative embodiment, feeding supplements can be carried out daily during the culture. Alternative culture feeding regimens are also contemplated.

[0099] Additional amino acid supplementation can also be carried out to provide a non - depleted medium, where the depleted amino acids are determined according to methods known in the prior art and described herein. When this regimen is applied, additional amino acids are supplemented or added at intervals according to the measured amino acid consumption, preferably at a frequency of daily or every 2 - 3 days during the production culture. In one embodiment, a mixture of additional amino acids for maintaining a non - depleted cell medium is added to the culture on or about day 1, on or about day 2, on or about day 3, on or about day 4, on or about day 5, on or about day 6, on or about day 7, on or about day 8, on or about day 9, on or about day 10, on or about day 11, on or about day 12, on or about day 13, on or about day 14, for a culture of 2 weeks or longer. Alternative culture feeding regimens are also contemplated.

[0100] Animal cells, such as CHO cells, can be cultured on a small scale, for example, in a 125 ml container with approximately 25 mL of medium, in a 250 ml container with approximately 50 to 100 mL of medium, in a 500 mL container with approximately 100 to 200 mL of medium. Alternatively, this culture can also be on a large scale, for example, in a 1000 mL container with approximately 300 to 1000 mL of medium, in a 3000 mL container with approximately 500 mL to 3000 mL of medium, in an 8000 mL container with approximately 2000 mL to 8000 mL of medium, in a 15000 mL container with approximately 4000 mL to 15000 mL of medium. The culture for production can contain 10,000 L of medium or more. Large - scale cell culture, such as for clinical production of protein therapeutics, typically lasts for several days or even weeks while the cells produce the desired protein. During this period, the culture can be supplemented with a concentrated feed medium containing, for example, nutrient and amino acid components, which are consumed during the culture. The concentrated feed medium can be based on any cell culture formulation. Such a concentrated feed medium can contain most of the components of the cell medium, for example, about 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even about 1000X their normal usage amounts. Concentrated feed media are typically used in fed - batch culture methods.

[0101] In certain embodiments, cell cultures containing taurine are further supplemented with "point-of-use additives", also referred to as additives, point-of-use components, or point-of-use chemicals, during cell growth or protein production. Point-of-use additives include any one or more 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 growth, or in some cases, cell differentiation. Growth factors are typically proteins (e.g., insulin), small molecule peptides, or steroid hormones such as estrogen, DHEA, testosterone, and the like. In some cases, growth hormones can be non-natural chemicals that promote cell proliferation or protein production, such as tetrahydrofolic acid (THF), methotrexate, and the like. Non-limiting examples of protein and peptide growth factors include angiopoietin, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), motility factor, myostatin (GDF-8), nerve growth factor (NGF) and other neurotrophic proteins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF), wnt signaling pathway agonists, placental growth factor (PlGF), fetal bovine serum (FBS), interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and so on. In one embodiment, the cell culture medium is supplemented with the point-of-use additive growth factor insulin. In one embodiment, the concentration of insulin in the medium, i.e., the amount of insulin in the cell culture medium after addition, is from about 0.1 μM to 10 μM.

[0102] Buffers are generally known in the art. The present invention is not limited to any particular buffer, and any person of ordinary skill in the art can select a suitable buffer for a particular cell line used for producing a particular protein. In one embodiment, the point-of-use additive buffer is NaHCO 3 . In a further embodiment, this buffer is HEPES. In other embodiments, this point-of-use additive buffer includes both NaHCO 3 and HEPES.

[0103] It is also well known in the prior art to use, as an energy source added at the point of use, in cell culture. Without limitation, in one embodiment, the energy source added at the point of use is glucose. If given the specific and designated requirements of a particular cell line and the production of a protein, in one embodiment this glucose can be added to the culture medium at a concentration of about 1 to 20 mM. In some cases, it can be added in a high amount of 20 g / L or higher.

[0104] Chelating agents are likewise well known in cell culture and protein production techniques. Although other chelating agents can be applied in practicing the present invention, tetrasodium EDTA dihydrate and citrate are two common chelating agents used in the prior art. In one embodiment, the chelating agent added at the point of use is tetrasodium EDTA dihydrate. In one embodiment, the chelating agent added at the point of use is citrate, such as Na 3 C 6 H 5 O 7 。

[0105] In one embodiment, the cell culture medium can be additionally supplemented with one or more amino acids added at the point of use as an energy source, such as glutamine. In one embodiment, the cell culture medium is supplemented with the amino acid glutamine added at the point of use at a final concentration of about 1 mM to 13 mM.

