Taurine-supplemented cell culture medium and its use

Abstract

Providing taurine-supplemented cell culture medium and its use. [Solution] This specification describes a composition comprising an improved eukaryotic cell culture medium that can be used for the production of a target protein. The production of the target protein can be increased by adding taurine to serum-free medium or chemically defined medium. This includes a method for recombinant expression of the protein at a high level using this medium composition. Various supply strategies including taurine increase the titer of the produced protein. Furthermore, the addition of taurine does not adversely affect the culture results or the quality of the resulting antibodies.

Description

Technical Field

[0001] (Field) The present invention relates to media and methods for cell culture and for recombinant protein production. Specifically, the present invention relates to a taurine-supplemented medium for culturing recombinant eukaryotic cells for producing protein biotherapeutics and a culturing method thereof.

Background Art

[0002] (Background) The organic acid taurine, often referred to as a β-amino acid, is found at high concentrations in most tissues and is a derivative of the amino acid cysteine (Huxtable, RJ., 1992, Physiol Rev, 72:101-163).

[0003]

Chemical Formula

[0004] Taurine is present in many tissues of humans and other mammalian species (e.g., brain, retina, myocardium, skeletal and smooth muscle, platelets, and neutrophils). Taurine has been recognized to assist in osmotic regulation, membrane stabilization, and anti-inflammation, and also controls mitochondrial protein synthesis by enhancing electron transport chain activity to protect against superoxide generation (Jong et al., 2010, Journal of Biomedical Science 17(Suppl 1):S25; Jong et al., 2012, Amino Acids 42:2223-2232sw). In primary neuronal cell culture, taurine has been characterized as a cytoprotectant due to its inhibitory effect on glutamate-induced toxicity. Various taurine-containing media for embryo culture have been developed.

[0005] Cell culture techniques involving amino acid feeds have long been used in recombinant protein production from cultured cells. Amino acids serve as biosynthetic precursors, energy sources, and osmolites, and their use in production cultures is strongly correlated with sustained cell growth and productivity.

[0006] However, there are countless physiological events that contribute to productivity and high-yield protein expression, and designing a supply strategy is difficult because metabolic activity and transport mechanisms compete. The type and timing of amino acid supplementation can also affect the quality of proteins produced by culture (Altamirano, et al., 2006, Electron. J. Biotechnol. 9:61-67). Byproduct accumulation is often a problem in producing cell cultures, and this accumulation is thought to result from nutrient imbalances in the cell culture, ultimately inhibiting cell growth (Fan, Y. et al., Biotechnol Bioeng. 2015 Mar;112(3):521-35). Hypotaurine or its analogs or precursors have been suggested to achieve the desirable result of reducing the color intensity of compositions containing recombinantly produced polypeptides in cell culture (WO2014145098A1, published September 18, 2014). Cell culture media containing taurine to promote the maturation of immature retinal pigment epithelial cells into mature retinal pigment epithelial cells have also been described (WO2013184809A1, published December 12, 2013). However, the optimization of recombinant protein productivity in taurine supplementation media is not recognized in this field. A cell culture process that increases the productivity of recombinant proteins while minimizing the production of potentially toxic cellular metabolic byproducts such as ammonia is highly desirable. Consistently increased productivity would allow for a significantly larger supply of biotherapeutic products on a commercial scale. Therefore, culture media and methods for culturing mammalian cells are needed in this field, where the media enable the growth and maintenance of healthy and robust cells, as well as the production of high-potency biopharmaceutical substances. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2014 / 145098 [Patent Document 2] International Publication No. 2013 / 184809 [Non-patent literature]

[0008] [Non-Patent Document 1] Altamirano, et al., 2006, Electron. J. Biotechnol. 9:61-67 [Non-Patent Document 2] Fan, Y. et al., Biotechnol Bioeng.2015 Mar;112(3):521-35) [Overview of the Initiative] [Means for solving the problem]

[0009] (Summary) The inventors have made the remarkable discovery that including taurine in cell culture medium increases cell-specific productivity and reduces the production of ammonia by-products by those cells. Various supply strategies including taurine increase the titer of the produced proteins. Furthermore, the addition of taurine does not adversely affect culture results or the quality of the resulting antibodies. The present invention provides a method for producing a therapeutic protein in high yield, comprising the step of culturing a recombinant cell line in a medium containing taurine, wherein the cell line contains nucleic acids stably incorporated into which the therapeutic protein is encoded.

[0010] The present invention relates to a serum-free cell culture medium containing about 0.1 mM to about 10 mM taurine. The present invention relates to a serum-free cell culture medium containing about 0.1 mM to about 1 mM taurine, about 0.2 mM to about 1 mM taurine, about 0.3 mM to about 1 mM taurine, about 0.4 mM to about 1 mM taurine, or about 0.5 mM to about 1 mM taurine. The present invention relates to a serum-free cell culture medium containing about 1 mM to about 10 mM taurine. The present invention relates to a serum-free cell culture medium containing about 1 mM to about 5 mM taurine, about 1 mM to about 6 mM taurine, about 1 mM to about 7 mM taurine, about 1 mM to about 8 mM taurine, or about 1 mM to about 9 mM taurine.

[0011] In some embodiments, the culture 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.

[0012] In some embodiments, the culture medium contains 16 g / L or less of hydrolysate. In some embodiments, the culture medium does not contain any hydrolysate.

[0013] In one embodiment, the culture medium includes a chemically defined (such as a custom formulation) basic medium or a commercially available basic medium. In one embodiment, the complete medium is chemically defined and does not contain serum or hydrolysates.

[0014] In some embodiments, all processes including the basal medium and the feed contain a mixture of amino acids or amino acid salts with a total amount of at least 115 mM. 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.

[0015] In some embodiments, the medium contains one or more fatty acids. In one particular 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 the group consisting of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid.

[0016] In some embodiments, the medium contains a mixture of nucleosides. In one embodiment, the medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0017] In some embodiments, the medium contains a mixture of salts. The salts include divalent cations (such as calcium and magnesium). In one embodiment, the medium contains calcium chloride and magnesium sulfate. Other salts may include phosphates.

[0018] In one embodiment, the medium contains (1) taurine at 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, (2) a hydrolyzate at 16 g / L or less, (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 calcium salts, magnesium salts, and phosphates.

[0019] The present invention provides a method for producing a protein of interest in high yield, comprising culturing a recombinant cell line in a cell culture medium containing at least about 0.1 mM to about 10 mM of taurine, wherein the cell line contains a stably integrated nucleic acid encoding the protein. In other embodiments, the medium embodies any of the aforementioned aspects of the present invention.

[0020] In another aspect, the present invention provides a method for culturing eukaryotic cells for producing an improved recombinant protein, comprising (a) growing or maintaining the cells in a specific cell culture medium during the growth phase, (b) supplementing the basal cell culture medium with about 0.1 mM to about 10 mM of L-taurine during the production phase to express the recombinant protein of interest, and (c) increasing the titer of the protein of interest by the addition of taurine. In some embodiments, the taurine supplementation is performed at least once, 2, 3, 4, or 5 times during the production phase, or daily during the duration of the production phase. In other embodiments, the method further comprises supplementing the culture medium with about 0.1 mM to about 10 mM of L-taurine during the growth phase. In some embodiments, the method improves recombinant protein production compared to eukaryotic cells under the same conditions but lacking taurine supplementation or supplemented with less than 0.1 mM of taurine.

[0021] In another embodiment, the present invention provides a method for culturing cells in a cell culture medium (such as any embodiment of the medium described in the preceding embodiment). In one embodiment, the method uses the step of growing or maintaining cells in a medium that (1) contains taurine at a concentration of at least 0.1 mM ± 0.015 mM, (2) contains or does not contain a hydrolysate of 16 g / L or less, (3) is serum-free, and (4) optionally contains amino acids selected from the group consisting of a mixture of amino acids selected from Table 1.

[0022] In one embodiment, the mixture of optional amino acid supplements is selected from the group of amino acids in Table 1 below: [Table 1]

[0023] In some embodiments, the cells are mammalian cells, bird cells, insect cells, yeast cells, or bacterial cells. In one embodiment, the cells are mammalian cells useful for recombinant protein production (e.g., CHO cells or induced CHO-K1 cells). In some embodiments, the cells express the protein of interest (e.g., a biotherapeutic protein). The biotherapeutic protein may be an antigen-binding protein, which may contain an Fc domain. In some embodiments, the protein of interest is an Fc-fusion protein (e.g., an ScFv molecule) or a trap molecule. Trap molecules include, but are not limited to, VEGFtrap and IL-1Trap proteins. In some embodiments, the protein of interest is an antibody (e.g., a human monoclonal antibody, a humanized monoclonal antibody, a bispecific antibody, or an antibody fragment).

[0024] Considering that the inclusion of taurine in various forms of serum-free media has a positive effect on protein production, cells cultured according to this method exhibit increased average protein titer. In one embodiment, compared to the protein titer of a medium without taurine supplementation, cells grown in the taurine-supplemented medium of this method produce proteins with at least 8% higher protein titer than a control culture (i.e., a culture without taurine supplementation). In one embodiment, cells grown in taurine-supplemented culture yield protein titers that are 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 those of control cultures grown in cultures without taurine supplementation.

[0025] Similarly, by including only taurine in serum-free medium, cultured cells can reduce ammonia byproducts compared to cultured cells without taurine. In a serum-free and hydrolysate-free embodiment of one taurine supplement medium, cell culture can reduce ammonia byproduct levels (mM NH3) by at least 4% and up to 32% compared to similar cell cultures in similar cell culture media without supplementation (i.e., taurine supplementation of less than 0.1 mM or no taurine supplementation).

[0026] In another embodiment, the method includes the step of adding one or more point-of-use additives to the cell culture medium. In some embodiments, the point-of-use additives are any one or more of NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, 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: NaHCO3, glutamine, insulin, glucose, CuSO4, ZnSO4, FeCl3, NiSO4, Na4EDTA, and sodium citrate. In some embodiments, the point-of-use additives can be included in the medium at the start of culture.

