Culture medium for cell culture

Abstract

Provided are a method, a reagent, and the like for improving the stability of a culture medium and improving the productivity of recombinant proteins. Specifically, the invention provides a method. Provided are: a culture medium containing N-acetylcysteine and L-cysteine; a method for culturing cells using the culture medium containing N-acetylcysteine and L-cysteine; a method for producing a recombinant protein using the culture medium containing N-acetylcysteine and L-cysteine; a method or reagent for stabilizing a culture medium comprising L-cysteine; and the like.

Description

Technical Field

[0001] The present invention relates to a culture medium. In addition, the present invention relates to a method for culturing cells in a culture medium or a method for producing a recombinant protein. In addition, the present invention relates to a method or an agent for improving the stability of a culture medium. Background Art

[0002] The culture medium used in cell culture is often physically and chemically unstable, and the composition changes during preparation, storage and / or cell culture, which has an adverse effect on the cell culture process. Therefore, in particular, when culturing cells on an industrial scale, it is crucial to develop a culture medium with high stability in order to improve productivity and reduce production costs.

[0003] The development of a culture medium with higher stability is particularly important in improving the productivity of products using recombinant proteins. For example, in the case of an antibody drug using a recombinant protein, by using a culture medium with higher stability, an antibody drug with greater social demand can be manufactured more stably or at a lower price. In addition, the research and development of antibody drugs that require a lot of time and cost can be carried out more efficiently.

[0004] Many attempts to improve culture media have been reported. For example, Japanese Patent Publication No. 2000-507812 describes a culture medium supplement component that is useful and stable for a culture medium that supports the proliferation of hematopoietic cells in culture.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Publication No. 2000-507812 Summary of the invention

[0008] The present invention can provide a culture medium with higher stability.

[0009] That is, according to the present invention, the following aspects are provided.

[0010] [1] A feed medium comprising 10 to 40 mM L-cysteine ​​and 1 to 40 mM N-acetylcysteine.

[0011] [2] A feed medium comprising L-cysteine ​​and N-acetylcysteine,

[0012] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.0.

[0013] [3] The feed medium according to [1] or [2], comprising 15 to 30 mM L-cysteine.

[0014] [4] The feed medium according to any one of [1] to [3], comprising 2 to 10 mM N-acetylcysteine.

[0015] [5] The feed medium according to any one of [1] to [4], wherein

[0016] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.0.

[0017] [6] The feed medium according to any one of [1] to [5], wherein

[0018] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.1 to 0.4.

[0019] [7] The feed medium according to any one of [1] to [6], wherein

[0020] The pH is 5.0~9.0.

[0021] [8] The feed medium according to any one of [1] to [7], wherein

[0022] The pH is 6.5~7.6.

[0023] [9] The feed medium according to any one of [1] to [8], which is a serum-free medium and / or an animal protein-free medium.

[0024]

[10] The feed medium according to any one of [1] to [9], which is a synthetic medium.

[0025]

[11] A powdered culture medium comprising L-cysteine ​​and N-acetylcysteine, wherein:

[0026] The powder culture medium is used to prepare a feed medium containing 10-40 mM L-cysteine ​​and 1-40 mM N-acetylcysteine.

[0027]

[12] A powdered culture medium comprising L-cysteine ​​and N-acetylcysteine, wherein:

[0028] The powder culture medium is used to prepare a feed culture medium in which the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04-1.0.

[0029]

[13] The powdered culture medium according to

[11] or

[12] , wherein

[0030] The feed medium contained 15-30 mM L-cysteine.

[0031]

[14] The powdered culture medium according to any one of

[11] to

[13] , wherein

[0032] The feed medium contained 2-10 mM N-acetylcysteine.

[0033]

[15] The powdered culture medium according to any one of

[11] to

[14] , wherein

[0034] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the feed medium is 0.04 to 1.0.

[0035]

[16] The powdered culture medium according to any one of

[11] to

[15] , wherein

[0036] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the feed medium is 0.1 to 0.4.

[0037]

[17] The powdered culture medium according to any one of

[11] to

[16] , wherein

[0038] The pH of the feed medium was 5.0~9.0.

[0039]

[18] The powdered culture medium according to any one of

[11] to

[17] , wherein

[0040] The pH of the feed medium was 6.5~7.6.

[0041]

[19] The powdered culture medium according to any one of

[11] to

[18] , wherein

[0042] The feed medium is serum-free and / or animal protein-free.

[0043]

[20] The powdered culture medium according to any one of

[11] to

[19] , wherein

[0044] The feed medium is a synthetic medium.

[0045]

[21] A method for culturing cells, comprising adding the feed medium described in any one of [1] to

[10] to a culture medium for culturing cells.

[0046]

[22] The method according to

[21] , wherein:

[0047] The cell is an animal cell, a plant cell, a bacterial cell or a fungal cell.

[0048]

[23] The method according to

[21] or

[22] , wherein:

[0049] The cells are animal cells.

[0050]

[24] The method according to any one of

[21] to

[23] , wherein

[0051] The cells are mammalian cells.

[0052]

[25] The method according to any one of

[21] to

[24] , wherein

[0053] The cells are Chinese hamster ovary (CHO) cells.

[0054]

[26] The method according to any one of

[21] to

[25] , wherein

[0055] Cells express recombinant proteins.

[0056]

[27] The method according to any one of

[21] to

[26] , wherein

[0057] The recombinant protein comprises an antibody or an antigen-binding fragment thereof.

[0058]

[28] The method according to

[27] , wherein:

[0059] The antibody or antigen-binding fragment thereof is derived from IgG.

[0060]

[29] The method according to any one of

[21] to

[28] , wherein

[0061] There is no precipitation of medium components due to heat load.

[0062]

[30] A method for producing a recombinant protein, comprising:

[0063] The feed medium described in any one of [1] to

[10] is added to a medium for culturing cells capable of producing a recombinant protein.

[0064]

[31] The method according to

[30] , wherein:

[0065] The cell is an animal cell, a plant cell, a bacterial cell or a fungal cell.

[0066]

[32] The method according to

[30] or

[31] , wherein:

[0067] The cells are animal cells.

[0068]

[33] The method according to any one of

[30] to

[32] , wherein

[0069] The cells are mammalian cells.

[0070]

[34] The method according to any one of

[30] to

[33] , wherein

[0071] The cells are Chinese hamster ovary (CHO) cells.

[0072]

[35] The method according to any one of

[30] to

[34] , wherein

[0073] The recombinant protein comprises an antibody or an antigen-binding fragment thereof.

[0074]

[36] The method according to

[35] , wherein:

[0075] The antibody or antigen-binding fragment thereof is derived from IgG.

[0076]

[37] The method according to any one of

[30] to

[36] , wherein

[0077] There is no precipitation of medium components due to heat load.

[0078]

[38] A method for improving the stability of a liquid culture medium, comprising:

[0079] L-cysteine ​​and / or N-acetylcysteine ​​are added to the medium so that the concentration of L-cysteine ​​is 10 to 40 mM and the concentration of N-acetylcysteine ​​is 1 to 40 mM.

[0080]

[39] A method for improving the stability of a liquid culture medium, comprising:

[0081] L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.0.

[0082]

[40] A method for inhibiting the generation and / or precipitation of cystine in a liquid culture medium, comprising:

[0083] L-cysteine ​​and / or N-acetylcysteine ​​are added to the medium so that the concentration of L-cysteine ​​is 10 to 40 mM and the concentration of N-acetylcysteine ​​is 1 to 40 mM.

[0084]

[41] A method for inhibiting the production and / or precipitation of cystine in a liquid culture medium, comprising:

[0085] L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.0.

[0086]

[42] The method according to any one of

[38] to

[41] , comprising:

[0087] L-cysteine ​​and N-acetylcysteine ​​were added to the culture medium.

[0088]

[43] The method according to any one of

[38] to

[41] , wherein

[0089] The liquid culture medium contains 10~40mM L-cysteine.

[0090] The method comprises: adding N-acetylcysteine ​​to a culture medium in such a manner that the concentration of N-acetylcysteine ​​is 1 to 40 mM.

[0091]

[44] The method according to any one of

[38] to

[41] , wherein

[0092] The liquid medium contains 1~40mM N-acetylcysteine.

[0093] The method comprises: adding L-cysteine ​​to a culture medium so that the concentration of L-cysteine ​​is 10 to 40 mM.

[0094]

[45] The method according to any one of

[38] to

[44] , comprising:

[0095] L-cysteine ​​and / or N-acetylcysteine ​​are added to the medium so that the concentration of L-cysteine ​​becomes 15 to 30 mM.