[0106] Other additives added at the point of use include one or more various metal salts, such as iron salts, nickel salts, zinc salts, and copper salts. In one embodiment, the cell culture medium is supplemented with any one or more of copper sulfate, zinc sulfate, iron chloride, and nickel sulfate.

[0107] In certain embodiments, the protein titer produced by cell culture supplemented with taurine is at least 4%, 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%, or at least 29% greater than the protein titer produced by cell culture not supplemented with taurine. In certain embodiments, the protein titer produced by cell culture supplemented with taurine is at least 2%, at least 3%, at least 4%, or at least 5% greater than the protein titer produced by a similar or identical cell culture not supplemented with taurine.

[0108] In certain embodiments, the ammonia concentration in cell cultures in a medium supplemented with taurine is reduced by more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 15%, or more than 20% compared to cell cultures in a medium not supplemented with taurine.

[0109] Protein production

[0110] In addition to the medium supplemented with taurine and the method of culturing cells in the medium supplemented with taurine, the present invention provides a method for increasing the production of proteins, such as therapeutically effective antibodies or other biopharmaceutical substances, in cell cultures in the medium supplemented with taurine. The present invention provides a method for producing therapeutic proteins in high yield, which comprises culturing a recombinant cell line in a medium containing taurine, wherein the cell contains a stably integrated nucleic acid encoding the therapeutic protein.

[0111] In certain embodiments, the protein titer (yield) of mammalian cells cultured in a medium containing taurine 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, at least 2.5 g / L greater than the protein titer of the same mammalian cells cultured in a medium not supplemented with taurine.

[0112] In certain embodiments, the protein yield or titer from cell cultures in a medium supplemented with taurine, which can be expressed as grams of protein per liter of medium, 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.

[0113] In certain embodiments, the protein titer produced by cells in a medium supplemented with taurine is at least 2%, at least 3%, at least 4%, 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% or at least 29% greater than the protein titer (yield) of similar or identical cells cultured in a medium not supplemented with taurine.

[0114] In certain embodiments, the protein product (protein of interest) 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 a tetrabody, a Fab fragment or an 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. 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.

[0115] In certain embodiments, the antibody is selected from: an anti-programmed cell death antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203579A1), an anti-programmed cell death ligand-1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203580A1), an anti-D114 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), an anti-angiopoietin-3-like antibody (e.g., an anti-AngPtl3 antibody as described in U.S. Patent No. 9,018,356), an anti-platelet-derived growth factor receptor antibody (e.g., an anti-PDGFR antibody, U.S. Patent No. 9,265,827), an anti-Erb3 antibody, an anti-prolactin receptor antibody (e.g., an anti-PRLR antibody as described in U.S. Patent No. 9,302,015), an anti-complement 5 antibody (e.g., an anti-C5 antibody as described in U.S. Patent Application Publication No. US2015 / 0313194A1), an anti-TNF antibody, an anti-epidermal growth factor receptor antibody (e.g., an anti-EGFR antibody as described in U.S. Patent No. 9,132,192 or an anti-EGFRvIII antibody as described in U.S. Patent Application Publication No. US2015 / 0259423A1), an anti-proprotein convertase subtilisin / kexin type 9 antibody (e.g., an anti-PCSK9 antibody as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. US2014 / 0044730A1), an anti-growth and differentiation factor-8 antibody (e.g., an anti-GDF8 antibody as described in U.S. Patent No. 8,871,209 or 9,260,515, also known as an anti-myostatin antibody), an anti-glucagon receptor (e.g., an anti-GCGR antibody as described in U.S. Patent Application Publication No. US2015 / 0337045A1 or US2016 / 0075778A1), an anti-VEGF antibody, an anti-IL1R antibody, an interleukin 4 receptor antibody (e.g., an anti-IL4R antibody as described in U.S. Patent Application Publication No. US2014 / 0271681A1 or U.S. Patent Nos. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g., antibody U.S. Patent Nos. 7,582,298, 8,043,617 or 9,173,the anti-IL6R), anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-interleukin 33 (e.g., the anti-IL33 antibody as described in U.S. Patent Application Publication Nos. US2014 / 0271658A1 or US2014 / 0271642A1), anti-respiratory syncytial virus antibody (e.g., the anti-RSV antibody as described in U.S. Patent Application Publication No. US2014 / 0271653A1), anti-cluster of differentiation 3 (e.g., the anti-CD3 antibody as described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1 and U.S. Application No. 62 / 222,605), anti-cluster of differentiation 20 (e.g., the anti-CD20 antibody as described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1 and U.S. Patent No. 7,879,984), anti-CD19 antibody, anti-CD28 antibody, anti-cluster of differentiation-48 (e.g., the anti-CD48 antibody as described in U.S. Patent No. 9,228,014), anti-Fel d1 antibody (e.g., described in U.S. Patent No. 9,079,948), anti-Middle East respiratory syndrome virus (e.g., the anti-MERS antibody as described in U.S. Patent Application Publication No. US2015 / 0337029A1), anti-Ebola virus antibody (e.g., described in U.S. Patent Application Publication No. US2016 / 0215040), anti-Zika virus antibody, anti-lymphocyte activation gene 3 (e.g., anti-LAG3 antibody or anti-CD223 antibody), and anti-activin A antibody. In certain embodiments, the bispecific antibody is selected from: anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966), anti-CD3x anti-mucin 16 bispecific antibody (e.g., anti-CD3 x anti-Muc16 bispecific antibody), and anti-CD3 x anti-prostate-specific membrane antigen bispecific antibody (e.g., anti-CD3 x anti-PSMA bispecific antibody). In certain embodiments, the protein of interest is selected from: alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab. All publications mentioned throughout the disclosure are incorporated herein by reference in their entirety.,