[0027] In one particular embodiment, the embodiment provides a method for culturing cells in a serum-free medium comprising (1) essentially taurine at a concentration of at least 0.1 mM; (2) a hydrolysate of 16 g / L or less; (3) serum-free; and (4) optionally further comprising a mixture of 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 in a total amount of 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.

[0028] In another embodiment, the present invention provides a method for producing a target protein by using the steps of (1) introducing a nucleic acid sequence encoding a target protein into cells; (2) selecting cells that express the target protein; (3) culturing the selected cells in one embodiment of the serum-free cell culture medium described in any of the preceding embodiments, or according to any embodiment of the method described herein; and (4) expressing the target protein in the cells, wherein the target protein is secreted into the culture medium. In some embodiments, the cells used for protein production are mammalian cells (such as CHO cells, 293 cells, and BHK cells) or any derivatives thereof that are capable of producing biopharmaceuticals. In one embodiment, the cells are CHO cells (such as CHO-K1 cells).

[0029] In some embodiments, the protein of interest is an antigen-binding protein. In some embodiments, the protein of interest is a protein having an Fc domain. In some cases, two proteins of interest may overlap (e.g., receptor-Fc fusion proteins, antibodies, and ScFv proteins). Thus, in some embodiments, the protein of interest is an antibody (human antibody or humanized antibody, antibody fragment (Fab or F(ab')2, etc.), bispecific antibody, trap molecule (VEGF-Trap or IL-1-Trap, etc.), ScFv molecule, or soluble TCR-Fc fusion protein, etc.).

[0030] In one embodiment, the target protein can be produced at an average titer on days 14, 15, 16, or 17 that is at least 8% higher than the average titer on days 14, 15, 16, or 17 produced by similar cells in serum-free cell culture media containing less than 0.1 mM taurine or without taurine supplementation. In one embodiment, it is possible to produce the protein of interest at an average titer on days 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 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 titer on days 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 produced by similar cells in serum-free cell culture media containing less than 0.1 mM taurine or without taurine supplementation.

[0031] In another embodiment, the target protein is produced by (1) introducing a nucleic acid sequence encoding the target protein (such as an antibody or other antigen-binding protein) into CHO cells; (2) selecting cells that stably express the target protein; and (3) culturing the selected cells in serum-free cell culture medium containing approximately 0.1 mM to approximately 10 mM taurine. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for culturing recombinant eukaryotic cells to produce an improved recombinant protein, comprising: (a) growing or maintaining the cells in a specified cell culture medium during the growth phase; (b) supplementing the basic cell culture medium with about 0.1 mM to about 10 mM L-taurine to express the recombinant protein of interest during the production phase; and (c) increasing the titer of the protein of interest by adding taurine. (Item 2) The method according to item 1, wherein the taurine supplementation described in (b) above is performed at least once during the production period. (Item 3) The method according to item 1, wherein the taurine supplementation described in (b) above is performed at least twice during the production period. (Item 4) The method according to item 1, wherein the taurine supplementation described in (b) above is performed at least three times during the production period. (Item 5) The method according to item 1, wherein the taurine supplementation described in (b) above is performed at least four times during the production period. (Item 6) The method according to item 1, wherein the taurine supplementation described in (b) above is performed at least five times during the production period. (Item 7) The method according to item 1, wherein the taurine supplementation described in (b) above is performed daily during the duration of the production phase. (Item 8) The method according to any one of items 1 to 7, further comprising the step of supplementing the culture in the aforementioned specified cell culture medium with approximately 0.1 mM to approximately 10 mM L-taurine during the growth phase. (Item 9) The method according to any one of items 1 to 8, wherein the eukaryotic cells are selected from the group consisting of mammalian cells, bird cells, insect cells, and yeast cells. (Item 10) The method according to item 9, wherein the cells are selected from the group consisting of CHO (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC5, Colo25, HB8065, HL-60, lymphocytes (e.g., Jurkat, Daudi), A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, PER.C6® cells, stem cells, tumor cells, and cell lines derived from the cells. (Item 11) The method according to item 10, wherein the cells are CHO cells. (Item 12) The method according to any one of items 1 to 11, wherein the target protein is an antigen-binding protein. (Item 13) The method according to any one of items 1 to 12, wherein the target protein comprises an Fc domain. (Item 14) The method according to item 12 or 13, wherein the target protein is selected from the group consisting of Fc-fusion proteins, receptor-Fc-fusion proteins (TRAPs), antibodies, antibody fragments, and ScFv-Fc fusion proteins. (Item 15) The target protein is anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody, anti- IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 15. The method of item 14, wherein the method is selected from the group consisting of antibodies, anti-CD3 / anti-CD20 bispecific antibodies, anti-CD3 / anti-MUC16 bispecific antibodies, and anti-CD3 / anti-PSMA bispecific antibodies. (Item 16) The method according to item 14, wherein the target protein is selected from the group consisting of alirocumab, sarilumab, facinumab, nesbakumab, dupilumab, trevoglumab, evinacumab, and linucumab. (Item 17) A method for producing a target recombinant protein, A method comprising: (a) introducing a nucleic acid containing a nucleotide sequence encoding a target protein into cells; (b) selecting cells that express the target protein; (c) culturing the selected cells in a cell culture medium containing about 0.1 mM to about 10 mM L-taurine; and (d) causing the cells to produce the target protein, wherein the target protein is secreted into the culture medium. (Item 18) The method according to item 17, wherein the titer of the target protein is increased by the addition of taurine to the culture medium. (Item 19) The method according to item 17 or 18, wherein the cells of step (c) are capable of producing the target protein in high yield. (Item 20) The method according to any one of items 17 to 19, wherein the cells are capable of producing a protein yield at least 3% higher than cells expressing the target protein in a cell culture medium containing less than 0.1 mM L-taurine. (Item 21) The method according to any one of items 17 to 20, wherein the cells are capable of producing a protein yield at least 8% higher than cells expressing the target protein in a cell culture medium containing less than 0.1 mM L-taurine. (Item 22) The method according to any one of items 17-21, wherein the cells are CHO cells, HEK293 cells, or BHK cells. (Item 23) The method according to any one of items 17 to 22, wherein the target protein is an antigen-binding protein. (Item 24) The method according to any one of items 17 to 22, wherein the target protein comprises an Fc domain. (Item 25) The method according to any one of items 17 to 24, wherein the target protein is selected from the group consisting of Fc-fusion proteins, receptor-Fc-fusion proteins (TRAPs), antibodies, and antibody fragments. (Item 26) The target protein is anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody, anti- IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 26. The method of item 25, wherein the method is selected from the group consisting of antibodies, anti-CD3 / anti-CD20 bispecific antibodies, anti-CD3 / anti-MUC16 bispecific antibodies, and anti-CD3 / anti-PSMA bispecific antibodies. (Item 27) The method according to item 25, wherein the target protein is selected from the group consisting of alirocumab, sarilumab, facinumab, nesbakumab, dupilumab, trevoglumab, evinacumab, and linucumab. (Item 28) A method for producing a target protein in taurine supplement culture medium, (a) The process of introducing nucleic acids containing a sequence encoding the target protein into cells; (b) A step of selecting cells that express the target protein; (c) A step of culturing the selected cells in a cell culture medium; (d) A step of supplementing the cell culture medium with taurine in an amount of approximately 0.1 mM to approximately 10 mM; (e) a step of maintaining the cell culture for at least 6 days under the taurine supplementation conditions of step (d), wherein the target protein is expressed in the cells at a higher titer compared to cells cultured in non-taurine supplementation culture, and the target protein is secreted into the culture medium; and (f) A method comprising the step of recovering the target protein. (Item 29) The method described in item 28, wherein the titer of the target protein is increased by taurine supplementation. (Item 30) The method according to item 29, wherein the protein titer of the recovered protein is at least 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or at least 20% higher than the titer of the control culture, and the control culture medium was not subjected to the taurine supplementation conditions of step (d). (Item 31) A method for producing a target recombinant protein in high yield, comprising the step of culturing a recombinant cell line in a cell culture medium containing at least about 0.1 mM L-taurine, wherein the cell line contains a stably incorporated nucleic acid encoding the recombinant protein. (Item 32) The method according to item 31, wherein the yield of the recombinant protein of the objective is increased by including taurine in the cell culture medium compared to a cell line cultured in a cell culture medium that does not contain L-taurine. (Item 33) The method according to item 31 or 32, wherein the target protein is an antigen-binding protein. (Item 34) The method according to any one of items 31 to 33, wherein the target protein contains an Fc domain. (Item 35) The method according to any one of items 31 to 34, wherein the target protein is selected from the group consisting of Fc-fusion proteins, receptor-Fc-fusion proteins (TRAPs), antibodies, and antibody fragments. (Item 36) The target protein is anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody, anti- IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 36. The method of item 35, wherein the method is selected from the group consisting of antibodies, anti-CD3 / anti-CD20 bispecific antibodies, anti-CD3 / anti-MUC16 bispecific antibodies, and anti-CD3 / anti-PSMA bispecific antibodies. (Item 37) The method according to item 35, wherein the target protein is selected from the group consisting of alirocumab, sarilumab, facinumab, nesbakumab, dupilumab, trevoglumab, evinacumab, and linucumab. (Item 38) The method according to any one of items 31 to 37, wherein the production method can increase the protein yield 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 taurine. (Item 39) The method according to any one of items 31 to 38, wherein the production method can increase the protein yield by at least 3% compared to a similar production method in a cell culture medium containing less than 0.1 mM taurine. (Item 40) The method according to any one of items 31 to 39, wherein the production method can reduce ammonia accumulation by at least 8% compared to a similar production method in a cell culture medium containing less than 0.1 mM taurine. (Item 41) The method according to any one of items 31 to 40, further comprising the step of adding one or more use-point additives to the cell culture medium. (Item 42) CHO cell culture medium for high-yield recombinant production of antibodies containing approximately 0.1 mM to 10 mM L-taurine. (Item 43) CHO cell culture medium as described in item 42, further containing ornithine. (Item 44) CHO cell culture medium as described in item 43, further containing putrescine. (Item 45) CHO cell culture medium according to any one of items 42-44, further comprising a mixture of 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. (Item 46) The CHO cell culture medium described in any one of items 42 to 45, wherein the culture medium is serum-free. (Item 47) The CHO cell culture medium described in any one of items 42 to 46, wherein the culture medium does not contain hydrolysates. (Item 48) The CHO cell culture medium described in any one of items 42 to 47, wherein the culture medium is chemically defined. (Item 49) The CHO cell culture medium according to any one of items 42 to 48, wherein the medium is provided to the production batch culture on day 0. (Item 50) CHO cell culture medium as described in any one of items 42-49, for supplying the feed to the production batch cultures on day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, and / or day 10. (Item 51) The CHO cell culture feed described in item 50, wherein the culture medium and feed provided to the aforementioned production batch cultures contain approximately 5 mM to approximately 10 mM total L-taurine during the production phase. (Item 52) A CHO cell culture medium according to any one of items 42 to 51, further comprising one or more fatty acids. (Item 53) The CHO cell culture medium according to item 52, wherein the one or more fatty acids are selected from the group consisting of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. (Item 54) CHO cell culture medium as described in any one of items 42 to 53, further comprising vitamins and cofactors selected from the group consisting of biotin, calcium D-pantothenate, choline chloride, folic acid, I-inositol, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and vitamin B12. (Item 55) A CHO cell culture medium according to any one of items 42 to 54, further comprising a mixture of nucleosides. (Item 56) The CHO cell culture medium according to item 55, wherein the nucleoside mixture comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine. (Item 57) A CHO cell culture medium according to any one of items 42 to 56, further comprising adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine. (Item 58) A CHO cell culture medium according to any one of items 42 to 57, further comprising one or more divalent cations. (Item 59) The CHO cell culture medium according to item 58, wherein the divalent cation is magnesium, calcium, or both. (Item 60) CHO cell culture medium as described in item 59, containing Ca2+ and Mg2+. (Item 61) A fed-batch culture medium and / or feed for protein production in CHO cell cultures, containing taurine in an amount of approximately 0.1 mM to 10 mM. (Item 62) A fed-batch culture medium and / or feed as described in item 61, further comprising ornithine. (Item 63) A fed-batch culture medium and / or feed as described in item 62, containing putrescine. (Item 64) A fed-batch culture medium and / or feed as described in any one of items 61 to 63, further comprising a mixture of 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. (Item 65) A method for culturing eukaryotic cells for the production of a target recombinant protein, comprising: (a) providing a cell culture medium as described in any one of items 42 to 64; and (b) growing or maintaining cells in the cell culture medium to form a cell culture, wherein the cells express the target protein. (Item 66) The method according to item 65, wherein the eukaryotic cells are selected from the group consisting of mammalian cells, bird cells, insect cells, and yeast cells. (Item 67) The method according to item 65 or 66, wherein the cells are CHO cells. (Item 68) The method according to any one of items 65 to 67, wherein the target protein is secreted into the culture medium by the cells. (Item 69) The method according to item 68, wherein the target protein is an antigen-binding protein. (Item 70) The method according to item 68 or 69, wherein the target protein comprises an Fc domain. (Item 71) The method according to any one of items 68 to 70, wherein the target protein is selected from the group consisting of Fc-fusion proteins, receptor-Fc-fusion proteins (TRAPs), antibodies, and antibody fragments. (Item 72) The target protein is anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody, anti- IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 72. The method of item 71, wherein the method is selected from the group consisting of antibodies, anti-CD3 / anti-CD20 bispecific antibodies, anti-CD3 / anti-MUC16 bispecific antibodies, and anti-CD3 / anti-PSMA bispecific antibodies. (Item 73) The method according to item 71, wherein the target protein is selected from the group consisting of alirocumab, sarilumab, facinumab, nesbakumab, dupilumab, trevoglumab, evinacumab, and linucumab. (Item 74) The method according to any one of items 65 to 73, wherein the cell culture is capable of reducing ammonia accumulation by at least 3% compared to a similar cell culture in a medium containing less than 0.1 mM taurine. (Item 75) The method according to any one of items 65 to 74, wherein the cell culture is capable of reducing ammonia accumulation by at least 8% compared to a similar cell culture in a medium containing less than 0.1 mM taurine. [Brief explanation of the drawing]