[0096]

[46] The method according to any one of

[38] to

[45] , comprising:

[0097] L-cysteine ​​and / or N-acetylcysteine ​​are added to the medium so that the concentration of N-acetylcysteine ​​is 2 to 10 mM.

[0098]

[47] The method according to any one of

[38] to

[46] , comprising:

[0099] L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.0.

[0100]

[48] ​​The method according to any one of

[38] to

[47] , comprising:

[0101] L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.1 to 0.4.

[0102]

[49] The method according to any one of

[38] to

[48] , comprising:

[0103] L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the pH of the liquid medium is 5.0 to 9.0.

[0104]

[50] The method according to any one of

[38] to

[49] , comprising:

[0105] L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the pH of the liquid medium is 6.5 to 7.6.

[0106]

[51] A reagent for improving the stability of a liquid culture medium, comprising L-cysteine ​​and N-acetylcysteine, wherein:

[0107] The liquid culture medium after adding the reagent contains 10-40 mM L-cysteine ​​and 1-40 mM N-acetylcysteine.

[0108]

[52] A reagent for improving the stability of a liquid culture medium, comprising L-cysteine ​​and N-acetylcysteine, wherein:

[0109] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.0.

[0110]

[53] A reagent containing N-acetylcysteine ​​for improving the stability of a liquid culture medium containing 10 to 40 mM L-cysteine, wherein:

[0111] The liquid culture medium after adding the reagent contains 1 to 40 mM N-acetylcysteine.

[0112]

[54] An agent containing N-acetylcysteine ​​for improving the stability of a liquid culture medium containing L-cysteine, wherein:

[0113] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.0.

[0114]

[55] A reagent for inhibiting the production and / or precipitation of cystine in a liquid culture medium, comprising L-cysteine ​​and N-acetylcysteine, wherein:

[0115] The liquid culture medium after adding the reagent contains 10-40 mM L-cysteine ​​and 1-40 mM N-acetylcysteine.

[0116]

[56] A reagent for inhibiting the production and / or precipitation of cystine in a liquid culture medium, comprising L-cysteine ​​and N-acetylcysteine, wherein:

[0117] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.0.

[0118]

[57] An agent comprising N-acetylcysteine ​​for inhibiting the production and / or precipitation of cystine in a liquid medium containing 10 to 40 mM L-cysteine, wherein:

[0119] The liquid culture medium after adding the reagent contains 1 to 40 mM N-acetylcysteine.

[0120]

[58] An agent comprising N-acetylcysteine ​​for inhibiting the production and / or precipitation of cystine in a liquid medium containing L-cysteine, wherein:

[0121] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.0.

[0122]

[59] The reagent according to any one of

[51] to

[58] , wherein

[0123] The liquid culture medium after adding the reagent contains 15-30 mM L-cysteine.

[0124]

[60] The reagent according to any one of

[51] to

[59] , wherein

[0125] The liquid culture medium after adding the reagent contains 2-10 mM N-acetylcysteine.

[0126]

[61] The reagent according to any one of

[51] to

[60] , wherein

[0127] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.0.

[0128]

[62] The reagent according to any one of

[51] to

[61] , wherein

[0129] The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.1 to 0.4.

[0130]

[63] The reagent according to any one of

[51] to

[62] , wherein

[0131] The pH of the liquid culture medium after adding the reagent is 5.0-9.0.

[0132]

[64] The reagent according to any one of

[51] to

[63] , wherein

[0133] The pH of the liquid culture medium after adding the reagent is 6.5-7.6.

[0134]

[65] A culture medium supplement comprising N-acetylcysteine ​​and L-cysteine, wherein:

[0135] The culture medium supplement is used to prepare the feed medium described in any one of [1] to

[10] or the powder culture medium described in any one of

[11] to

[20] .

[0136]

[66] A culture medium supplement comprising N-acetylcysteine, wherein:

[0137] The culture medium supplement is used to prepare the feed medium described in any one of [1] to

[10] or the powder culture medium described in any one of

[11] to

[20] .

[0138] The invention can improve the stability of the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] [ Figure 1 ] Figure 1 It is a graph showing the changes over time in the concentrations of L-cysteine, cystine, N-acetylcysteine, N-acetylcystine, and N,N′-diacetylcystine in a liquid culture medium.

[0140] [ Figure 2 ] Figure 2 These are photographs showing the effect of N-acetylcysteine ​​on the precipitation of medium components in a medium containing L-cysteine.

[0141] [ Figure 3 ] Figure 3 This is a graph showing the effect of N-acetylcysteine ​​on the viable cell density in a medium containing L-cysteine. The graph shows the viable cell density on the 9th and 14th day after the start of culture.

[0142] [ Figure 4 ] Figure 4 This is a graph showing the effect of N-acetylcysteine ​​on the amount of antibody produced in a medium containing L-cysteine. The graph shows the amount of antibody produced on days 7, 9, 11, and 14 after the start of culture.

[0143] [ Figure 5 ] Figure 5This is a graph showing the effect of L-cysteine ​​concentration on the amount of antibody produced in the culture medium. The relative value of the amount of antibody produced on the 14th day after the start of culture is shown. DETAILED DESCRIPTION

[0144] [Explanation of terms]

[0145] Unless otherwise specified, each term in the present disclosure has the following meanings.

[0146] The term "cell culture medium" as used herein refers to a substance that provides a cell growth environment for proliferation or maintenance of cells. Typically, a cell culture medium contains components necessary for cell proliferation and survival, as well as useful components.

[0147] The "feed medium" in the present disclosure refers to the components of the medium that are added to the cell culture in an additional manner after the cell culture step is started.

[0148] As for the feed medium, typically, it contains the necessary components and useful components for achieving cell proliferation and survival. The feed medium may be only one of these components, or it may be a composition comprising multiple components. For example, in the present disclosure, a liquid containing only L-cysteine ​​and / or N-acetylcysteine ​​is also included in the feed medium. The feed medium may contain components of a cell culture medium (e.g., sugar (glucose), amino acids, etc.). It should be noted that the expression "culture medium" may be used in the sense of a cell culture medium, may be used in the sense of a feed medium, or may include the meanings of both.

[0149] As for the feed medium, it is typically used for cell culture based on the fed batch culture method (fed-batch culture method). The fed batch culture method is a culture method in which a basal medium containing all the components for cell culture (including cells and all nutrient sources) is supplied to a culture container at the beginning of the culture process, and then the feed medium is continuously or stagedly added to the culture container. Here, the basal medium is a medium used from the beginning of the cell culture process. If it can be used as a basal medium, it is not particularly limited and can be used as a basal medium.

[0150] The feed medium is usually provided and sold in the form of a liquid medium or a powder medium. The feed medium is typically stored in a plastic bottle or a stainless steel tank, but is not limited thereto.

[0151] The period of adding the feed medium to the culture vessel is not particularly limited, and can be, for example, the beginning of cell culture, the induction period, the logarithmic growth period, the stationary period and / or the death period. In addition, the feed medium can be used in other culture methods, such as continuous culture methods. The continuous culture method can also be referred to as the perfusion culture method, which is a culture method in which the additional culture medium (feed medium, etc.) is supplied to the culture vessel at a certain speed, and an equal amount of culture fluid is taken out from the culture vessel at the same time.

[0152] Non-limiting examples of components necessary for cell proliferation and survival and useful components include carbohydrates such as sugars (e.g., glucose), lipids, nucleic acids, vitamins (e.g., thiamine), essential and non-essential amino acids, ammonium salts, nitrates, minerals (e.g., phosphorus, sulfur, potassium, magnesium, iron, magnesium and their salts), trace metals (e.g., cobalt, copper, zinc, nickel and their salts), serum and its substitutes, animal proteins, animal-derived components (e.g., hydrolysates), plant proteins, plant-derived components (e.g., hydrolysates), microbial proteins, microbial-derived components (e.g., hydrolysates), nutrients / growth factors, hormones, enzyme cofactors, pH buffers, sodium chloride, calcium chloride, antibiotics, preservatives (e.g., antioxidants) and pH indicators (e.g., phenol red).

[0153] Non-limiting examples of essential and non-essential amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, cystine, hydroxyproline, thyroxine, phosphoserine, desmosine, β-alanine, sarcosine, ornithine, citrulline and γ-aminobutyric acid, and salts thereof, and hydrates thereof.

[0154] The concentration of essential and non-essential amino acids in the medium is appropriately selected according to the purpose of culture and the cells. When the medium is a liquid medium and is used as a feed medium, the total concentration of essential and non-essential amino acids contained in the medium is typically 10 to 1000 mM.