[0116] In certain embodiments, the protein of interest is a recombinant protein (e.g., an Fc-fusion protein) that contains an Fc portion and an additional region. In certain embodiments, the Fc-fusion protein is a receptor Fc-fusion protein that contains the extracellular region of one or more receptors that bind to the Fc portion. In certain embodiments, this Fc portion comprises a hinge region followed by the CH2 and CH3 regions of IgG. In certain embodiments, this receptor Fc-fusion protein contains two or more different receptor chains that bind to a single ligand or multiple ligands. For example, this Fc-fusion protein is a Trapping Receptor Affibody Protein (TRAP) protein, such as an IL-1 TRAP (e.g., rilonacept, which contains the IL-1RAcP ligand-binding region fused to the extracellular region of Il-1R1, and the extracellular region of Il-1R1 is fused to the Fc domain of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF TRAP (e.g., aflibercept, which contains the Ig 2 region of VEGF receptor Flt1 fused to the Ig 3 region of VEGF receptor Flk1, and the Ig 3 region of VEGF receptor Flk1 is fused to the Fc domain of hIgG1; see U.S. Patents Nos. 7,087,411 and 7,279,159). In other embodiments, the Fc-fusion protein is a ScFv-Fc-fusion protein that contains one or more antigen-binding regions, such as variable heavy chain and variable light chain fragments of an antibody that bind to the Fc portion.

[0117] For protein production, the present invention is not limited to any particular type of cell. Examples of cell types suitable for protein production include mammalian cells, insect cells, avian cells, bacterial cells, and yeast cells. The cells can be stem cells or recombinant cells transformed with a vector expressing a recombinant gene, or cells transfected with a virus that produces viral products. The cells can contain a recombinant heterologous polynucleotide construct encoding a protein of interest. This construct can be an episome or it can be a component that is naturally integrated into the cell genome. The cells can also produce the protein of interest without having the protein encoded on a heterologous polypeptide construct. In other words, the cells can inherently encode the protein of interest, such as B-cells producing antibodies. The cells can also be primary cells such as chicken embryo cells, or primary cell lines. Examples of available cells include BSC 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-21 cells, chicken embryo cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 293 cells, RK cells, Per.C6 cells, and CHO 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 lec mutant.

[0118] In one embodiment, the cell is a CHO cell that ectopically expresses a protein. In one embodiment, the protein comprises an immunoglobulin heavy chain region, such as a CH1, CH2, or CH3 region. In one embodiment, the protein comprises human or rodent immunoglobulin CH2 and CH3 regions. In one embodiment, the protein comprises human or rodent immunoglobulin CH1, CH2, and CH3 regions. In one embodiment, the protein comprises a hinge region and CH1, CH2, and CH3 regions. In one embodiment, the protein comprises an immunoglobulin heavy chain variable domain. In one embodiment, the protein comprises an immunoglobulin light chain variable domain. In one embodiment, the protein comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain. In one embodiment, the protein is an antibody, such as a human antibody, a rodent antibody, or a chimeric human / rodent antibody (e.g., human / mouse, human / rat, or human / hamster).