[0032] [Figure 1]Figure 1 shows the protein titer (yield) from samples collected on each production culture day in Ab3-producing cell cultures, comparing taurine-supplemented cultures (black squares connected by solid lines) with non-taurine-supplemented cultures (x connected by dotted lines). The advantage of taurine-supplemented cultures in terms of protein yield is observed as early as day 6 of production culture. [Modes for carrying out the invention]

[0033] (Detailed explanation) It should be understood that the present invention is not limited to the specific methods and experimental conditions described, for such methods and conditions may vary. Similarly, it should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit them, for the scope of the present invention is defined by the claims.

[0034] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include references to the plural unless the context otherwise specifies otherwise. Thus, for example, a reference to “a method” includes one or more methods and / or processes of the type described herein and / or that would become apparent to those skilled in the art by reading this disclosure.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of this invention, specific methods and materials are described herein. All publications referenced herein are incorporated herein by reference in their entirety.

[0036] The applicant has made the remarkable discovery that the addition of taurine to cell culture media improves protein production by recombinant cells in cell culture compared to cell culture media containing very little taurine or no taurine at all.

[0037] Before describing the cell cultures and methods described herein, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and therefore the methods and conditions may vary. It should also be understood that the terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. Unless otherwise indicated, the methods and techniques described herein shall generally follow conventional methods known in the art and as described in various general literature and more specific literature cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (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, NY (1990), and Julio E. Celis, Cell Biology: A Laboratory Handbook, 2 ndSee ed., Academic Press, New York, NY (1998), and Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1995). All publications referenced throughout this disclosure are incorporated herein by reference in their entirety.

[0039] (definition) "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. Although taurine is an organic acid containing an amino group, it is not considered an "amino acid" as is traditionally known to those skilled in the art, since an amino acid contains both an amino group and a carboxyl group. Taurine is biosynthesized when hypotaurine, a derivative of cysteine, is converted to taurine by oxidation.

[0040] The terms "supplementation," "supplementing," and "supplemented" refer to the addition of components, components, molecules, etc., to a cell culture medium that can be used to maintain and / or promote cell growth and / or differentiation, to enhance or improve the overall characteristics of a culture or cells, or to compensate for deficiencies. To achieve this purpose, taurine supplementation involves adding a specific concentration of taurine solution to the culture medium.

[0041] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably throughout this specification, and these terms refer to molecules containing two or more amino acid residues interconnected by peptide bonds. Peptides, polypeptides, and proteins may also include modifications such as glycosylation, lipid attachment, sulfated material, γ-carboxylation, alkylation, hydroxylation, and ADP-ribosylation of glutamate residues. Peptides, polypeptides, and proteins may be scientifically or commercially useful (including protein-based drugs). Peptides, polypeptides, and proteins include, in particular, antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins are produced by recombinant animal cell lines using cell culture methods.

[0042] The term "heterogeneous polynucleotide sequence," as used herein, refers to a nucleic acid polymer encoding a target protein, such as a chimeric protein (e.g., a trap molecule), an antibody, or a part of an antibody (e.g., VH, VL, CDR3), produced as a biopharmaceutical drug substance. Heterogeneous polynucleotide sequences can be manufactured by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, intronless sequences, etc.) and introduced into cells, where they can exist as episomes or be incorporated into the cellular genome. Heterogeneous polynucleotide sequences may be naturally occurring sequences introduced ectopically within the producing cellular genome. Heterogeneous polypeptide sequences may be naturally occurring sequences derived from another organism (e.g., sequences encoding human orthologs).

[0043] An "antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region contains one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term “antibody” refers to both glycosylated and nonglycosylated immunoglobulins of any isotype or subclass. The term “antibody” includes antibody molecules prepared, expressed, produced, or isolated by recombinant means (such as antibodies isolated from host cells transfected to express antibodies). The term “antibody” also includes bispecific antibodies, which include heterotetrameric immunoglobulins capable of binding to more than one different epitopes. Bispecific antibodies are generally described in U.S. Patent Application Publication 2010 / 0331527 (incorporated herein by reference).

[0044] The term "antigen-binding portion" (or "antibody fragment") of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. Examples of binding fragments included in the term "antigen-binding portion" of an antibody include: (i) Fab fragments (monovalent fragments consisting of VL, VH, CL, and CH1 domains); (ii) F(ab')2 fragments (bivalent fragments consisting of two Fab fragments linked by disulfide crosslinking at the hinge region); (iii) Fd fragments consisting of a VH domain and a CH1 domain; (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); (vi) isolated CDRs; and (vii) scFv, which consists of two domains (VL and VH) of an Fv fragment, where these fragments are linked by a synthetic linker to form a single protein chain, in which the VL and VH regions pair to form a monovalent molecule. Other forms of single-chain antibodies (such as diabodies) are also included in the term "antibody" (see, for example, Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0045] Furthermore, an antibody or its antigen-binding portion may be part of a larger immunoadhesion molecule formed by the covalent or non-covalent association of the antibody or a portion of the antibody with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to construct tetrameric scFv molecules (Kipriyanov et al. (1995) Human antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to construct divalent biotinylated scFv molecules (Kipriyanov et al. (1994) Mol.Immunol.31:1047-1058). Parts of an antibody (such as Fab fragments and F(ab')2 fragments) can be prepared from the whole antibody using conventional techniques (such as papain digestion or pepsin digestion of the whole antibody). Furthermore, antibodies, parts of antibodies, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques commonly known in the field (see Sambrook et al., 1989).

[0046] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of this invention may, for example, contain amino acid residues not encoded by human germline immunoglobulin sequences in the CDR, particularly CDR3 (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence.

[0047] The term "recombinant human antibody" is intended to encompass, in this specification, any human antibody prepared, expressed, produced, or isolated by recombinant means, including antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice) that are transgenic of human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. Vol. 20: pp. 6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means involved in the splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, the recombinant human antibody is subjected to in vitro mutagenesis (or, if an animal transgenic of the human Ig sequence is used, in vivo somatic mutagenesis), so that the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to the human germline VH and VL sequences, but do not need to be naturally present in the human antibody germline repertoire in vivo.