[0155] Non-limiting examples of pH buffers include sodium carbonate buffer, potassium carbonate buffer, sodium phosphate buffer, phosphate buffer, tris buffer, citric acid buffer, and Good's buffer (e.g., HEPES buffer and MES buffer). When the culture medium contains a pH buffer, the concentration of the pH buffer is typically 10 to 500 mM.

[0156] Substratum can be suitably selected from the known substratum that can be commercially available, document and put down in writing according to the cell of cultivation, purpose.Can change composition according to the purpose of cultivation, cell, or reagent, substratum additive are added to substratum or mix and use more than 2 kinds of substratum.In addition, in cell culture, the substratum, reagent and / or substratum additive that can be added can be added to substratum.

[0157] The culture medium can be a synthetic culture medium. In addition, the culture medium can be a serum-free culture medium, can be a culture medium without animal protein. In addition, the culture medium can be a liquid culture medium or a powder culture medium.

[0158] Non-limiting examples of commercially available culture media include Dulbecco's modified Eagle's medium (DMEM), DME / F12, Eagle's minimum essential medium (MEM), basic medium Eagle (BME), F-10 medium, F-12 medium, α-minimum essential medium (α-MEM), Glasgow's minimum essential medium (G-MEM), PF CHO medium, Iscove's modified Dulbecco's medium, AmpliCHO CD medium, Dynamis TM Culture medium, EX-CELL TM Advanced TM CHO fed-batch medium, CD FortiCHO TM Culture medium, CP OptiCHO TM 、BalanCD TM CHO Proliferation Medium A, ActiPro TM 、RPMI-1640 (Roswell Park Memorial Institute 1640) medium、CELLiST TM Basal Media (BASAL3, BASAL10) and Feed Media (FEED2), HyClone TM Cell Boost TM 7a Supplement, Cellvento TM Feed-200, Cellvento TM Feed-210, Cellvento TM Feed-220 and Cellvento TM 4CHO COMP BalanCD CHO Feed 4. These media can be used as cell culture media or feed media. In addition, these media are provided and sold in the form of liquid media and / or powder media.

[0159] The term "synthetic medium" as used herein refers to a medium prepared using only chemically defined components. Synthetic medium is typically a medium to which serum and its substitutes, unpurified animal protein, unpurified animal-derived components (e.g., hydrolyzates), unpurified vegetable protein, unpurified vegetable-derived components (e.g., hydrolyzates), unpurified microbial protein, and unpurified microbial-derived components (e.g., hydrolyzates) are not added.

[0160] The "serum-free medium" in the present disclosure refers to a medium that does not contain unregulated or unpurified serum. A medium mixed with purified blood-derived components or animal tissue-derived components (e.g., growth factors) is included in the serum-free medium as long as it does not contain unregulated or unpurified serum.

[0161] The "animal protein-free medium" in the present disclosure refers to a medium that does not contain animal proteins (e.g., proteins of animal origin such as albumin, transferrin, insulin, and growth factors). The animal protein-free medium may contain small peptides and / or oligopeptides.

[0162] The "powdered culture medium" in the present disclosure refers to a culture medium in powder form. Powdered culture medium also includes culture medium in granular form. In addition, powdered culture medium also includes dry culture medium. Powdered culture medium can be dissolved in an appropriate solvent (e.g., water) as a liquid culture medium. Any culture medium in the present disclosure can be prepared or provided in the form of a powdered culture medium.

[0163] The "liquid culture medium" in the present disclosure refers to a culture medium in liquid form. Any culture medium in the present disclosure can be prepared or provided in the form of a liquid culture medium. In addition, the liquid culture medium can be prepared by dissolving any powder culture medium in the present disclosure in an appropriate solvent (e.g., water). The liquid culture medium can be used as a feed medium.

[0164] The "recombinant protein" in the present disclosure refers to a polymer of amino acids of any length produced using any genetic recombination technology. The polymer may be linear or branched, may contain modified amino acids, or may contain components other than amino acids. In addition, the recombinant protein may be chemically or biologically modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, amidation, derivatization based on protecting groups, etc., cleavage, or binding to a labeled component (e.g., a radioactive isotope) or a drug (e.g., a toxin, a cytotoxic drug, or a radioactive isotope). All molecules that contain a recombinant protein portion in a part of their structure are included in the recombinant protein in the present disclosure. Genetic recombination technology is not particularly limited as long as it is a technology for artificially manipulating genes. As a typical example, the introduction of nucleic acids encoding recombinant proteins into host cells can be cited.

[0165] As non-limiting examples of recombinant proteins, antibodies and antigen-binding fragments thereof, antibody mimetics, immunoadhesins, enzymes, hormones, cytokines, coagulation factors (or blood coagulation-related proteins), extracellular proteins, and membrane proteins produced using genetic recombination techniques can be cited. These recombinant proteins are mainly used in medicine, pesticides, food, and other chemical industry fields, but are not limited to specific uses.

[0166] "Antibody" in the present disclosure refers to an immunoglobulin molecule that has the ability to bind to an antigen. Antibodies include full-length antibodies and fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, and VHH fragments) having polypeptide chains containing light chains and heavy chains. Antibodies may include heavy chains and / or light chains. Heavy chains and / or light chains may include variable regions (involved in the recognition and binding of antigens) and constant regions (involved in localization and cell-to-cell interactions). The most common full-length antibodies include 2 heavy chain constant regions (CH), 2 heavy chain variable regions (VH), 2 light chain constant regions (CL), and 2 light chain variable regions (VL). The variable region includes complementarity determining regions (CDRs) and framework regions (FRs) that are sequences that confer antigen specificity to antibodies.

[0167] Antibodies include monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed by two or more antibodies or antigen-binding sites, antibody fragments with desired biological activity, and fusion proteins comprising the antigen-binding sites of antibodies. In addition, antibodies include chimeric antibodies, mammalian antibodies (e.g., human antibodies), mammalized antibodies (e.g., humanized antibodies), multivalent antibodies, and modified antibodies.

[0168] The antibodies can be of any class (eg, IgG, IgA, IgM, IgD, IgE), and of any subclass (isotype).

[0169] Antibodies may be chemically or biologically modified by binding to other molecules (eg, low-molecular or high-molecular toxins (cytotoxic drugs) or radioactive isotopes), and the antibodies of the present invention include antibodies that have undergone such modifications.

[0170] "Antibody mimics" in the present disclosure refer to proteins that can specifically bind to antigens but are not structurally related to antibodies. Specific examples of antibody mimics include the Z domain of protein A (affine; Affibody), and more specific examples include ZHER2 affibody.

[0171] As examples of the enzyme, there can be mentioned alkaline phosphatase, β-lactamase, β-galactosidase, glucosidase, glucocerebrosidase, superoxide dismutase and DNase.

[0172] Examples of hormones include renin, growth hormone (human growth hormone or bovine growth hormone, etc.), parathyroid hormone, thyroid stimulating hormone, calcitonin, gonadotropin (e.g., follicle stimulating hormone, luteinizing hormone), glucagon, insulin (insulin A chain and insulin B chain), and proinsulin.

[0173] Examples of cytokines include tumor necrosis factor (e.g., TNF-α and β), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neurotrophic factors (e.g., NT-3 to NT-6), nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (e.g., aFGF, bFGF), epidermal growth factor (EGF), transforming growth factor (e.g., TGF-α, TGF-β1 to TGF-β5), activin (e.g., Activin A, activin C, activin E), insulin-like growth factors (e.g., IGF-I, IGF-II), hepatocyte growth factor (HGF), keratinocyte growth factor, stem cell factor (SCF), osteogenic protein (BMP), RANTES, erythropoietin (EPO), thrombopoietin (TPO), interferon-α, -β and -γ, monocyte chemotactic factors (e.g., M-CSF, GM-CSF, G-CSF) and interleukins (e.g., IL-1~IL-13).

[0174] Examples of coagulation factors and blood coagulation-related proteins include factor VII, factor VIII, factor VIIIC, factor IX, factor X, factor XI, factor XII, tissue factor, von Willebrand factor, protein C, protein S, plasminogen activators (e.g., urokinase, human urine or human tissue plasminogen activator (t-PA)), thrombin, prothrombin, thrombopoietin, thrombomodulin, antithrombin III, fibrinogen and serum albumin.

[0175] Examples of the extracellular protein include fibronectin, vitronectin, collagen, osteopontin, laminin, and fragments thereof.

[0176] Examples of membrane proteins include membrane receptor proteins, membrane transport proteins, membrane enzymes, cell adhesion factors, and fragments thereof.