[0119] The production phase can be carried out at any scale of cultivation, from shaker flasks or rocking bags, to 1-liter bioreactors and large-scale industrial bioreactors. Similarly, the inoculum expansion phase can be carried out at any scale of cultivation, from shaker flasks or rocking bags, to 1-liter or large bioreactors. Large-scale processing can be carried out at volumes of approximately 100 liters to 20,000 liters or greater. One or several methods can be used to control protein production, such as temperature shift or chemical induction. The growth phase can occur at a higher temperature than the production phase. For example, the growth phase can occur at a first temperature of approximately 35°C to 38°C, while the production phase can occur at a second temperature of approximately 29°C to 37°C, optionally from approximately 30°C to 36°C or from approximately 30°C to 34°C. In addition, chemical inducers of protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, hexamethylene bisacetamide (HMBA) can be added simultaneously with, before, or after the temperature shift. If the inducer is added after the temperature shift, it can be added from 1 hour to 5 days after the temperature shift, such as 1 to 2 days after the temperature shift. The production cell culture can operate as a continuous feed culture system, such as in a chemostat (see C. Altamirano et al., 2001 supra) or according to the fed-batch method (Huang, 2010 supra).

[0120] The present invention can be used to increase cell yields via cell culture procedures. The cell lines used in the present invention can be genetically engineered to express polypeptides of commercial or scientific interest. Genetically engineering cells involves transfecting, transforming or converting cells with recombinant polynucleotide molecules, or otherwise altering (e.g., by homologous recombination and gene activation or by fusion of recombinant cells with non-recombinant cells) such that the host cells express the desired recombinant polypeptide. Methods and vectors for genetically engineering cells or cell lines to express polypeptides of interest are well known to those skilled in the art; for example, the various techniques shown in the Molecular Biology Experiment Manual. 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, R.J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69. A wide variety of cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and commercial suppliers. Examples of cell lines commonly used industrially include VERO, BHK, HeLa, CV1 (including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NS1), PC12, WI38 cells and Chinese hamster ovary (CHO) cells. The CHO cell line is widely used to produce complex recombinant proteins, such as cytokines, clotting factors and antibodies (Brasel et al. (1996), Blood 88: 2004-2012; Kaufman et al. (1988), J. Biol Chem 263: 6352-6362; McKinnon et al. (1991), J Mol Endocrinol 6: 231-239; Wood et al. (1990), J Immunol. 145: 3011-3016). Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77: 4216-4220), DXBI1 and DG-44 and the desired CHO host cells allow high levels of recombinant protein expression in these cells because of the selectable and amplifiable gene expression of this defective DHFR (Kaufman RJ. (1990), Meth Enzymol 185: 537-566). In addition, these cells are easily manipulated for adherent or suspension culture and exhibit fairly good genetic stability.CHO cells and the proteins recombinantly expressed thereby have been widely characterized and approved by regulatory authorities for clinical and commercial production. In certain embodiments, the CHO cell line is a cell line as described in U.S. Patent Application Publication Nos. 2010 / 0304436 A1, 2009 / 0162901 A1, and 2009 / 0137416 A1, as well as U.S. Patent Nos. 7,455,988 B2, 7,435,553 B2, and 7,105,348 B2.

[0121] The invention is not limited to the scope of the specific embodiments described herein, which are intended to be illustrative of individual aspects or embodiments of the invention. Functionally equivalent methods and components are within the scope of the invention. Various modifications of the invention, in addition to those described herein, will be apparent to those of ordinary skill in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the invention. Examples

[0122] Example 1: Since taurine supplementation increased antibody titers

[0123] Example 1A - High - throughput shake - flask culture: Inoculate 250 mL shake - flasks with a seeded culture of monoclonal antibody (Ab1) - producing cells derived from CHO - K1. Allow the inoculated cells to grow at 35.5 °C for 17 days and supply glucose and other supplemented nutrients as needed. The cell line is grown in a chemically defined (hydrolysate - free and serum - free) basal medium.

[0124] Each culture flask was non - supplemented (flask 1a) or supplemented with 1 mM taurine (flask 1b) on day 0.

[0125] Table 2: Average 17 - day antibody titers (g / L) and approximate titer increase relative to baseline (%)

[0126]

[0127] *Baseline control for % titer increase; compare titer in flask 1b to titer in non - supplemented medium (flask 1a).

[0128] A The difference in final titers between non - supplemented and supplemented cultures was statistically significant (p < 0.05).

[0129] Titer values were calculated from the protein harvested on day 17 and were statistically significant compared to the baseline (p < 0.05). Cultures supplemented with taurine showed an overall 8% increase in final protein titer over non - supplemented cultures.