[0048] An "Fc fusion protein" comprises part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, and these proteins are not found together in nature. The preparation of fusion proteins containing certain heterologous polypeptides fused to various portions (including the Fc domain) of an antibody-inducing polypeptide is described, for example, Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins," in Current Protocols in Immunology, Suppl. 4, pages 10.19.1 - 10.19.11, 1992. A "receptor Fc fusion protein" in some embodiments comprises one or more extracellular domains of receptors coupled to the Fc portion, which includes the hinge region of the immunoglobulin and subsequent CH2 and CH3 domains. In some embodiments, the Fc-fusion protein comprises two or more separate receptor chains that bind to one or more ligands. For example, the Fc-fusion protein is a trap such as an IL-1 trap (e.g., lilonacept, which contains an IL-1RAcP ligand-binding domain fused to an IL-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004) or a VEGF trap (e.g., aflibercept, which contains an Ig domain 2 of VEGF receptor Flt1 fused to an Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patents No. 7,087,411 and 7,279,159).

[0049] (cell culture) The terms "cell culture medium" and "culture medium" typically refer to a nutrient solution used for the growth of mammalian cells that provides the nutrients necessary to enhance cell growth (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.). Cell culture mediums may contain extracts (e.g., serum or peptone (hydrolyzed)) that supply the raw materials supporting cell proliferation. The medium may contain yeast extracts or soybean extracts instead of animal extracts. A chemically defined medium is a cell culture medium in which all chemical components are known (i.e., have known chemical structures). A chemically defined medium is completely free of animal components (e.g., serum or animal-derived peptone). In one embodiment, the culture medium is a chemically defined culture medium.

[0050] This solution may also contain components (including hormones and growth factors) that enhance growth and / or survival in proportions greater than the minimum required. The solution is preferably formulated so that the pH and salt concentration are optimal for cell survival and proliferation.

[0051] A "cell line" refers to cells induced from a specific lineage through continuous cell passage or subculturing. The term "cell" is used interchangeably with "cell population."

[0052] The term “cell” includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include eukaryotic cells (e.g., non-human animal cells, mammalian cells, human cells, bird cells, insect cells, yeast cells, or cell fusions (e.g., hybridomas or quadromas)). In certain embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In other embodiments, the cells are selected from the following cells: CHO cells (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS cells (e.g., COS-7), retinal cells, Vero cells, CV1 cells, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa cells, HepG2 cells, WI38 cells, MRC 5 cells, Colo25 cells, HB 8065 cells, HL-60 cells, lymphocytes (e.g., Jurkat cells (T lymphocytes) or Daudi cells (B lymphocytes)), A431 cells (epidermal), CV-1 cells, U937 cells, 3T3 cells, L cells, C127 cells, SP2 / 0 cells, NS-0 cells, MMT cells, stem cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cells contain one or more viral genes (e.g., retinal cells expressing viral genes (e.g., PER.C6® cells)). In some embodiments, the cells are CHO cells. In other embodiments, the cells are CHO K1 cells.

[0053] One aspect of the present invention relates to a seed culture that expands the cell population before protein production and harvesting in a production culture. Taurine can be added to the basic medium in the seed culture formulation according to the invention described herein.

[0054] Another aspect of the present invention relates to production cultures for producing and harvesting proteins. Prior to the production phase, there is typically a growth phase (also known as a seed train or seed culture), during which all components for cell culture are supplied to the culture vessel at the start of the culture process, and the cell population is then expanded to production scale. Thus, cells are inoculated into the culture vessel at an appropriate seeding density during the initial cell growth phase, depending on the cell line to be started. In some aspects, taurine may be added to the basal culture medium in the seed culture formulation according to the inventions described herein in order to further improve or enhance the productivity of cells in the subsequent production phase.

[0055] One aspect of the present invention relates in particular to a production culture in which cell culture conditions are modified to improve the production of one or more recombinant proteins of interest by recombinant eukaryotic cells and to enhance the growth of recombinant eukaryotic cells while maintaining cell viability, by adding taurine to the production culture medium and / or seed train culture. In a production culture vessel or bioreactor, basal culture medium and cells are supplied to the culture vessel after seed culture or growth phase. In certain embodiments, the cell supernatant or cell lysate is recovered after production culture. In other embodiments, the polypeptide or protein of interest is recovered from the culture medium or cell lysate (which may depend in any case on the location of the protein of interest) using techniques known in the said part.

[0056] Examples of culture vessels include, but are not limited to, well plates, T-flasks, shaking flasks, stirring vessels, spinner flasks, hollow fiber containers, and air-lift bioreactors. A suitable cell culture vessel is a bioreactor. A bioreactor is any culture vessel manufactured or operated to handle or control environmental conditions. Such culture vessels are well known in the field.

[0057] Bioreactor processes and systems have been developed to optimize gas exchange, supply sufficient oxygen to sustain cell growth and productivity, and remove CO2. Maintaining gas exchange efficiency is a critical criterion for ensuring the successful scaling up of cell culture and protein production. Suitable systems are well known to those skilled in the art.

[0058] In the polypeptide production phase, “fed-batch cell culture” or “fed-batch culture” refers to a batch culture in which animal cells and culture medium are initially supplied to the culture vessel, and further culture nutrients are slowly supplied to the culture continuously or discontinuously in increments during the culture (with or without periodic cell and / or product collection before the end of the culture). Fed-batch culture includes “semi-continuous fed-batch culture” in which the entire culture (which may include cells and medium) is periodically removed and replaced with fresh medium. Fed-batch culture is distinguished from simple “batch culture” in that all components for cell culture (including animal cells and all culture nutrients) are supplied to the culture vessel at the start of the culture process in batch culture. Fed-batch culture can be further distinguished from perfusion culture in that the supernatant is not removed from the culture vessel during the process described above, whereas in perfusion culture, the cells are retained in the culture, for example by filtration, and the culture medium is introduced continuously or intermittently and removed from the culture vessel. However, it is intended that samples be taken for testing purposes during fed-batch cell culture. The above feed-in process continues until it is determined that the maximum working volume and / or protein production has been achieved.

[0059] The term "continuous cell culture," as used herein, refers to a technique used to continuously grow cells, typically within a specific growth phase. For example, if a constant supply of cells is required, or if the production of a polypeptide or protein of a particular purpose is required, such cell culture may require maintenance within a specific growth phase. Accordingly, the conditions must be continuously monitored and adjusted accordingly to maintain the cells within that particular phase. ( Culture medium ) This invention provides cell culture media containing approximately 0.1 mM to 10 mM taurine in serum-free form. “Serum-free” applies to cell culture media that do not contain animal serum (such as fetal bovine serum). Serum-free media may contain 16 g / L or less of hydrolysates (such as soybean hydrolysate). This invention also provides chemically defined media that are not only serum-free but also hydrolysate-free. “Hydrolysate-free” applies to cell culture media that do not contain exogenous protein hydrolysates such as animal or plant protein hydrolysates (e.g., peptone and tryptone). “Basic medium” is the initial medium (present during seed training and / or on day 0 of cell culture production) that promotes cell growth and contains all necessary nutrients (including a basic mixture of amino acids). Various recipes (i.e., formulations) for basic medium can be manufactured or purchased in commercially available lots. Similarly, “basic feed medium” contains a mixture of supplemental nutrients commonly consumed during production culture and utilized in feeding strategies (so-called “fed-batch” culture). A variety of basic feed media are commercially available. "Feed" includes planned additions or additions to the culture medium at regular intervals according to protocols (including continuous feed culture systems such as chemostats (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec; 17(6): 1032-41)) or fed-batch processes (YMHuang et al., Biotechnol Prog. 2010 Sep-Oct; 26(5): 1400-10). For example, the culture may be supplied once a day, once every other day, once every three days, or when the concentration of a specific culture medium component being monitored falls outside the desired range.

[0060] Removing serum from cell culture media and reducing or eliminating hydrolysates while reducing lot-to-lot variability and strengthening downstream processing steps unfortunately leads to decreased cell growth, viability, and protein expression. Therefore, chemically defined serum-free and hydrolysate-free or hydrolysate-free media require additional components to improve cell growth and protein production.

[0061] Therefore, the cell culture medium of the present invention comprises a basic medium containing all the nutrients necessary for live cell culture. Taurine can be added to the basic medium in the seed culture formulation according to the invention described herein. Furthermore, taurine can be added to the basic medium in the production culture formulation, and then, depending on the requirements of the cells to be cultured or the desired cell culture parameters, further components such as polyamines or increased concentrations of amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, and trace elements may or may not be supplied periodically (e.g., in so-called "fed-batch" culture).

[0062] In the present invention, taurine-supplemented cell culture medium may be depleted of amino acids through protein production culture (when no further amino acid supplementation is performed), or it may be "non-supplemented" (when depleted amino acids (listed below) are supplemented). The inventors have observed that recombinant protein production under the various culture conditions described above is improved by cultures supplemented with taurine during the production phase.

[0063] The present invention provides a taurine supplement medium containing 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 / liter).

[0064] In one embodiment, the culture medium further comprises 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 culture medium comprises 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.

[0065] Putrescine can be optionally added to supplemental media. Putrescine is included in very low concentrations as a component in several cell culture medium formulations; for example, WO2005 / 028626 (listed as 0.02-0.08 mg / L putrescine); U.S. Patent No. 5,426,699 (0.08 mg / L); U.S. Patent RE30,985 (0.16 mg / L); U.S. Patent No. 5,811,299 (0.27 mg / L); U.S. Patent No. 5,12 See Patent No. 2,469 (0.5635 mg / L); U.S. Patent No. 5,063,157 (1 mg / L); WO2008 / 154014 (approx. 100 μM to approx. 1000 μM); U.S. Patent Application Publication No. 2007 / 0212770 (0.5 to 30 mg / L polyamine; 2 mg / L putrescine; 2 mg / L putrescine + 2 mg / L ornithine; 2 mg / L putrescine + 10 mg / L ornithine).