[0177] The recombinant protein is not limited to the above examples, for example, the recombinant protein also includes receptors such as the cytokines, transferrin, various CD proteins, tumor-associated antigens (such as HER2, HER3 or HER4 receptors, CA125), viral antigens for vaccines (such as viral envelope proteins), etc. In addition, the recombinant protein also includes a fusion protein containing any of the above proteins.

[0178] In the present disclosure, "having the ability to produce recombinant proteins" means having the ability to produce proteins by genetic recombination technology. The produced recombinant proteins may be secreted outside the cells (ie, in the culture medium) or accumulated inside the cells, preferably secreted outside the cells.

[0179] Typically, by introducing a nucleic acid encoding a recombinant protein into a host cell, the host cell can be endowed with the ability to produce a recombinant protein. That is, a typical cell having the ability to produce a recombinant protein is a cell comprising a nucleic acid encoding a recombinant protein. Various methods are known in the art as methods for introducing nucleic acids into host cells. In the present disclosure, these various methods are included, and known methods can be used arbitrarily at the time point of implementing the present invention. As a non-limiting example of such a method, a method of introducing a vector comprising a nucleic acid encoding a recombinant protein into a host cell can be cited. The vector is preferably an expression vector comprising a nucleic acid encoding a recombinant protein in a manner that can be expressed (e.g., a manner that can be operably connected to regulatory sequences such as promoters, enhancers, ribosome binding sequences and / or transcription termination sequences), and can be a plasmid.

[0180] As a non-limiting example of a method for introducing a vector into a host cell, a method for physically or chemically introducing a vector into a host cell and a method for infecting a host cell with a viral vector can be cited by electroporation, gene gun method, calcium phosphate method or lipofection method. In addition, the nucleic acid in the vector introduced into the host cell can be introduced into the genome in the host cell by genome editing techniques such as CRISPR / Cas9. Alternatively, instead of introducing a nucleic acid encoding a recombinant protein into the host cell, the gene sequence on the genomic DNA can be directly modified by genome editing techniques such as CRISPR / Cas9 to encode the target recombinant protein, or the production of the target endogenous protein can be controlled by modifying the control sequence, etc. (for example, it can be increased). The recombinant protein in the present disclosure includes all proteins produced in this way.

[0181] The "cell" in the present disclosure includes animal cells, plant cells, bacterial cells and fungal cells. The cell is preferably a strain of cells suitable for the production of recombinant proteins.

[0182] The animal cells may be cells of any animal origin, preferably cells of mammal (mammals), birds, amphibians, fish or insect origin, more preferably cells of mammal or insect origin, and most preferably cells of mammal origin.

[0183] Non-limiting examples of mammal-derived cells include cells derived from primates such as humans and monkeys, and cells derived from rodents such as mice, rats, and hamsters.

[0184] Non-limiting examples of mammalian-derived cells include cell lines such as HL-60 (ATCC No. CCL-240), HT-1080 (ATCC No. CCL-121), HeLa (ATCC No. CCL-2), 293 (ECACC No. 85120602), Hep G2 (ATCC No. HB8065), VERO (ATCC No. CCL-1651), CV1 (ATCC No. CCL70), COS-7 (ATCC No. CRL-1651), NIH3T3 (ATCC No. CRL-1658), NS0 (ATCC No. CRL-1827), Chinese hamster ovary (CHO) cells, BHK21 (also referred to as BHK cells. ATCC No. CRL-10), and MDCK (ATCC No. CCL-34), and substrains induced from these cell lines (for example, BHK substrains of BHK cells). TK-cells, CHO-K1 cells which are a substrain of CHO cells, CHO-S cells, CHO-DXB11 cells (also called CHO-DUKX cells or DuxB11 cells), and CHO-DG44 cells).

[0185] Non-limiting examples of bird-derived cells include chicken cell line SL-29, non-limiting examples of amphibian-derived cells include Xenopus laevis oocytes, and non-limiting examples of fish-derived cells include zebrafish cell line ZF4.

[0186] Non-limiting examples of cells derived from insects include cells derived from Spodoptera frugiperda. Non-limiting examples of cells derived from Spodoptera frugiperda include Sf9 cells, Sf21 cells, and SF+ cells.

[0187] Plant cells are not particularly limited as long as the collected cells and / or tissues can be cultured in liquid. Non-limiting examples of plant cells include plants that produce drugs (e.g., saponins, alkaloids, berberine, scopolamine, or phytosterols) (e.g., medicinal ginseng, catharanthus roseus, henbane, coptis root, or belladonna), plants that produce pigments and polysaccharides (e.g., anthocyanins, safflower pigments, madder pigments, saffron pigments, or flavonoids) as raw materials for cosmetics / foods (e.g., blueberries, safflower, madder, or saffron), or plants that produce raw materials for drugs.

[0188] Bacterial cells are cells of true bacteria (Gram-positive or Gram-negative) or archaea. Non-limiting examples of bacterial cells include bacterial cells of the genus Escherichia (e.g., Escherichia coli), bacterial cells of the genus Bacillus (e.g., Bacillus subtilis), bacterial cells of the genus Pseudomonas (e.g., Pseudomonas fluorescens), bacterial cells of the genus Corynebacterium, and bacterial cells of the genus Lactococcus (e.g., Lactococcus lactis).

[0189] Fungal cells are large cells with differentiated organelles and a typical nucleus belonging to eukaryotic organisms. Non-limiting examples of fungal cells include fungi of the genus Saccharomyces (e.g., Saccharomyces cerevisiae), fungi of the genus Schizosaccharomyces (e.g., Schizosaccharomyces pombe), and fungi of the genus Pichia (e.g., Pichia pastoris).

[0190] "L-cysteine" in the present disclosure includes L-cysteine ​​salts, hydrates of L-cysteine, and hydrates of L-cysteine ​​salts that function as L-cysteine ​​in a solution (e.g., a solution using water, a buffer solution, or a culture medium as a solvent). However, when referring to the concentration of L-cysteine, the concentration refers to the concentration of L-cysteine ​​that functions in a solution.

[0191] The concentration of L-cysteine ​​in the solution is typically expressed as the concentration of L-cysteine ​​at the time point when the solution is prepared (preferably a calculated value, but may also be a measured value immediately after preparation). However, in the case of referring to the concentration change over time after the preparation of the solution, the concentration of L-cysteine ​​refers to the concentration of L-cysteine ​​at the time point after a given time has passed since the preparation of the solution.

[0192] As non-limiting examples of L-cysteine ​​salts, L-cysteine ​​hydrochloride, L-cysteine ​​sodium salt and L-cysteine ​​ammonium salt can be mentioned. As non-limiting examples of L-cysteine ​​hydrates, L-cysteine ​​hydrochloride monohydrate, L-cysteine ​​sodium salt monohydrate and L-cysteine ​​ammonium salt monohydrate can be mentioned. As L-cysteine, multiple types of L-cysteine, L-cysteine ​​salts and hydrates thereof can be used in combination.

[0193] "N-acetylcysteine" in the present disclosure includes N-acetylcysteine ​​salts, hydrates of N-acetylcysteine, and hydrates of N-acetylcysteine ​​salts that function as N-acetylcysteine ​​in a solution (e.g., a solution using water, a buffer solution, or a culture medium as a solvent). However, when referring to the concentration of N-acetylcysteine, the concentration refers to the concentration of N-acetylcysteine ​​that functions in a solution.

[0194] N-acetylcysteine ​​may be in the L form or the D form. Furthermore, it may be a mixture of the L form and the D form.

[0195] The concentration of N-acetylcysteine ​​in a solution is typically expressed as the concentration of N-acetylcysteine ​​at the time when the solution is prepared (preferably a calculated value, but may also be a measured value immediately after preparation). However, when referring to the concentration change over time after the preparation of a solution, the concentration of N-acetylcysteine ​​refers to the concentration of N-acetylcysteine ​​at a time when a given time has passed after the preparation of the solution.

[0196] As non-limiting examples of N-acetylcysteine ​​salts, N-acetylcysteine ​​sodium salt and N-acetylcysteine ​​ammonium salt can be cited. As non-limiting examples of L-cysteine ​​hydrates, N-acetylcysteine ​​sodium salt monohydrate and N-acetylcysteine ​​ammonium salt monohydrate can be cited. As N-acetylcysteine, multiple types of N-acetylcysteine, N-acetylcysteine ​​salts and hydrates thereof can be used in combination.