[0130] Example 1B - Bench-top bioreactor: In a similar example and on a larger scale of production, an inoculum of cells producing monoclonal antibodies (Ab2, Ab3 or Ab4) derived from CHO-K1 was inoculated into a 2 L bioreactor. The inoculated cells were grown at 35.5 °C with a setpoint of 40.4% and an air injection of 22 ccm for 14 days. The Ab2 and Ab3 treatments had a pH setpoint of 7.0 ± 0.15, while the Ab4 treatment had a pH setpoint of 7.13 ± 0.27. Glucose, antifoam and basal feed were supplied to the bioreactor as needed. The cultures were grown either without a supplemented medium (bioreactors 2a, 3a, 4a) or in a medium supplemented with approximately 1 mM taurine (Ab2 and Ab3) or in a medium supplemented with approximately 3 mM taurine (Ab4), which was added on day 0 of production (bioreactors 2b, 3b and 4b, respectively).

[0131] For the Ab2-producing cells, the antibody yield (titer) was 6.4 g / L, while the cells grown with taurine produced 8 g / L of protein. The 24% increase compared to the cells grown without taurine supplementation was statistically significant (p < 0.05). After 14 days (11% and 20% respectively), the final titers produced by the Ab3-producing cultures and the Ab4-producing cultures were also significantly higher (p < 0.05) compared to the non-supplemented taurine cultures. See Table 3.

[0132] Table 3: Average 14-day antibody titers (g / L) and approximate titer increase relative to baseline (%)

[0133]

[0134] *The non-supplemented medium was used as a baseline control for the % titer increase, where the % titer increase for bioreactors 2b, 3b or 4b was compared to the titer in the non-supplemented medium (bioreactors 2a, 3a or 4a, respectively).

[0135] A The difference in final titers between the non-supplemented and supplemented cultures was statistically significant (p < 0.05).

[0136] For the Ab3-producing cell cultures, a plot of the protocol against the protein titer was made, and a significant increase in protein titer was observed daily in the cultures starting from day 6 due to taurine supplementation.

[0137] Table 4: Improvement in antibody titers (g / L) caused by taurine supplementation at representative time points

[0138]

[0139] *Approximate increase (%) compared to non-supplemented medium collected on the same day

[0140] A The increase in titer with taurine addition was statistically significant compared to non-supplemented cultures (p < 0.05).

[0141] A significant increase in titer was seen as early as day 6 in production cultures (12% increase compared to the same cultures without taurine supplementation). See also Figure 1 . In this protocol, the largest difference was seen at day 9 (significant (p < 0.05), 29% increase in bioreactor 3b compared to 3a), and a significant (p < 0.05) 11% increase in titer was observed on the last day of culture (14).

[0142] Benefits in protein production with taurine supplementation were observed via different scales (Examples 1A and 1B) and different cell lines (Example 1B).

[0143] Example 2: Constant productivity at various taurine concentrations in high-throughput shake flask cultures

[0144] The constancy of protein titer was tested by adding different amounts of taurine to the cultures on day 0 of production. 250 mL shake flasks were inoculated with cultures of monoclonal antibody (Ab1)-producing cells derived from CHO-K1. The inoculated cells were grown at 35.5 °C for 14 days and glucose and other supplementary nutrients were given as needed. The cell line was grown in a chemically defined (hydrolysate-free and serum-free) basal medium.

[0145] Each culture line either contained no taurine (unsupplemented) or contained taurine at concentrations of 0.1 mM, 0.3 mM, 0.5 mM, 0.7 mM, 1 mM, 3 mM, 5 mM, 7.5 mM, or 10 mM.

[0146] Table 5: Average 14-day antibody titers (g / L) of cultures supplemented with 0.1 to 10 mM taurine

[0147]

[0148] * The unsupplemented medium was the baseline control for % titer increase.

[0149] # The difference in final titer was statistically significant compared to the non-added control (P < 0.1).

[0150] A The difference in final titer was statistically significant compared to the unsupplemented control (P < 0.05).

[0151] It shows that when taurine is supplemented in the range of at least 0.1 mM to 10 mM, different taurine-supplementation amounts consistently produce high titers. The final titers of the taurine-supplemented conditions are statistically different from the non-supplemented conditions. For 0.1 mM taurine, p < 0.1, and for 0.3 mM to 10 mM taurine, p < 0.05.