[0066] In some embodiments, the culture medium is further supplemented with a combination of ornithine and putrescine, where the putrescine concentration may be at least about 150–720 μM. In some embodiments, the culture medium is further supplemented with about 170–230 μM of putrescine. In one embodiment, the culture medium contains 200 μM ± 30 μM of putrescine in addition to 90 μM ± 15 μM or more of ornithine. In one embodiment, the culture medium contains 30 mg / L ± 4.5 mg / L or less of putrescine-2HCl in addition to 15 mg / L ± 2.25 mg / L or less of ornithine-2HCl (see International Publication No. WO2014 / 144198A1, published September 18, 2014, which is incorporated herein by reference in its entirety).

[0067] In further embodiments, ornithine is present in the culture medium at concentrations ranging from 0.09±0.014 mM to 0.9±0.14 mM (e.g., 0.09±0.014 mM, 0.3±0.05 mM, 0.6±0.09 mM, or 0.9±0.14 mM ornithine). In some embodiments, the culture medium also contains at least 0.20±0.03 mM putrescine. In some embodiments, further putrescine concentrations range from 0.20±0.03 mM to 0.714±0.11 mM (e.g., 0.20±0.03 mM, 0.35±0.06 mM, or 0.714±0.11 mM putrescine).

[0068] Various other supplements can be added to the culture medium, and further additions to determine appropriate conditions are within the scope of the art of the art. In some embodiments, if the medium is not depleted or supplemental nutrients are required, 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 is added to the medium.

[0069] In one embodiment, the culture medium is further supplemented with approximately 170 μM to 175 μM of nucleosides. In one embodiment, the culture medium contains purine derivatives with a cumulative concentration of at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, at least 65 μM, at least 70 μM, at least 75 μM, at least 80 μM, at least 85 μM, at least 90 μM, at least 95 μM, at least 100 μM, or at least 105 μM. In one embodiment, the culture medium contains approximately 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 a pyrimidine derivative with a cumulative concentration of at least 30 μM, at least 35 μM, at least 40 μM, at least 45 μM, at least 50 μM, at least 55 μM, at least 60 μM, or at least 65 μM. In one embodiment, the culture medium contains about 65 μM to 75 μM of pyrimidine derivatives. Pyrimidine derivatives include thymidine, uridine, and cytidine, which are nucleosides. In one particular embodiment, the culture medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0070] In addition to the inclusion of 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, the fatty acids include one or more of the following: linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid. In one embodiment, the culture medium includes tocopherols, linoleic acid, and thioctic acid.

[0071] In one embodiment, the culture medium may be further supplemented with a mixture of vitamins (including other nutrients and essential nutrients) at a cumulative concentration of at least about 700 μM or at least about 2 mM. In one embodiment, the mixture of vitamins includes one or more D-biotins, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine hydrochloride, D-pantothenic acid (hemiCa), riboflavin, thiamine hydrochloride, and vitamin B12. In one embodiment, the mixture of vitamins includes all of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, pyridoxine hydrochloride, D-pantothenic acid (hemiCa), riboflavin, thiamine hydrochloride, and vitamin B12.

[0072] Various embodiments of the culture medium of the present invention include any combination of the above embodiments (including serum-free media that do not contain chemically defined hydrolysates, particularly (a) amino acids; (b) optionally nucleosides; (c) divalent cation salts; (d) fatty acids and tocopherols; and (e) vitamins, in the indicated amounts of taurine, as indicated in the amounts of taurine). In some embodiments, small amounts of total hydrolysates can be added to the taurine supplement medium.

[0073] The applicant envisions that taurine may be used in any one or more or a combination of various basic media when carrying out the present invention. These basic media are commonly known in the art, and include, in particular, Eagle MEME (Minimal Essential Medium) (Eagle, Science, 1955, 112(3168):501-504), Ham F12 (Ham, Proc. Nat'l. Acad. Sci. USA, 1965, 53:288-293), F-12 K medium, Dulbecco's medium, Dulbecco's modified Eagle medium (Proc. Nat'l. Acad. Sci. USA, 1952 August; 38(8):747-752), DMEM / Ham F12 1:1, Trowell T8, and A2 medium (Holmes). (and Wolf, Biophys. Biochem. Cytol., 1961, 10:389-401), Weymouth 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, Iskov modified Dulbecco medium, McCoy 5A medium, Leibovitz L-15 medium, and serum-free medium (EX-CELL(trademark) 300 series (JRH) Biosciences, Lenexa, Kansas, etc.), protamine-zinc-insulin medium (Weiss et al., 1974, U.S. Patent No. 4,072,565), biotin-folate medium (Cartaya, 1978, U.S. Reissue Patent No. 30,985), transferrin-fatty acid medium (Baker, 1982, U.S. Patent No. 4,560,655), transferrin-EGF medium (Hasegawa, 1982, U.S. Patent No. 4,615,977; Chessebeuf, 1984, U.S. Patent No. 4,786,599), and permutations of other media (Inlow, U.S. This includes patents such as 6,048,728; Drapeau, 7,294,484; Mather, 5,122,469; Furukawa, 5,976,833; Chen, 6,180,401; Chen, 5,856,179; Etcheverry, 5,705,364; Etcheverry, 7,666,416; Ryll, 6,528,286; Singh, 6,924,124; and Luan, 7,429,491, etc.

[0074] In certain embodiments, the culture medium is chemically defined and, in addition to taurine, includes: amino acid mixture as defined herein, CaCl22H2O; HEPES buffer, KCl; MgSO4; NaCl; Na2HPO4 or other phosphates; pyruvate; D-biotin; choline chloride; folic acid; myo-inositol; niacinamide; pyridoxine hydrochloride; D-pantothenic acid; riboflavin; thiamine hydrochloride; vitamin B12; ρ-aminobenzoic acid; ethanolamine HCl; poloxamer 188; DL-α-tocopherol phosphate; linoleic acid; Na2SeO3; thioctic acid; and glucose; as well as optionally, adenosine; guanosine; cytidine; uridine; thymidine; and hypoxanthine disodium.

[0075] In one embodiment, the initial volumetric osmolality of the culture medium of the present invention is 200–500, 250–400, 275–350, or about 300 mOsm. During cell growth in the culture medium of the present invention, in particular, the volumetric osmolality of the culture after any feeding according to the fed-batch protocol may increase to about 350, 400, 450, 500, or about 550 mOsm.

[0076] In some embodiments where the volumetric osmolality of a particular medium is less than about 300, the volumetric osmolality is increased to about 300 by the addition of a specific excess amount of one or more salts. In one embodiment, the volumetric osmolality is increased to a desired level by the addition of one or more osmolites selected from sodium chloride, potassium chloride, magnesium salts, calcium salts, amino acid salts, fatty acid salts, sodium bicarbonate, sodium carbonate, potassium carbonate, chelating agents such as salts, sugars (e.g., galactose, glucose, sucrose, fructose, fucose, etc.), and combinations thereof. In one embodiment, the osmolite is added at a concentration exceeding the osmolite concentration in the components already present in the particular medium (e.g., the sugar is added at a concentration exceeding the concentration of the specified sugar component).

[0077] All embodiments of the above-described media are referred to as taurine supplementation media, and any other serum-free media containing at least about 0.1 mM taurine are also referred to as taurine supplementation media. Conversely, media that do not contain taurine (i.e., media containing less than 0.1 mM taurine) are hereafter referred to as non-taurine supplementation media or non-taurine supplements.

[0078] ( Fed-batch culture ) The supply strategy for cell culture aims to ensure optimal growth and proliferation of cells outside of multicellular organisms or tissues. Appropriate culture conditions for mammalian cells are well known in the field. 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, rotary bottles, shaking flasks, or agitated tank bioreactors (with or without microcarriers), and operation in batch, fed-boil, continuous, semi-continuous, or perfusion modes are available for mammalian cell culture. Cell culture medium or concentrated feed medium can be added to the culture continuously or at intervals during culture. For example, the culture can be supplied once daily, once every other day, once every three days, or when the concentration of a specific medium component being monitored falls outside the desired range.

[0079] In addition to including taurine, in one embodiment, the medium may be further supplemented with amino acids in a cumulative (total) concentration of at least 20 mM. In one embodiment, the initial amino acid concentration contained in the initial cell culture medium is not included in such cumulative (total) supplemented amino acid concentration. In one embodiment of the cell culture medium, i.e., a method for culturing cells or a method for producing a protein of interest, the medium may be supplemented in amounts 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 this specification. In one embodiment, the amount of amino acids added to the medium is about 30 mM ± 10 mM or greater.

[0080] Those skilled in the art can optimize supplement supply regimes to support cell growth, minimize cell stress, or provide a "non-depleted medium" during the production phase.

[0081] "Non-depletion media" include cell culture media that are deemed to contain nutrients, particularly amino acids necessary for the production of the target recombinant protein. Amino acid feeds typically supplement amino acids required as building blocks for recombinant protein production in cell culture. However, depending on the requirements of the specific protein produced by the cultured cells, some amino acids may be depleted faster than others. In non-depletion media, the supply regime is specified so that the required amino acids are replenished when they are consumed. Therefore, depletion and the subsequent optimal consumption rate (pg / cell-day) can be determined by the following steps: culturing eukaryotic cells expressing the target protein in the cell culture medium; measuring the concentration of each amino acid in the culture medium at each time point to establish the depletion level; identifying the depletion point at which the amino acid concentration falls below the depletion level; calculating the consumption rate of each amino acid; and determining the optimal consumption rate as the consumption rate at the time immediately preceding the depletion point. Then, to prevent depletion of the culture medium, the cell culture is supplemented with the appropriate concentration of the specific amino acid to maintain the determined optimal consumption rate.

[0082] It is understood that the present invention provides a taurine-supplemented cell culture medium that improves protein titer in depleted cultures as well as in non-depleted cultures.

[0083] The present invention provides cell cultures comprising cell lines expressing a target protein in the taurine supplement medium described above. Examples of cell lines routinely used to produce protein biotherapeutic drugs include, among others, primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells, and chicken embryo cells. In one embodiment, the cell line is a CHO cell line or one or more of several specific CHO cell variants optimized for large-scale protein production (e.g., CHO-K1).

[0084] In one embodiment, the taurine-supplemented cell culture contains insulin, which can be added to the culture medium as a use-point component or included in the culture medium formulation. In one embodiment, the cell line contains cells capable of producing a biotherapeutic protein.