[0197] "Heat load" in the present disclosure refers to heat at a temperature that can adversely affect the culture medium when applied to the culture medium during preparation, storage and / or cell culture. Non-limiting examples of such adverse effects include precipitation of culture medium components in the culture medium during preparation, storage and / or cell culture, and reduction in the yield and / or quality of recombinant proteins produced in the culture medium. The heat at a temperature that can adversely affect the culture medium can be a temperature that can be normally used for the culture medium during preparation, storage and / or cell culture (typically, 4 to 40°C), and can be a temperature that includes higher temperatures (e.g., 4°C to 120°C).

[0198] "Stability" in the present disclosure refers to the property that, when a culture medium is used, physical or chemical changes do not occur or are not likely to occur during preparation, storage, and / or cell culture. For example, when the production rate of byproducts in the culture medium (e.g., cystine generated from L-cysteine) is slow, when a culture medium component (e.g., cystine) is not precipitated, or when the time until precipitation (number of days, etc.) is long, the stability of the culture medium can be considered to be high.

[0199] The term "medium additive" as used herein refers to any substance or composition containing a substance added to a medium. A medium additive may be a substance that supplements components already present in an added medium, or may be a substance that newly adds components not present in an added medium. Typically, medium additives are provided in a liquid form or a powder form (including a granular form). In addition, the purpose of addition is not particularly limited.

[0200] As used in this disclosure, the singular forms "a," "an," and "the" may include plural referents unless expressly stated otherwise.

[0201] “And / or” used in the present disclosure includes both the relationship represented by “and” and the relationship represented by “or”. “And” is sometimes expressed as “and” or “as well as”. “Or” is sometimes expressed as “or”. “Include” in the present disclosure includes: “mainly include”, “substantially include” and “consist of”, and “mainly include” includes: “substantially include” and “consist of”, and “substantially include” includes: “consist of”.

[0202] For each numerical range in the present disclosure, the upper limit and lower limit indicated by "~" and "to" are included respectively. For example, the record of "A~B" or "A to B" using the numerical values ​​A and B means that it is above A and below B. In addition, the records of "A~B", "A to B" or "above A and below B" in the numerical ranges recorded in stages in the present disclosure are independent, including both "preferably above A" and "preferably below B", and these lower limits or upper limits can be replaced by the upper limits or lower limits of other numerical ranges. In addition, the lower limit or upper limit of the numerical range recorded in the present disclosure is a numerical value within the numerical range, and can also be replaced by the numerical value shown in the embodiments.

[0203] [Implementation Method]

[0204] Hereinafter, the present invention will be described in detail using specific embodiments of the present invention, but the present invention is not limited thereto.

[0205] These embodiments may be implemented individually or in combination. For the definitions and details in the embodiments, please refer to the above "Explanation of Terms".

[0206] With respect to the well-known techniques and steps used in the present disclosure, those skilled in the art can fully understand and implement them according to routine methods.

[0207] In one embodiment, a feed medium (or liquid culture medium) comprising L-cysteine ​​and N-acetylcysteine ​​is provided. In addition, in other embodiments, a powdered culture medium comprising N-acetylcysteine ​​and L-cysteine ​​for preparing a feed medium (or liquid culture medium) is provided. In addition, in other embodiments, a method for preparing a feed medium (or liquid culture medium) or a powdered culture medium is provided. In addition, in other embodiments, a culture medium additive for preparing a feed medium (or liquid culture medium) or a powdered culture medium is provided. In these embodiments, the liquid culture medium is preferably a feed medium.

[0208] In the liquid culture medium of these embodiments (hereinafter, a feed medium in the form of a liquid culture medium),

[0209] (1) the concentration of L-cysteine ​​is 1 to 40 mM, preferably 10 to 40 mM, 10 to 30 mM or 15 to 40 mM, more preferably 15 to 30 mM, and / or the concentration of N-acetylcysteine ​​is 1 to 40 mM, preferably 1 to 30 mM, 1 to 20 mM or 1 to 10 mM, more preferably 2 to 10 mM, and / or

[0210] (2) The molar ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 2.0, preferably 0.04 to 1.0, 0.04 to 0.4, 0.1 to 2.0, 0.1 to 1.0 or 0.1 to 0.4, and more preferably 0.2 to 0.4.

[0211] The liquid culture medium in these embodiments preferably suppresses the formation and / or precipitation of cystine from L-cysteine ​​by containing N-acetylcysteine ​​at a suitable concentration and ratio, and / or improves stability. In addition, the liquid culture medium in these embodiments preferably contains a high concentration of L-cysteine ​​that is beneficial to the growth or proliferation of cultured cells. By using such a liquid culture medium, the process of cell culture can be stably implemented. In addition, it is preferred to reduce the cost of cell culture. In addition, by using such a liquid culture medium in the production of recombinant protein, the productivity of the product using the recombinant protein can be improved. In addition, it is preferred to reduce the manufacturing cost of the product using the recombinant protein. In addition, preferably, the research and development efficiency of the product using the recombinant protein is improved.

[0212] The liquid culture medium in these embodiments is not limited to a specific composition as long as it contains L-cysteine ​​and N-acetylcysteine, and any liquid culture medium can be used. The pH of the liquid culture medium can be 5.0 to 9.0, preferably 6.0 to 8.0, more preferably 6.5 to 7.5, and most preferably 6.5 to 7.0. The liquid culture medium can be a serum-free medium and / or an animal protein-free medium. In addition, the liquid culture medium can be a synthetic medium. The liquid culture medium can be used for large-scale processes (e.g., cell culture on a 500L scale).

[0213] When the liquid culture medium in these embodiments is used in the culture of cells with the ability to produce recombinant proteins, the ability to produce recombinant proteins can be improved. That is, when the various conditions of cell culture are optimized, typically, 1 to 20 g / L of recombinant protein can be produced, preferably 2 to 16 g / L, more preferably 6 to 14 g / L, further more preferably 8 to 12 g / L, and most preferably 10 to 12 g / L of recombinant protein. In addition, the components of the liquid culture medium are preferably not precipitated due to heat load during preparation, storage and / or cell culture.

[0214] The powder culture medium in these embodiments comprises L-cysteine ​​and N-acetylcysteine. As long as the powder culture medium comprises L-cysteine ​​and N-acetylcysteine, it is not limited to a specific composition, and any powder culture medium can be used. The powder culture medium can be dissolved in a solvent (e.g., water) and used to prepare the liquid culture medium of the embodiments of the present disclosure. In this case, the powder culture medium comprises L-cysteine ​​and N-acetylcysteine ​​in an amount such that the concentration of L-cysteine ​​and N-acetylcysteine ​​in the liquid culture medium becomes the desired concentration. The powder culture medium can be accompanied by instructions or instructions on a website for diluting the concentration of L-cysteine ​​and N-acetylcysteine ​​in the liquid culture medium to the desired concentration.

[0215] Powdered media are generally easier to handle than liquid media, and are advantageous in that, for example, they can be stored more stably and can be circulated.

[0216] The method for preparing a liquid culture medium or a powder culture medium comprises: mixing L-cysteine, N-acetylcysteine ​​and components necessary for cell proliferation and survival, useful components, etc., to obtain any liquid culture medium or powder culture medium in the embodiments of the present disclosure. As L-cysteine ​​and / or N-acetylcysteine, any culture medium additive or reagent in the embodiments of the present disclosure can be used. According to the method, a person skilled in the art can appropriately prepare a liquid culture medium or a powder culture medium.

[0217] The culture medium additive contains L-cysteine ​​and / or N-acetylcysteine. The culture medium additive can be added to any culture medium of the cell culture medium and the feed medium, or can be added to both culture media. As long as the culture medium additive contains L-cysteine ​​and / or N-acetylcysteine, it is not limited to a specific composition, and any composition can be used. The culture medium additive can contain only L-cysteine ​​and / or N-acetylcysteine, or can be a composition containing a carrier, a pH adjusting agent, a culture medium, etc. The culture medium additive can be in liquid form, or can be in solid form, such as powder form (including granular form).

[0218] The culture medium additive can be added to any liquid culture medium. In this case, as far as the culture medium additive is concerned, typically, it includes L-cysteine ​​and / or N-acetylcysteine ​​in an amount that makes the concentration of L-cysteine ​​and N-acetylcysteine ​​in the liquid culture medium become the desired concentration. In addition, the culture medium additive can be added to any powder culture medium. In this case, as far as the culture medium additive is concerned, typically, it includes L-cysteine ​​and / or N-acetylcysteine ​​in an amount that makes the concentration of L-cysteine ​​and N-acetylcysteine ​​in the liquid culture medium become the desired concentration when the powder culture medium added with the culture medium additive is dissolved in a solvent (e.g., water) to prepare the liquid culture medium.

[0219] Furthermore, the medium supplement may be accompanied by instructions or instructions on a website for diluting the concentrations of L-cysteine ​​and N-acetylcysteine ​​in the liquid medium to a desired concentration.