[0152] Example 3: Culturing various taurine administration regimens in high-throughput shake flask

[0153] Example 3A: Adding taurine during the inoculation amplification period: Evaluating the benefit of adding taurine to the culture during the amplification inoculation period using a high-throughput shake flask model. In flask 6a (Table 6), CHO cells producing Ab1 were dissolved in a chemically defined (no hydrolysates and no serum) basal medium supplemented with 1 mM taurine. Throughout the amplification period, the taurine concentration in the basal medium was maintained at 1 mM. During production, the culture basal medium was supplemented with 1 mM taurine on day 0. During the 17-day production period, glucose and nutrient basal feeds were supplied as needed.

[0154] Throughout the inoculation amplification period, the cells in flask 6b grew in a taurine-free (unsupplemented) chemically defined (no hydrolysates and no serum) basal medium. On day 0 of production, the culture basal medium was supplemented with 1 mM taurine. For the 17-day production, glucose and nutrient basal feeds were supplied as needed.

[0155] Table 6: Average 17-day antibody titers of cultures supplemented with 1 mM taurine

[0156] Flask Addition of 1 mM taurine Ab1 titer 6a Inoculation training and production phase 7.7 g / L 6b Only production phase 7.8 g / L

[0157] The difference in the final titers (day 17) was not statistically significant (p > 0.1).

[0158] The final (day 17) titer values were similar for both conditions (taurine supplementation only during production or combined supplementation during inoculation and production). The titers produced were not statistically significant (p > 0.1). The benefit of adding taurine during the inoculation amplification period was similar to that of supplementing taurine during the production stage.

[0159] Example 3B: Various taurine feeding regimens during the production stage: To determine whether any of the different standard feeding regimens have an impact on the protein titers of the taurine-supplemented medium, additional experiments were conducted by growing CHO cells producing Ab1 in a similar shake flask culture. Similar to Example 2, the cells were subjected to different culture conditions, where the feeding regimens were the same as before, and glucose / nutrient basal feeds were added as needed.

[0160] The Ab1-production culture system was supplemented with a total of 5 mM taurine. Similar productivities (7.1 g / L, 6.8 g / L, and 7.0 g / L) were observed with different taurine feeding regimens. The titer values compared in this experiment were not statistically different (p > 0.1) (see Table 7).

[0161] Table 7: Average 14-day antibody titers (g / L) of cultures supplemented with 5 mM

[0162]

[0163] The differences in titer values between days 14 were not statistically significant (p > 0.1).

[0164] The taurine feeding regimen did not have any negative effects and did not change the favorable outcome of taurine supplementation on product yield. Therefore, taurine can be supplemented once at day 0, or at subsequent production stage days, or can be supplemented at multiple time intervals during the production stage.

[0165] Example 4: Measuring ammonia by-products in high-throughput shake flask cultures

[0166] After 14-day cultures performed in a manner similar to Example 2, ammonia by-products were measured in cultures of Ab1-producing CHO cells supplemented with taurine. The cells were subjected to various culture conditions similar to those described above, with glucose / nutrient basal media added as needed.

[0167] Table 8: Average 17-day ammonia (mM) and reduction (%)

[0168]

[0169] * As a baseline control for ammonia reduction %; the ammonia in the taurine-supplemented condition was compared to that in the unsupplemented culture.

[0170] ^ The reduction in ammonia concentration was statistically significant (p < 0.1).

[0171] In cells producing Ab1, taurine supplemented in the medium supported healthy continuous cultures, in which the ammonia by-product had decreased by 32%. The decrease in ammonia concentration with taurine supplementation was statistically significant (p < 0.1).

[0172] The present invention may be embodied in other specific embodiments.

Claims

1. A method for culturing CHO cells expressing dupilumab, comprising: (a) providing a serum-free basal cell culture medium supplemented with 0.1 mM to 10 mM L-taurine, wherein the basal cell culture medium is chemically defined; (b) proliferating or maintaining CHO cells in the basal cell culture medium to form a cell culture; and (c) producing dupilumab from the cell culture; wherein supplementing the basal cell culture medium with L-taurine increases the titer of dupilumab by at least 3% compared to the titer of dupilumab from cells proliferated or maintained in a cell culture medium containing less than 0.1 mM L-taurine.

2. The method according to claim 1, wherein the basal cell culture medium does not contain hydrolysates.

3. The method according to claim 1, wherein in (b), the basal cell culture medium is supplemented with ornithine, putrescine, or a combination thereof.

4. The method according to claim 3, wherein the basal cell culture medium is supplemented with 0.09 mM to 0.9 mM ornithine.