[0085] In one embodiment, cells are supplemented with culture medium at intervals during cell culture according to a fed-batch process. Fed-batch culture is commonly known in the field and is used to optimize protein production (see YMHuang et al., Biotechnol Prog. 2010 Sep-Oct;26(5):1400-10).

[0086] Typically, after a cell proliferation phase or seed culture (i.e., a first cell culture) without changing the culture medium, a separate second culture known as the polypeptide production phase is performed. A fed-batch process is typically used during the production phase.

[0087] The present invention provides a cell culture medium containing approximately 0.1 mM to approximately 10 mM taurine at the start of production cell culture (day 0). Alternatively, a cell culture medium containing approximately 0.1 mM to approximately 10 mM taurine can be supplemented on days 1, 2, 3, 4, 5, 6, 7, 8, 9, and / or 10 of production cell culture. The cell culture medium added to the production culture on multiple days contains a total amount of approximately 0.1 mM to approximately 10 mM taurine. Cell culture medium containing approximately 0.1 mM to approximately 10 mM total taurine can be added in any order.

[0088] Taurine can also be supplemented to the basal culture medium during the seed train expansion phase.

[0089] To include additional nutrients such as the vitamins, amino acids, and other nutrients mentioned above, supplements can be supplied daily or at intervals of every 2-3 days during the production culture period. Supplements can be supplied (addition of supplemental media containing nutrients) at least twice, or at least eight times, throughout the duration of the production culture of two weeks or longer. In another embodiment, supplements can be supplied daily throughout the duration of the culture. Other culture supply schedules are also conceivable.

[0090] Further amino acid supplementation may be performed to provide a non-depleted medium, where the depleted amino acids are known in the art and determined according to the methods described herein. When using this regime, further amino acids are supplemented or added at regular intervals, preferably daily or every 2-3 days, during the duration of the production culture, depending on the determined amino acid depletion. In one embodiment, a mixture of further amino acids to maintain a non-depleted cell culture medium is added to the culture around day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, and day 14 during a culture of two weeks or more. Other culture supply schedules are also possible.

[0091] Animal cells (such as CHO cells) can be cultured in small quantities (e.g., a 125 mL container with approximately 25 mL of medium, a 250 mL container with approximately 50–100 mL of medium, a 500 mL container with approximately 100–200 mL of medium). Alternatively, cultures may be large-scale (e.g., a 1000 mL container with approximately 300–1000 mL of medium, a 3000 mL container with approximately 500–3000 mL of medium, an 8000 mL container with approximately 2000–8000 mL of medium, and a 15000 mL container with approximately 4000–15000 mL of medium). Production cultures may contain 10,000 L or more of medium. Large-scale cell cultures, such as those for the clinical production of protein therapeutics, are typically maintained for several days or weeks, during which time the cells produce the desired protein. During this time, the culture can be supplemented with a concentrated feed medium containing the components (nutrients and amino acids, etc.) consumed during culture. The concentrated feed medium can be obtained based on any cell culture medium formulation. Such a concentrated feed medium can contain most of the components of the cell culture medium in amounts equivalent to, for example, about 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 30, 50, 100, 200, 400, 600, 800, or even about 1000 times their normal useful amounts. Concentrated feed mediums are often used in fed-batch culture processes.

[0092] In some embodiments, cell cultures containing taurine are further supplemented with “use-point additives” (also known as additives, use-point components, or use-point chemicals) during cell growth or protein production. Use-point additives include any one or more of 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 commonly 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 peptides, or steroid hormones (e.g., estrogen, DHEA, and testosterone). In some cases, growth factors may be non-natural chemicals (e.g., tetrahydrofolate (THF) and methotrexate) that promote cell proliferation or protein production. Non-exclusive examples of protein and peptide growth factors include angiopoietin, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth and differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatome-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), and migration-stimulating factors. This includes IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7, among others.In one embodiment, insulin, a use-point added growth factor, is supplemented to the cell culture medium. In one embodiment, the insulin concentration in the medium after addition (i.e., the amount of insulin in the cell culture medium) is approximately 0.1 μM to 10 μM.

[0093] The buffers are generally known in the art. The present invention is not limited to any particular buffer, and any person skilled in the art can select a buffer or buffer system suitable for use with a particular cell line producing a specific protein. In one embodiment, the use-point buffer is NaHCO3. In another embodiment, the buffer is HEPES. In yet another embodiment, the use-point buffer includes both NaHCO3 and HEPES.

[0094] Energy sources for use as use-point additives in cell culture are also well known in the field. In one embodiment, though not limited, the use-point additive energy source is glucose. Depending on the specific requirements of the particular cell line and the protein to be produced, in one embodiment glucose can be added to the culture medium to a concentration of about 1 to 20 mM. In some cases, glucose can be added at a high level of 20 g / L or higher.

[0095] Chelating agents are also well known in the fields of cell culture and protein production. EDTA tetrasodium dihydrate and citrate are two common chelating agents used in these fields, but other chelating agents can be used in the implementation of the present invention. In one embodiment, the use-point chelating agent is EDTA tetrasodium dihydrate. In one embodiment, the use-point chelating agent is citrate (e.g., Na3C6H5O7).

[0096] In one embodiment, the cell culture medium may be further supplemented with one or more use-point added amino acids (e.g., glutamine) as an energy source. In one embodiment, the cell culture medium is supplemented with use-point added glutamine at a final concentration of approximately 1 mM to 13 mM.

[0097] Other use-point additives include one or more different metal salts (such as salts of iron, nickel, zinc, and copper). In one embodiment, the cell culture medium is supplemented with any one or more of copper sulfate, zinc sulfate, ferric chloride, and nickel sulfate.

[0098] In some embodiments, the protein titer obtained from cells in taurine-supplemented medium 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% higher than the protein titer obtained from cells cultured in taurine-supplemented medium. In some embodiments, the protein titer obtained from cells in taurine-supplemented medium is at least 2%, at least 3%, at least 4%, or at least 5% higher than the protein titer obtained from similar or identical cells cultured in taurine-supplemented medium.

[0099] In some embodiments, ammonia accumulation during cell culture 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% in taurine-supplemented medium compared to cell culture in non-taurine-supplemented medium.

[0100] (Protein production) In addition to taurine supplement medium and methods for culturing cells in taurine supplement medium, the present invention provides an improved method for producing proteins (such as therapeutically effective antibodies or other biopharmaceutical substances) in cells cultured in taurine supplement medium. The present invention provides a method for producing therapeutic proteins in high yield, comprising the step of culturing a recombinant cell line in a taurine-containing medium, wherein the cell line contains a stably incorporated nucleic acid encoding the therapeutic protein.

[0101] In some embodiments, the protein titer (yield) of mammalian cells cultured in a taurine-containing medium (taurine supplement medium) is at least 100 mg / L, at least 0.5 g / L, at least 1 g / L, at least 1.2 g / L, at least 1.4 g / L, at least 1.6 g / L, at least 1.8 g / L, at least 2 g / L, and at least 2.5 g / L higher than the protein titer of the same mammalian cells cultured in a non-taurine supplement medium.

[0102] In some embodiments, the protein yield or titer, which can be expressed in grams of protein product per liter of culture medium, derived from cells cultured in taurine supplement 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.

[0103] In some embodiments, the protein titer obtained from cells in taurine supplement medium 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% higher than the protein titer (yield) obtained from similar or identical cells cultured in non-taurine supplement medium.

[0104] In some embodiments, the protein product (the protein of interest) is an antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, multispecific antibody, bispecific antibody, antigen-binding antibody fragment, single-chain antibody, diabody, triabody or tetrabody, Fab fragment or F(ab')2 fragment, IgD antibody, IgE antibody, IgM antibody, IgG antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0105] In some embodiments, the antibody is an anti-programmed cell death 1 antibody (e.g., the anti-PD1 antibody described in U.S. Patent Application Publication No. 2015 / 0203579A1), an anti-programmed cell death ligand-1 (e.g., the anti-PD-L1 antibody described in U.S. Patent Application Publication No. 2015 / 0203580A1), an anti-Dll4 antibody, an anti-angiopoetin-2 antibody (e.g., the anti-ANG2 antibody described in U.S. Patent No. 9,402,898), an anti-angiopoetin-like 3 antibody (e.g., Anti-AngPtl3 antibody (as described in U.S. Patent No. 9,018,356), anti-platelet-derived growth factor receptor antibody (e.g., anti-PDGFR antibody as described in U.S. Patent No. 9,265,827), anti-Erb3 antibody, anti-prolactin receptor antibody (e.g., anti-PRLR antibody as described in U.S. Patent No. 9,302,015), anti-complement 5 antibody (e.g., anti-C5 antibody as described in U.S. Patent Publication No. 2015 / 0313194A1), anti-TNF antibody, anti-epidermal growth factor receptor antibody (e.g., anti-EG antibody as described in U.S. Patent No. 9,132,192) FR antibody (or anti-EGFRvIII antibody as described in U.S. Patent Application Publication No. 2015 / 0259423A1), anti-proprotein convertase subtilisin kexin-9 antibody (e.g., anti-PCSK9 antibody as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. 2014 / 0044730A1), anti-growth differentiation factor-8 antibody (e.g., also known as anti-myostatin antibody, anti-GDF8 antibody as described in U.S. Patent No. 8,871,209 or 9,260,515), anti-glucagon receptor (e.g. For example, anti-GCGR antibodies described in U.S. Patent Application Publication No. 2015 / 0337045A1 or No. 2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin-4 receptor antibodies (for example, anti-IL4R antibodies described in U.S. Patent Application Publication No. 2014 / 0271681A1 or U.S. Patent No. 8,735,095 or No. 8,945,559), anti-interleukin-6 receptor antibodies (for example, U.S. Patent No. 7,582,298, No. 8,043,617 or No. 9,173,Anti-IL6R antibody (as described in Patent No. 880), 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., anti-IL33 antibody as described in U.S. Patent Application Publication No. 2014 / 0271658A1 or 2014 / 0271642A1), anti-respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Patent Application Publication No. 2014 / 0271653A1), anti-differentiation antigen group 3 (e.g., U.S. Patent Application Publication No. 20 Anti-CD3 antibodies described in U.S. Patent Publication No. 14 / 0088295A1 and No. 20150266966A1 and U.S. Patent Application No. 62 / 222,605), anti-differentiation antigen group 20 (e.g., anti-CD20 antibodies described in U.S. Patent Publication No. 2014 / 0088295A1 and No. 20150266966A1 and U.S. Patent No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-differentiation antigen group-48 (e.g., anti-CD48 antibodies 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., anti-MERS antibody described in U.S. Patent Publication No. 2015 / 0337029A1), anti-Ebola virus antibody (e.g., described in U.S. Patent Publication No. 2016 / 0215040), anti-Zika virus antibody, anti-lymphocyte activator gene 3 antibody (e.g., anti-LAG3 antibody or anti-CD223 antibody), anti-nerve growth factor antibody (e.g., U.S. Patent Publication No. 2016 / 0017029 and U.S. Patents No. 8,309,088 and No. 9,353,The anti-NGF antibody (as described in Patent No. 176) and the anti-activin A antibody are selected from the group. In some embodiments, the bispecific antibody is selected from the group consisting of anti-CD3 × anti-CD20 bispecific antibody (as described in U.S. Patent Publication Nos. 2014 / 0088295A1 and 20150266966A1), anti-CD3 × anti-mucin 16 bispecific antibody (e.g., anti-CD3 × anti-Muc16 bispecific antibody), and anti-CD3 × anti-prostate-specific membrane antigen bispecific antibody (e.g., anti-CD3 × anti-PSMA bispecific antibody). In some embodiments, the protein of interest is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab. All publications referenced through this disclosure are incorporated herein by reference in their entirety.