[0220] The liquid culture medium or powder culture medium according to the embodiment of the present disclosure can be obtained by adding culture medium additives.

[0221] The medium additive is used to prepare a liquid medium or a powder medium. The use is preferably described in a sales brochure, an operating manual, etc. of the medium additive. In this case, the use can be described as an effect of the medium additive.

[0222] In one embodiment, a method for culturing cells is provided, comprising: adding a liquid culture medium of an embodiment of the present disclosure to a culture medium for culturing cells. In addition, in other embodiments, a method for producing a recombinant protein is provided, comprising: adding a liquid culture medium of an embodiment of the present disclosure to a culture medium for culturing cells capable of producing a recombinant protein.

[0223] For the culture medium of culturing cells, typically, it is a culture medium placed in a culture container in cell culture. In addition, for the culture medium of culturing cells, typically, it is a basal medium or a basal medium with a feed medium added thereto. As the culture medium and feed medium for culturing cells, any liquid culture medium (including a liquid culture medium prepared by a powdered culture medium and a liquid culture medium with a culture medium additive) in the embodiments of the present disclosure can be used respectively. For the liquid culture medium added to the culture medium for culturing cells, typically, it is a feed medium.

[0224] The method for culturing cells preferably enables stable cell culture by using a medium with improved stability and / or a medium in which the formation and / or precipitation of cystine is suppressed. In addition, the method for culturing cells preferably reduces the cost of culture.

[0225] The method for producing a recombinant protein can improve the productivity of a product using the recombinant protein by using a medium with improved stability and / or a medium in which the formation and / or precipitation of cystine is suppressed. In addition, the method for producing a recombinant protein preferably reduces the manufacturing cost of the product using the recombinant protein. In addition, the method for producing a recombinant protein preferably contributes to the efficiency of the research and development of the product using the recombinant protein.

[0226] The cell can be an animal cell, a plant cell, a bacterial cell or a fungal cell, preferably an animal cell, more preferably a mammalian cell, and even more preferably a Chinese hamster ovary (CHO) cell. In addition, the cell can express a protein. In terms of protein, typically, it is a recombinant protein. The recombinant protein can include an antibody or an antigen-binding fragment thereof. The antibody or its antigen-binding fragment can be derived from IgG.

[0227] The number of times of adding liquid culture medium to the culture medium of cultured cells is not particularly limited, and can be once or multiple times. The period of adding liquid culture medium to the culture medium of cultured cells is not particularly limited, and can be any period before the start of culture, when the culture starts, and after the start of culture, or multiple periods. In addition, liquid culture medium can be added to the cells of cultured cells continuously, or can be added to the cells of cultured cells in stages. In addition, liquid culture medium can be added to the culture medium of cultured cells alone, or can be added to the culture medium of cultured cells together with other culture mediums, reagents or culture medium additives. In addition, when adding liquid culture medium, an equal amount of culture solution can be taken out at the same time.

[0228] The amount of the liquid medium added to the culture medium for culturing cells is typically 1 to 20%, preferably 2 to 10%, of the amount of the culture medium for culturing cells. The concentration of L-cysteine ​​in the culture medium for culturing cells after adding the liquid medium is typically 0.01 to 8 mM, preferably 0.02 to 4 mM. The concentration of N-acetylcysteine ​​in the culture medium for culturing cells after adding the liquid medium is typically 0.01 to 8 mM, preferably 0.02 to 4 mM.

[0229] The method for culturing cells and the method for producing a recombinant protein can be used for large-scale processes (eg, 500 L scale cell culture).

[0230] The method for culturing cells may include: preparing a culture medium. In addition, the method for culturing cells may include: culturing cells. As for the method for culturing cells, typically, a fed-batch culture method is used, and other culture methods (such as continuous culture methods) may also be used.

[0231] The method for producing a recombinant protein may include: preparing a culture medium. In addition, the method for producing a recombinant protein may include: culturing cells. In addition, the method for producing a recombinant protein may include: producing a recombinant protein. In addition, the method for producing a recombinant protein may include: recovering the produced recombinant protein. In addition, the method for producing a recombinant protein may include other additional steps. As non-limiting examples of additional steps, purification of recombinant proteins and chemical or biological modification of recombinant proteins can be cited. In the purification of recombinant proteins, a combination of multiple methods is usually used, and chromatography is preferably used. In particular, when the recombinant protein is an antibody, affinity chromatography, protein A chromatography, etc. are preferably used.

[0232] As non-limiting examples of chemical or biological modifications of recombinant proteins, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, amidation, derivatization based on protecting groups, cleavage, or binding with labeling components or drugs (toxins, etc.) can be cited. The method for producing a recombinant protein also includes a method for producing an antibody bound to a drug such as a toxin. In this case, the method for producing a recombinant protein may further include, as an additional step, binding the antibody to the drug. The recombinant protein disclosed herein includes an antibody bound to a drug.

[0233] According to the method for producing a recombinant protein disclosed herein, a recombinant protein with a higher production amount can be produced. That is, according to the method for producing a recombinant protein disclosed herein, when the culture conditions are optimized, typically, 1 to 20 g / L of recombinant protein can be produced, preferably 2 to 16 g / L, more preferably 6 to 14 g / L, further more preferably 8 to 12 g / L, and most preferably 10 to 12 g / L of recombinant protein can be produced. In addition, according to the method for producing a recombinant protein disclosed herein, the culture medium components of the liquid culture medium preferably do not precipitate due to heat load during preparation, storage and / or cell culture.

[0234] In one embodiment, a method or reagent for improving the stability of a liquid culture medium (or a feed medium) is provided, comprising: adding L-cysteine ​​and / or N-acetylcysteine ​​to the culture medium. In other embodiments, a method or reagent for inhibiting the generation and / or precipitation of cystine in a liquid culture medium (or a feed medium) is provided, comprising: adding L-cysteine ​​and / or N-acetylcysteine ​​to the culture medium. In other embodiments, the use of L-cysteine ​​and / or N-acetylcysteine ​​for improving the stability of a liquid culture medium (or a feed medium) is provided. In other embodiments, the use of L-cysteine ​​and / or N-acetylcysteine ​​for inhibiting the generation and / or precipitation of cystine in a liquid culture medium (or a feed medium) is provided. The liquid culture medium in these embodiments is preferably a feed medium.

[0235] In these embodiments, L-cysteine ​​and / or N-acetylcysteine ​​are added to the liquid culture medium such that:

[0236] (1) the concentration of L-cysteine ​​is 1 to 40 mM, preferably 10 to 40 mM, 10 to 30 mM or 15 to 40 mM, more preferably 15 to 30 mM, and / or the concentration of N-acetylcysteine ​​is 1 to 40 mM, preferably 1 to 30 mM, 1 to 20 mM or 1 to 10 mM, more preferably 2 to 10 mM; and / or

[0237] (2) the molar ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 2.0, preferably 0.04 to 1.0, 0.04 to 0.4, 0.1 to 2.0, 0.1 to 1.0 or 0.1 to 0.4, more preferably 0.2 to 0.4,

[0238] Or the concentration or ratio in the liquid culture medium after adding the reagent is within the range.

[0239] According to the methods, reagents or uses of these embodiments, the stability of the liquid culture medium containing L-cysteine ​​can be improved by including N-acetylcysteine ​​at an appropriate concentration and a certain ratio, and / or the formation and / or precipitation of cystine in the liquid culture medium can be inhibited. Therefore, according to the methods, reagents or uses of these embodiments, industrial-scale cell culture can be stably implemented.

[0240] In addition, when the methods, reagents or uses of these embodiments are used in the production process of recombinant proteins, the productivity of products using recombinant proteins can be improved. In addition, it is preferred to reduce the manufacturing cost of products using recombinant proteins. In addition, it is preferred to efficiently carry out research and development of products using recombinant proteins.

[0241] With regard to the methods, reagents or uses of these embodiments, the stability of the liquid culture medium containing L-cysteine ​​and N-acetylcysteine ​​can be improved compared to the liquid culture medium containing L-cysteine ​​but not containing N-acetylcysteine. The improvement of the stability of the liquid culture medium can be evaluated by a known method, or it can be that the improvement of stability is the goal without evaluation. As a non-limiting example of a method for evaluating stability, the precipitation of the culture medium components (such as cystine) can be detected by visual or spectral means (such as a turbidimeter), the method for evaluating stability by the time (days, etc.) until precipitation occurs, and the method for evaluating stability by using a physicochemical analysis means to analyze the concentration changes of the culture medium components (such as cystine). As a non-limiting example of a physicochemical analysis means, the means of using a liquid chromatography-tandem mass spectrometer, such as described in International Publication No. 2021 / 060517, can be cited.