5. The method according to claim 1, wherein the basal cell culture medium contains 0.1 mM to 1 mM L-taurine, 0.5 mM to 1 mM L-taurine, 1 mM to 5 mM L-taurine, 1 mM to 7 mM L-taurine, or 1 mM to 10 mM L-taurine.

6. The method according to claim 1, further comprising a growth phase before step (b), wherein the growth phase comprises proliferating or maintaining CHO cells in a basal cell culture medium not supplemented with L-taurine.

7. The method according to claim 1, further comprising a growth phase before step (b), wherein the growth phase comprises proliferating or maintaining CHO cells in a basal cell culture medium supplemented with 0.1 mM to 10 mM L-taurine.

8. The method according to claim 7, wherein the growth phase comprises proliferating or maintaining CHO cells in a basal cell culture medium containing 0.1 mM to 1 mM L-taurine, 0.5 mM to 1 mM L-taurine, 1 mM to 5 mM L-taurine, 1 mM to 7 mM L-taurine, or 1 mM to 10 mM L-taurine.

9. The method according to claim 1, further comprising supplementing the basal cell culture medium with L-taurine at least once, at least twice, at least 3 times, at least 4 times, or at least 5 times during step (b).

10. The method according to claim 1, further comprising supplementing the basal cell culture medium with L-taurine daily during step (b).

11. The method according to claim 1, wherein the basal cell culture medium contains (a) a mixture of amino acids, (b) one or more fatty acids, (c) vitamins and cofactors, (d) a mixture of nucleosides, or (e) one or more divalent cations.

12. The method according to claim 11, wherein: (a)The mixture of amino acids comprises one or more amino acids selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan; (b)One or more fatty acids are selected from linolenic acid, linoleic acid, lipoic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, capric acid, dodecanoic acid, caproic acid, lignoceric acid, myristic acid, and caprylic acid; (c)The vitamins and cofactors are selected from biotin, calcium D-pantothenate, choline chloride, folic acid, inositol, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and vitamin B12; (d)The mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, and inosine; or (e) One or more divalent cations include Ca 2+ , Mg 2+ or both.

13. The method according to claim 1, wherein the CHO cell is a CHO-K1 cell, a CHO DUX B-11 cell, a CHO DG-44 cell, a Veggie-CHO cell, a GS-CHO cell, an S-CHO cell, or a CHO lec mutant cell.

14. The method according to claim 1, wherein the CHO cell is a CHO-K1 cell.

15. A method for producing dupilumab, comprising the steps of: (a)introducing a nucleic acid comprising a nucleotide sequence encoding dupilumab into a CHO cell; (b)isolating the CHO cell expressing dupilumab; (c)culturing the isolated CHO cell in a serum-free cell culture medium containing 0.1 mM to 10 mM L-taurine to produce a CHO cell population; (d)expressing dupilumab from the CHO cell population, wherein dupilumab is secreted into the culture medium; and (e)harvesting dupilumab; wherein adding 0.1 mM to 10 mM L-taurine to the cell culture medium increases the titer of dupilumab by at least 3% compared to the titer of dupilumab from CHO cells cultured in a cell culture medium containing less than 0.1 mM L-taurine.

16. The method according to claim 15, wherein adding at least 0.7 mM taurine increases the titer of dupilumab by at least 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 20% compared to cells cultured in a cell culture medium containing less than 0.1 mM L-taurine.

17. The method according to claim 15 or 16, wherein the cell population is capable of producing at least 8% more protein of dupilumab compared to cells expressing dupilumab in a cell culture medium containing less than 0.1 mM L-taurine.

18. The method according to claim 15 or 16, wherein culturing a cell population in a cell culture medium having L-taurine can increase the production of dupilumab by at least 0.1 g / 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, at least 2.2 g / L, at least 2.4 g / L, or at least 2.5 g / L compared to culturing the cell population in a similar cell culture medium containing less than 0.1 mM L-taurine.

19. The method according to claim 15 or 16, wherein the cell culture medium contains 0.1 mM to 1 mM L-taurine, 0.5 mM to 1 mM L-taurine, 1 mM to 5 mM L-taurine, 1 mM to 7 mM L-taurine, or 1 mM to 10 mM L-taurine.

20. The method according to claim 15 or 16, wherein the CHO cells are CHO-K1 cells, CHO DUX B-11 cells, CHO DG-44 cells, Veggie-CHO cells, GS-CHO cells, S-CHO cells, or CHO lec mutant cells.

21. The method according to claim 15 or 16, comprising culturing the cell population in a cell culture medium containing 0.1 mM to 10 mM L-taurine for at least 6 days in (c).