[0106] In some embodiments, the protein of interest is a recombinant protein (e.g., an Fc-fusion protein) comprising an Fc moiety and another domain. In some embodiments, the Fc-fusion protein is a receptor Fc-fusion protein comprising one or more extracellular domains of a receptor coupled to the Fc moiety. In some embodiments, the Fc moiety comprises CH2 and CH3 domains after the hinge region of IgG. In some embodiments, the receptor Fc-fusion protein comprises two or more separate receptor chains that bind to either a single ligand or multiple ligands. For example, the Fc-fusion protein is a TRAP protein (e.g., an IL-1 trap (e.g., lilonacept (containing an IL-1RAcP ligand-binding domain fused to the Il-1R1 extracellular domain fused to the Fc 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 or ziv-aflibercept (containing the Ig domain 2 of VEGF receptor Flt1 fused to the Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1); see U.S. Patents No. 7,087,411 and 7,279,159)). In other embodiments, the Fc-fusion protein is an ScFv-Fc-fusion protein, which comprises one or more antigen-binding domains (such as heavy-chain and light-chain variable fragments of an antibody coupled to the Fc portion).

[0107] The present invention is not limited to any particular type of cell for protein production. Examples of cell types suitable for protein production include mammalian cells, insect cells, bird cells, bacterial cells, and yeast cells. Cells may be stem cells, recombinant cells transformed with recombinant gene expression vectors, or cells transfected with viruses for viral product production. Cells may contain recombinant heterologous polynucleotide constructs encoding the protein of interest. These constructs may be episomes or elements physically integrated into the cell's genome. Cells may also produce the protein of interest without the protein being encoded by a heterologous polypeptide construct. In other words, cells may naturally encode the protein of interest (e.g., B cells that produce antibodies). Cells may also be primary cells (e.g., chicken embryo cells) or primary cell lines. Examples of useful 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 CHOlec mutants).

[0108] In one embodiment, a cell that is a CHO cell ectopically expresses the protein. In one embodiment, the protein includes an immunoglobulin heavy chain region (such as a CH1, CH2, or CH3 region). In one embodiment, the protein includes human or rodent immunoglobulin CH2 and CH3 regions. In one embodiment, the protein includes human or rodent immunoglobulin CH1, CH2, and CH3 regions. In one embodiment, the protein includes a hinge region as well as the CH1, CH2, and CH3 regions. In one embodiment, the protein includes an immunoglobulin heavy chain variable domain. In one embodiment, the protein includes an immunoglobulin light chain variable domain. In one embodiment, the protein includes 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)).

[0109] The production phase can be carried out in cultures of any scale, from shaking flasks or wave bags to 1-liter bioreactors and large-scale industrial bioreactors. Similarly, the seed train expansion phase can be carried out in cultures of any scale, from shaking flasks or wave bags to 1-liter or larger bioreactors. Large-scale processes can be carried out in volumes from approximately 100 liters to 20,000 liters or more. Protein production can be controlled using one or more of several means (such as temperature changes or chemical induction). The growth phase can occur at higher temperatures than the production phase. For example, the growth phase can occur at a first temperature of approximately 35°C to 38°C, and the production phase can occur at a second temperature of approximately 29°C to 37°C, or optionally, approximately 30°C to 36°C or approximately 30°C to 34°C. Furthermore, chemical inducers of protein production (such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene bisacetamide (HMBA)) can be added simultaneously with, before, or after the temperature change. When the inducer is added after the temperature change, it can be added from 1 hour to 5 days after the temperature change (e.g., 1-2 days after the temperature change). Cell culture of the producing cells can be carried out using a continuous supply culture system such as a chemostat (see C. Altamirano et al., 2001, cited above) or according to a fed-batch process (Huang, 2010, cited above).

[0110] The present invention is useful for improving protein production via cell culture processes. Cell lines used in the present invention can be genetically engineered to express commercially or scientifically interesting polypeptides. Genetic engineering of cell lines includes transfection, transformation, or introduction of cells with recombinant polynucleotide molecules, or other modifications to host cells to express a desired recombinant polypeptide (e.g., by homologous recombination and gene activation or fusion of recombinant cells with non-recombinant cells). Methods and vectors for genetically engineering cells or cell lines to express a target polypeptide are well known to those skilled in the art; for example, various techniques are exemplified in Current Protocols in Molecular Biology. Ausubel et al., eds. (Wiley & Sons, New York, 1988 and quarterly updates); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69. A wide variety of cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and its distributors. Examples of cell lines commonly used in industry include VERO, BHK, HeLa, CVl (including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NSL), PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO cells are widely used for the production of complex recombinant proteins (such as cytokines, coagulation 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 MoI 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), DXBl1, and DG-44 are desirable CHO host cell lines because they exhibit effective DHFR selectivity and allow for high levels of recombinant protein expression in these cells via amplified gene expression systems (Kaufman RJ. (1990), Meth Enzymol 185:537-566). Furthermore, these cells are easy to handle as adherent or suspension cultures and exhibit relatively good gene stability. CHO cells and proteins recombinantly expressed by CHO cells have been widely characterized and approved by regulatory authorities for use in clinical and commercial manufacturing. In some embodiments, the CHO cell line is the cell line described in U.S. Patent Publication Nos. 2010 / 0304436A1, 2009 / 0162901A1, and 2009 / 0137416A1, and U.S. Patent Nos. 7,455,988B2, 7,435,553B2, and 7,105,348B2.

[0111] The present invention is not limited to the scope of the specific embodiments described herein, and these embodiments are intended to be illustrative of each aspect or embodiment of the invention. Functionally equivalent methods and components are within the scope of the invention. In addition to the inventions described herein, various modifications of the invention are obvious to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are within the scope of the invention. [Examples]

[0112] (Example 1: Improvement of antibody titer by taurine supplementation) Example 1A - High-throughput shaking flask culture: Seed cultures of a monoclonal antibody (Ab1)-producing cell line derived from CHO-K1 were inoculated into a 250 mL shaking flask. The inoculated cells were grown at 35.5°C for 17 days, with glucose and other supplemental nutrients supplied as needed. The cells were grown in chemically defined (hydrolyzate-free and serum-free) basic medium.

[0113] Each culture flask was either unsupplemented (Flask 1a) or supplemented with 1 mM taurine on day 0 (Flask 1b). [Table 2] * Baseline control for titer increase (%); Flask 1b was compared to the titer in unsupplemented medium (Flask 1a). The difference in final titer between the unsupplemented culture and the supplemented culture was statistically significant (p<0.05).

[0114] The titer was calculated from the protein recovered on day 17, and this titer was statistically significant compared to baseline (p<0.05). Taurine-supplemented cultures showed an overall 8% increase in final protein titer compared to unsupplemented cultures.

[0115] Example 1B - Benchtop-scale bioreactor: A similar example, but for large-scale production, seed cultures of CHO-K1-derived monoclonal antibody (Ab2, Ab3, or Ab4) producing cell lines were inoculated into a 2 L bioreactor. The inoculated cultures were grown for 14 days at a temperature of 35.5°C, a DO setpoint of 40.4%, and an air injection of 22 ccm. The pH setpoint for the Ab2 and Ab3 processes was 7.0 ± 0.15, and the pH setpoint for the Ab4 process was 7.13 ± 0.27. Glucose, antifoaming agents, and basal feeds were supplied to the bioreactor as needed. Cultures were grown in unsupplemented medium (bioreactors 2a, 3a, and 4a) or in approximately 1 mM taurine supplemented medium (Ab2 and Ab3) or approximately 3 mM taurine supplemented medium (Ab4) (added on day 0 of production (bioreactors 2b, 3b, and 4b, respectively)).

[0116] The antibody yield (titer) was 6.4 g / L for Ab2-producing cells, while cells grown with taurine produced 8 g / L of protein. The 24% increase in titer compared to cells grown without taurine supplementation was statistically significant (p<0.05). The final titers of the resulting Ab3-producing and Ab4-producing cultures were also significantly higher at 14 days compared to the taurine-unsupplemented cultures (p<0.05) (11% and 20%, respectively). See Table 3. [Table 3] * The unsupplemented medium serves as a baseline control for the percentage increase in titer, where the percentage increase in titer in bioreactors 2b, 3b, or 4b is compared to the titer in the unsupplemented medium (bioreactors 2a, 3a, or 4a, respectively). The difference in final titer between supplemented and unsupplemented cultures is statistically significant (p<0.05).