[0242] In another embodiment of the present disclosure, with respect to the method or reagent for improving the stability of the liquid culture medium, the time (number of days, etc.) until precipitation occurs in the liquid culture medium containing L-cysteine ​​and N-acetylcysteine ​​is prolonged compared with the liquid culture medium containing L-cysteine ​​but not containing N-acetylcysteine. The prolonged time is, for example, more than 1 day, preferably more than 5 days, more preferably more than 10 days, and further more preferably more than 15 days. In addition, in another embodiment, with respect to the method, reagent or use for inhibiting the generation and / or precipitation of cystine in the liquid culture medium, the generation rate of the byproduct (e.g., cystine) in the liquid culture medium containing L-cysteine ​​and N-acetylcysteine ​​is reduced by more than 10%, preferably more than 20%, more preferably more than 30%, and further more preferably more than 40% compared with the liquid culture medium containing L-cysteine ​​but not containing N-acetylcysteine. In another embodiment, in the method, reagent or use for inhibiting the production and / or precipitation of cystine in a liquid culture medium, a by-product (e.g., cystine) is not precipitated in a liquid culture medium containing L-cysteine ​​and N-acetylcysteine ​​for a period of 2 weeks or more, preferably 3 weeks or more, more preferably 1 month or more, further more preferably 2 months or more, and most preferably 3 months or more.

[0243] For the method, reagent or use for suppressing the generation and / or precipitation of cystine in the liquid culture medium, the generation and / or precipitation of cystine in the liquid culture medium containing L-cysteine ​​and N-acetylcysteine ​​can be suppressed compared with the liquid culture medium containing L-cysteine ​​but not containing N-acetylcysteine. The generation and / or precipitation of cystine in the liquid culture medium can be evaluated by a known method, or it can be not evaluated and only the generation and / or precipitation of cystine in the liquid culture medium can be targeted. As a non-limiting example of the method for evaluating the generation and / or precipitation of cystine, the precipitation of cystine can be detected by visual or spectral means (such as turbidimeter), the method for evaluating the generation and / or precipitation of cystine by the time (days, etc.) until precipitation occurs, and the method for evaluating the generation and / or precipitation of cystine by using physicochemical analysis means to analyze the concentration change of cystine and the method for evaluating the generation and / or precipitation of cystine. As a non-limiting example of the physicochemical analysis means, for example, there can be mentioned a means using liquid chromatography-tandem mass spectrometry as described in International Publication No. 2021 / 060517.

[0244] In another embodiment of the present disclosure, with respect to the method, reagent or use for inhibiting the generation and / or precipitation of cystine in a liquid culture medium, the number of days until cystine is precipitated in a liquid culture medium containing L-cysteine ​​and N-acetylcysteine ​​is prolonged compared to a liquid culture medium containing L-cysteine ​​but not containing N-acetylcysteine. The prolonged time is, for example, more than 1 day, preferably more than 5 days, more preferably more than 10 days, and further more preferably more than 15 days. In addition, in another embodiment, with respect to the method, reagent or use for inhibiting the generation and / or precipitation of cystine in a liquid culture medium containing L-cysteine ​​and N-acetylcysteine, the rate of generation and / or precipitation of cystine in a liquid culture medium containing L-cysteine ​​and N-acetylcysteine ​​is reduced by more than 10%, preferably more than 20%, more preferably more than 30%, and further more preferably more than 40% compared to a liquid culture medium containing L-cysteine ​​but not containing N-acetylcysteine.

[0245] The pH of the liquid culture medium after adding L-cysteine ​​and / or N-acetylcysteine ​​to the culture medium may be 5.0 to 9.0, preferably 6.0 to 8.0, more preferably 6.5 to 7.5, and most preferably 6.5 to 7.0. The characteristics of the liquid culture medium after adding L-cysteine ​​and / or N-acetylcysteine ​​to the culture medium may be the same as the characteristics of the liquid culture medium described in any one of the present disclosures.

[0246] Adding L-cysteine ​​and / or N-acetylcysteine ​​to a liquid culture medium includes all of the following: (1) adding L-cysteine ​​and / or N-acetylcysteine ​​or a composition comprising these to a liquid culture medium; (2) mixing L-cysteine ​​and / or N-acetylcysteine ​​or a composition comprising these with other components or other compositions (culture medium, etc.) comprising other components, and then adding the mixture to a culture medium; and (3) mixing N-acetylcysteine ​​or a composition comprising these with other components or other compositions (culture medium, etc.) comprising other components to prepare a culture medium.

[0247] The liquid culture medium to which L-cysteine ​​and / or N-acetylcysteine ​​or the reagent is added may contain L-cysteine ​​and / or N-acetylcysteine ​​before addition. In this case, the desired concentration of L-cysteine ​​and N-acetylcysteine ​​in the liquid culture medium may be achieved after addition of L-cysteine ​​and / or N-acetylcysteine ​​or the reagent.

[0248] The reagent is not limited to a specific form as long as it contains L-cysteine ​​and / or N-acetylcysteine, and may be in the form of a composition containing only L-cysteine ​​and / or N-acetylcysteine, or may be in the form of a carrier, a pH adjuster, a culture medium, etc. In addition, the reagent may be in a liquid form or a solid form, such as a powder form (including a granular form).

[0249] The reagent preferably has the purpose of improving the stability of the liquid culture medium or inhibiting the generation and / or precipitation of cystine in the liquid culture medium. The purpose is preferably recorded in the sales brochure, operating instructions, website related to the product, etc. of the reagent. In this case, the purpose can be recorded as the effect of the reagent.

[0250] It should be noted that the culture medium, method, reagent, additive, use, etc. described in the embodiments are not limited to the present invention, but are disclosed for illustration. The technical scope of the present invention is determined by the description of the patent claims, and those skilled in the art can make various design changes within the scope of the invention described in the scope of the patent claims.

[0251] For example, in the description of the above embodiments, the description is centered on the feed medium, but the embodiments of the present disclosure are not limited to the feed medium, and can be applied to any liquid medium that is not used for feeding, etc. That is, other embodiments of the present disclosure are any of the above embodiments in which the feed medium is replaced with a liquid medium (including a medium that is not a feed medium).

[0252] In addition, for example, in one embodiment, a recombinant protein produced in an embodiment of the present disclosure is provided. In addition, another embodiment relates to a pharmaceutical composition or drug comprising the recombinant protein, the use of the recombinant protein in the manufacture of a drug, or a method for treating a disease comprising the step of administering the recombinant protein. In addition, another embodiment relates to a method for producing a pharmaceutical composition or a pharmaceutical preparation, which comprises any of the steps in the embodiments of the present disclosure or any of the methods in the embodiments of the present disclosure, the production method comprising: thereby obtaining a recombinant protein, and the production method further comprising: mixing the recombinant protein with a carrier.

[0253] Example

[0254] Hereinafter, the present invention is described by way of examples, which are not intended to limit the present invention. It should be noted that commercially available reagents, equipment, etc. mentioned in the examples were used according to the manufacturer's instructions or standard methods unless otherwise specified.

[0255] [Example 1]

[0256] Effect of N-acetylcysteine ​​on the liquid stability of L-cysteine ​​high concentration fed medium

[0257] CELLiST TM Feed media (AJINOMOTO Co., Ltd.: FEED2) was used as the base to prepare Feed medium A. Feed medium A does not contain N-acetylcysteine.

[0258] To Feed medium A containing L-cysteine ​​at a final concentration of 18 to 26 mM and glucose at 60 g / L, N-acetylcysteine ​​at a final concentration of 0 to 5.5 mM was added, and a liquid medium at pH 6.7 to 6.9 was prepared according to the procedure described in the product manual of FEED2. The prepared liquid medium was filtered using 150 mL of Stericup-GP (Merck: S2GPU01RE) per 200 mL, and stored at 4°C or 25°C under light-shielding conditions for 18 days.

[0259] The results of visually inspecting the precipitation after storage are shown in Table 1. In Feed medium A to which N-acetylcysteine ​​was not added, precipitation was detected after 18 days. On the other hand, when 2.7 to 5.5 mM N-acetylcysteine ​​was further added to FEED medium A to which 18 mM L-cysteine ​​was added, no precipitation was detected.

[0260] Furthermore, when 2.7 to 5.5 mM N-acetylcysteine ​​was further added to FEED medium A supplemented with 22 mM L-cysteine, no precipitation was detected.