22. The method according to claim 15 or 16, wherein the nucleic acid encoding dupilumab is stably integrated in the cells.

23. The method according to claim 15 or 16, comprising the step of adding one or more point-of-use additives to the cell culture medium, wherein the point-of-use additives are NaHCO 3 , glutamine, insulin, glucose, CuSO 4 , ZnSO 4 , FeCl 3 , NiSO 4 , Na 4 EDTA and any one or more of sodium citrate.

24. A method for culturing CHO cells to increase the production of dupilumab, comprising the following steps: (a) proliferating CHO cells in a chemically defined cell culture medium during the growth phase, wherein the chemically defined cell culture medium is serum-free; and (b) supplementing the chemically defined cell culture medium with 0.1 mM to 10 mM L-taurine and expressing dupilumab during the production phase, wherein adding L-taurine increases the titer of dupilumab by at least 7% compared to CHO cells expressing dupilumab in a cell culture medium containing less than 0.1 mM L-taurine, and wherein the proliferating cells are carried out at a temperature of 35°C to 38°C during the growth phase, while the expressing of dupilumab is carried out at a temperature of 29°C to 37°C during the production phase, wherein the temperature of the growth phase is higher than the temperature of the production phase.

25. The method according to claim 24, wherein L-taurine is supplemented 1 to 5 times during the production phase.

26. The method according to claim 24, wherein L-taurine is supplemented daily during the production phase.

27. The method according to claim 24, which further comprises supplementing the chemically defined cell culture medium with 0.1 mM to 10 mM L-taurine during the growth phase.

28. The method according to claim 24, wherein the chemically defined cell culture medium is supplemented with 0.09 mM to 0.9 mM ornithine.

29. A method for producing dupilumab, comprising the following steps: (a) Culturing CHO cells expressing dupilumab in a serum-free cell culture medium containing 0.1 mM to 10 mM L-taurine, wherein adding L-taurine increases the titer of dupilumab by at least 3% compared to cells expressing dupilumab in a cell culture medium containing less than 0.1 mM L-taurine; and (b) Producing dupilumab in the cells, wherein dupilumab is secreted into the culture medium, wherein the cells are cultured at a temperature of 35°C to 38°C during the growth phase and at a temperature of 29°C to 37°C during the production phase, and the temperature during the growth phase is higher than that during the production phase.

30. The method according to claim 29, wherein the cells are capable of increasing the titer of dupilumab by about 7% or more compared to cells expressing dupilumab in a cell culture medium containing less than 0.1 mM L-taurine.

31. The method according to claim 29, further comprising supplementing the cell culture medium with 0.09 mM to 0.9 mM ornithine.

32. A method for producing dupilumab, comprising: Culturing a recombinant CHO cell line in a serum-free cell culture medium containing at least 0.1 mM L-taurine, wherein the recombinant cell line contains a stably integrated nucleic acid encoding dupilumab, and producing dupilumab from the cells, wherein adding L-taurine to the cell culture medium increases the titer of dupilumab by at least 3% to 7% compared to culturing the recombinant cell line in a cell culture medium containing less than 0.1 mM L-taurine, and wherein the recombinant cell line is cultured at a temperature of 35°C to 38°C during the growth phase and at a temperature of 29°C to 37°C during the production phase, and the temperature during the growth phase is higher than that during the production phase.

33. The method according to claim 32, wherein the production method is capable of increasing the titer of dupilumab by at least 0.1 g / 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, at least 2.2 g / L, at least 2.4 g / L or at least 2.5 g / L compared to a similar production method in a cell culture medium containing less than 0.1 mM L-taurine.

34. The method according to claim 32, wherein the production method is capable of increasing the titer of dupilumab by about 7% or more compared to a similar production method in a cell culture medium containing less than 0.1 mM L-taurine.

35. The method according to claim 32, wherein the recombinant cell line is cultured in a cell culture medium supplemented with L-taurine for at least 6 days and expresses a higher titer of dupilumab compared to a recombinant cell line expressing dupilumab cultured in a cell culture medium without L-taurine for at least 6 days.

36. The method according to claim 32, further comprising supplementing the cell culture medium with 0.09 mM to 0.9 mM ornithine.

37. The method according to claim 35, wherein the titer of dupilumab from cells expressing dupilumab cultured in cell culture medium supplemented with L-taurine for at least 6 days is at least 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or at least 20% higher than the titer of dupilumab from cells cultured in cell culture medium for at least 6 days in the absence of L-taurine.

Images