[0117] The time course of protein titer in Ab3-producing cell cultures was plotted, and a significant improvement in protein titer with taurine supplementation was observed on each culture day starting from day 6. [Table 4] * Approximate increase (%) compared to unsupplemented medium collected on the same day The increase in titer with taurine supplementation was statistically significant compared to the medium without supplementation (p<0.05).

[0118] A significant improvement in titer was observed as early as day 6 in the production culture (12% increase compared to the same culture without taurine supplementation). See also Figure 1. The greatest difference in this time course was observed on day 9 (a significant 29% increase (p<0.05) in bioreactor 3b compared to 3a), and a significant 11% increase in titer (p<0.05) was observed on the final day of culture (day 14).

[0119] The advantages of taurine supplementation in protein production are observed across different scales (Examples 1A and 1B) and different cell lines (Example 1B).

[0120] (Example 2: Consistency of productivity in taurine concentration fluctuations in high-throughput shaking flask culture) The consistency of protein titer was tested by varying the amount of taurine added to the culture on day 0 of production. Seed cultures of a monoclonal antibody (Ab1)-producing cell line derived from CHO-K1 were inoculated into 250 mL shaking flasks. The inoculated cells were grown at 35.5°C for 14 days, with glucose and other supplemental nutrients supplied as needed. The cells were grown in chemically defined (hydrolyzate-free and serum-free) basal medium.

[0121] Each culture either contained no taurine (no supplementation) 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. [Table 5] * The unsupplemented medium serves as a baseline control for the percentage increase in titer. # The difference in final titer compared to the unsupplemented control was statistically significant (p<0.1). The difference in final titer compared to the unenhanced control group was statistically significant (p<0.05).

[0122] When taurine is supplemented in a concentration of at least 0.1 mM to 10 mM, consistently high titer is observed even when the amount of supplemented taurine is varied. The final titer under the taurine supplementation condition was statistically different from that under the no-supplement condition. p<0.1 was observed for 0.1 mM taurine, and p<0.05 for 0.3 mM to 10 mM taurine.

[0123] (Example 3: Testing of taurine supply fluctuation schedule in high-throughput shaking flask culture) Example 3A: Taurine addition during the seed train expansion phase:

[0124] The benefits of taurine supplementation to cultures during the expansion seed train phase were evaluated using a high-throughput shaking flask model. In flask 6a (Table 6), Ab1-producing CHO cells were thawed in chemically defined (hydrolyzate-free and serum-free) basal medium supplemented with 1 mM taurine. The taurine concentration in the basal medium was maintained at 1 mM throughout the expansion phase. During production, 1 mM taurine was supplemented to the culture basal medium on day 0. Glucose and nutrient basal feeds were supplied as needed during the 17-day production period.

[0125] Flask 6b cells were grown in chemically defined (no hydrolysates and serum-free) basal medium without taurine supplementation throughout the seed train expansion phase. On day 0 of production, 1 mM taurine was supplemented to the culture basal medium. During day 17 of production, glucose and nutrient basal feeds were supplied as needed. [Table 6] The difference in final titer (day 17) was not statistically significant (p>0.1).

[0126] The final (day 17) titers for both conditions (production phase only, or taurine supplementation in a combination of seed train and production phase) are similar. The obtained titers are not statistically significant (p>0.1). The benefits of taurine supplementation during the seed train expansion phase are similar to those of taurine supplementation during the production phase.

[0127] Example 3B: Taurine Supply Fluctuation Schedule During Production: Further experiments were performed in similar shaking flask cultures growing Ab1-producing CHO cells to determine whether a standard taurine supply fluctuation schedule had any effect on the protein titer of taurine-supplied cultures. The cells were subjected to fluctuation culture conditions similar to Example 2, with the same supply schedule as described above and glucose / nutrient basal feed added as needed.

[0128] Ab1-producing cultures were supplemented with a total of 5 mM taurine. Similar productivity (7.1 g / L, 6.8 g / L, and 7.0 g / L) was observed under taurine supply fluctuation schedules. The potency values ​​compared in this experiment were not statistically different (p>0.1) (see Table 7). [Table 7] The difference in power values ​​on the 14th was not statistically significant (p>0.1).

[0129] The taurine supply schedule has no negative impact and does not alter the finding that taurine supplementation is advantageous for product yield. Therefore, taurine supplementation can be added once on day 0, during subsequent production phases, or at multiple intervals during production phases.

[0130] (Example 4: Measurement of ammonia by-products in high-processing, high-throughput shaking flask culture) Ammonia byproducts were measured in taurine-supplemented cultures of Ab1-producing CHO cells after 14 days, using a method similar to that of Example 2. Cells were subjected to variable culture conditions similar to those described above, with glucose / nutrient basal feed added as needed. [Table 8] * Baseline control for ammonia reduction percentage; ammonia under taurine supplementation conditions was compared to ammonia in unsupplemented medium. The decrease in ammonia concentration is statistically significant (p<0.1).

[0131] In Ab1-producing cells, taurine supplementation in the culture medium supported healthy and sustainable culture and reduced ammonia byproducts by 32%. The reduction in ammonia concentration due to taurine supplementation was statistically significant (p<0.1).

[0132] The present invention can be embodied in other specific embodiments.

Claims

1. A method for culturing recombinant eukaryotic cells for improved production of dupilumab, (a) A step of growing recombinant eukaryotic cells in a prescribed cell culture medium during the growth phase, wherein the cell culture medium does not contain glutamine, serum, and hydrolysates. (b) A step of supplementing the specified cell culture medium with 0.1 mM to 10 mM L-taurine, and a step of expressing dupilumab during the production phase. Includes, A method wherein the addition of L-taurine increases the titer of dupilumab by at least 7% compared to cells expressing dupilumab in a cell culture medium that does not contain L-taurine, the step of growing the cells during the proliferation phase is carried out at a temperature of 35°C to 38°C, and the step of expressing dupilumab during the production phase is carried out at a temperature of 29°C to 37°C, wherein the temperature during the proliferation phase is higher than the temperature during the production phase.

2. The method according to claim 1, wherein the L-taurine is supplemented 1 to 5 times during the production period.

3. The method according to claim 1, wherein the L-taurine is supplemented daily during the duration of the production period.

4. The method according to claim 1, further comprising the step of supplementing the prescribed cell culture medium with 0.1 mM to 10 mM L-taurine during the growth phase.

5. The method according to claim 1, wherein the defined cell culture medium is supplemented with 0.09 mM to 0.9 mM ornithine.

6. The method according to claim 1, wherein the eukaryotic cell is a CHO cell.

7. A method for producing dupilumab, (a) A step of culturing cells expressing dupilumab in a cell culture medium containing 0.1 mM to 10 mM L-taurine, wherein the addition of L-taurine increases the titer of dupilumab by at least 3% compared to cells expressing dupilumab in a cell culture medium without L-taurine. (b) A step of producing dupilumab in the cells, wherein the dupilumab is secreted into the culture medium. A method comprising the following, wherein the cells are cultured at a temperature of 35°C to 38°C during the proliferation phase and at a temperature of 29°C to 37°C during the production phase, the temperature during the proliferation phase being higher than the temperature during the production phase.

8. The method according to claim 7, wherein the cells are capable of a 7% or higher increase in dupilumab titer compared to cells expressing dupilumab in a cell culture medium that does not contain L-taurine.

9. The method according to claim 7, wherein the cells are CHO cells.

10. The method according to claim 7, further comprising the step of supplementing the cell culture medium with 0.09 mM to 0.9 mM ornithine.

11. A method for producing dupilumab, A step of culturing a recombinant cell line in a cell culture medium containing at least 0.1 mM L-taurine, wherein the recombinant cell line contains a stably incorporated nucleic acid encoding dupilumab. A step of producing dupilumab from the cells, wherein the addition of L-taurine to the cell culture medium increases the titer of the dupilumab by at least 3% compared to a step of culturing the recombinant cell line in a cell culture medium that does not contain L-taurine. A method comprising the recombinant cell line being cultured at a temperature of 35°C to 38°C during the growth phase and 29°C to 37°C during the production phase, wherein the temperature during the growth phase is higher than the temperature during the production phase.

12. The method according to claim 11, wherein the production method is capable of increasing the dupilumab titer by at least 0.1 g / L compared to a similar production method in a cell culture medium that does not contain L-taurine.

13. The method according to claim 11, wherein the production method is capable of increasing the dupilumab titer by 7% or more compared to a similar production method in a cell culture medium that does not contain L-taurine.

14. The method according to claim 11, wherein the recombinant cell line is cultured in L-taurine-supplemented cell culture medium for at least 6 days and expresses a higher titer of dupilumab compared to a recombinant cell line expressing dupilumab cultured in L-taurine-free cell culture medium for at least 6 days.

15. The method according to claim 11, further comprising the step of supplementing the cell culture medium with 0.09 mM to 0.9 mM ornithine.

16. A method for producing dupilumab in taurine-supplemented cell culture medium, (a) A step of culturing CHO cells expressing dupilumab in a cell culture medium, (b) A step of capturing the cell culture medium with 0.1 mM to 10 mM L-taurine to produce L-taurine-supplemented cell culture medium, (c) A step of culturing the CHO cells expressing dupilumab in the taurine-supplemented cell culture medium of (b) for at least 6 days, wherein the dupilumab is secreted into the medium and the addition of L-taurine increases the titer of the dupilumab by at least 3% compared to CHO cells expressing dupilumab in a cell culture medium that does not contain L-taurine. (d) A step of recovering the dupilumab A method comprising culturing the CHO cells at a temperature of 35°C to 38°C during the proliferation phase and 29°C to 37°C during the production phase, wherein the temperature during the proliferation phase is higher than the temperature during the production phase.

17. The method according to claim 16, wherein the titer of dupilumab from CHO cells expressing dupilumab cultured in the L-taurine-supplemented cell culture medium (a) for at least six days is at least 8% higher than the titer of dupilumab from CHO cells cultured in the L-taurine-free cell culture medium (a) for at least six days.

18. The method according to claim 16, wherein the production method is capable of increasing the dupilumab titer by at least 0.1 g / L compared to a similar production method in a cell culture medium that does not contain L-taurine.

19. The method according to claim 16, wherein the taurine-supplemented cell culture medium is further supplemented with 0.09 mM to 0.9 mM ornithine.

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