[0261] Furthermore, when 3.7 mM of N-acetylcysteine ​​was added to FEED medium A supplemented with 24 mM or 26 mM of L-cysteine, precipitation was detected. On the other hand, when 5.5 mM of N-acetylcysteine ​​was added, precipitation was not detected.

[0262] [Table 1]

[0263]

[0264] The above results show that the addition of N-acetylcysteine ​​suppresses the generation of precipitates in a liquid culture medium containing a high concentration of L-cysteine, thereby improving the stability of the liquid culture medium, compared with the case where N-acetylcysteine ​​is not added.

[0265] [Example 2]

[0266] Effect of N-acetylcysteine ​​on the liquid stability of L-cysteine-enriched culture medium and the cystine concentration during culture medium storage

[0267] CELLiST TM Feed media (AJINOMOTO Co., Ltd.: FEED2) was used as the base to prepare Feed medium A. Feed medium A does not contain N-acetylcysteine.

[0268] To Feed medium A supplemented with 18mM or 24mM L-cysteine ​​and 60g / L glucose, 0-5.5mM N-acetylcysteine ​​was added as a final concentration, and a liquid medium with a pH of 6.7-6.9 was prepared according to the order described in the product manual of FEED2. The prepared liquid medium was filtered using Stericup-GP, and 25mL or 50mL of it was placed in a 60mL PETG square medium bottle (Thermo Fisher Scientific: 2019-0030) and stored at 4°C under light-shielding conditions for 21 days.

[0269] After storage, the results of visually inspecting the precipitation of crystals are shown in Table 2. When N-acetylcysteine ​​was not added to the feed medium A containing 18 mM L-cysteine, precipitation was detected after 18 or 13 days. On the other hand, when 5.5 mM N-acetylcysteine ​​was added, no precipitation was detected.

[0270] In addition, when N-acetylcysteine ​​was not added to FEED medium A containing 24 mM L-cysteine, precipitation was detected after 7 days. On the other hand, when 2.8 mM N-acetylcysteine ​​was added, precipitation was detected after 8 or 13 days. In addition, when 5.5 mM N-acetylcysteine ​​was added, precipitation was detected after 21 days under the condition of 25 mL of liquid volume, and no precipitation was detected under the condition of 50 mL of liquid volume.

[0271] [Table 2]

[0272]

[0273] Next, for the five culture media with a liquid volume of 25 mL used in Table 2, the concentration changes of L-cysteine, cystine, N-acetylcysteine, N-acetylcysteine, and N,N'-diacetylcysteine ​​in the liquid were analyzed over time according to the method described in International Publication No. 2021 / 060517.

[0274] The results are as follows Figure 1 In the medium without N-acetylcysteine ​​( Figure 1A, C), the cystine concentration in the liquid increased and precipitation was detected. On the other hand, in the medium with N-acetylcysteine ​​added ( Figure 1 B, D, E), N-acetylcysteine ​​was produced, and the rate of cysteine ​​production was slowed down compared with the medium without N-acetylcysteine ​​supplementation.

[0275] Furthermore, for (A) a liquid medium containing 18 mM L-cysteine ​​but not N-acetylcysteine, (B) a liquid medium containing 24 mM L-cysteine ​​but not N-acetylcysteine, and (C) a liquid medium to which 24 mM L-cysteine ​​and 5.5 mM N-acetylcysteine ​​were added (the liquid volume of each liquid medium was 50 mL), 10 mL of the liquid medium was taken out on the 13th day after preparation, and the liquid medium was continuously stored until the 63rd day, and precipitation was visually observed.

[0276] The results are as follows Figure 2 In the liquid culture medium (A) and (B) not containing N-acetylcysteine, precipitation was detected. On the other hand, in the liquid culture medium (C) containing N-acetylcysteine, precipitation was not detected.

[0277] The above results show that, in the liquid medium containing L-cysteine ​​to which N-acetylcysteine ​​is added, the generation of precipitates is suppressed and the rate of cystine generation is slowed down.

[0278] [Example 3]

[0279] Effect of N-acetylcysteine ​​on the liquid stability of L-cysteine ​​high concentration culture medium

[0280] CELLiST, a commercially available culture medium, was used. TM Feed media (AJINOMOTO Co., Ltd.: FEED2). FEED2 does not contain N-acetylcysteine. To FEED2 containing 72 g / L of glucose, 18 mM of L-cysteine ​​and 0, 3, or 9 mM of N-acetylcysteine ​​were further added as a final concentration. The prepared liquid culture medium was filtered using Stericup-GP, and 10 or 40 mL was placed in a 50 mL FALCON tube and stored under light-shielding conditions at 25°C.

[0281] After storage, the results of visually inspecting the precipitation of crystals are shown in Table 3. In the medium to which 18 mM L-cysteine ​​was added to FEED2, precipitation was detected after 2 days under the condition of 10 mL of liquid volume and after 5 days under the condition of 40 mL of liquid volume.

[0282] In the medium to which 18 mM L-cysteine ​​and 3 mM N-acetylcysteine ​​were added to FEED2, precipitation was detected after 5 days under the condition of 10 mL of liquid volume and after 10 days under the condition of 40 mL of liquid volume.

[0283] In the medium to which 18 mM L-cysteine ​​and 9 mM N-acetylcysteine ​​were added to FEED2, precipitation was observed after 10 days under the condition of 10 mL of liquid volume and after 19 days under the condition of 40 mL of liquid volume.

[0284] [Table 3]

[0285]

[0286] The above results show that the addition of N-acetylcysteine ​​suppresses the generation of precipitates in the L-cysteine ​​high-concentration medium and improves the liquid stability.

[0287] [Example 4]

[0288] Effect of N-acetylcysteine ​​on liquid stability of feed medium

[0289] CELLiST, a commercially available culture medium, was used. TM Feed media (AJINOMOTO Co., Ltd.: FEED2). FEED2 does not contain N-acetylcysteine. 3, 6, 9, 13.5 or 18 mM N-acetylcysteine ​​was added to FEED2 containing 72 g / L of glucose. The prepared liquid medium was filtered using Stericup-GP, and 10 mL was placed in a 50 mL FALCON tube under conditions that promoted oxidation reaction and stored at 25°C under light-shielding conditions for 32 days.

[0290] After storage, the results of visually inspecting the precipitation of crystals are shown in Table 4. In the FEED2 medium without N-acetylcysteine, precipitation was detected after 22 days. On the other hand, in the medium with 3, 6, 9, 13.5 or 18 mM N-acetylcysteine ​​added to FEED2, no precipitation was detected.

[0291] [Table 4]

[0292]

[0293] The above results show that the addition of N-acetylcysteine ​​suppresses the generation of precipitates in the L-cysteine ​​high-concentration medium and improves the liquid stability.

[0294] [Example 5]

[0295] Effects of adding N-acetylcysteine ​​to feed medium on the proliferation and antibody production of CHO cells

[0296] CELLiST TM Basal media (AJINOMOTO Co., Ltd.: BASAL3) was supplemented with LR3-IGF-1 (SIGMA-ALDRICH: I1271-1MG, final concentration 50 μg / mL) and L-glutamine (Thermo Fisher SCIENTIFIC: 25030081, final concentration 6 mM) to prepare a basal medium.

[0297] The six types of culture media having different N-acetylcysteine ​​concentrations used in Example 4 were used immediately after preparation.

[0298] The prepared basal medium was used to adjust the concentration to 3 × 10 5 A CHO cell suspension of cells / mL was inoculated into a 6-well plate (6-well plate) (CORNING, Cat. No. CLS3471), and culture was started with 4 mL for 14 days. All experimental groups were implemented with n=2. The culture temperature was 37°C and the stirring speed was 110 rpm. On the 4th, 7th, 9th and 11th days after the start of culture, a 7% feed medium was added relative to the culture medium placed. On the 7th, 9th, 11th and 14th days after the start of culture, the cell culture fluid was sampled, and the number of live cells was determined using the live / dead cell automatic analyzer Vi-CELLTM XR (BECKMAN COULTER), and the antibody production was determined using Octet QK (FORTEBIO).

[0299] The results are as follows Figure 3 and 4 Sufficient cell proliferation and antibody production were also achieved in a medium supplemented with 3, 6, 9, 13.5 or 18 mM N-acetylcysteine ​​to the feed medium.

[0300] These results show that even when N-acetylcysteine ​​is added, the excellent cell proliferation ability and recombinant protein production ability brought about by the medium containing high concentration of L-cysteine ​​are maintained.

[0301] [Example 6]

[0302] Effects of L-cysteine-enriched feed medium on the proliferation and antibody production of CHO cells

[0303] CELLiST TMA basal medium was prepared by adding L-glutamine (Thermo Fisher SCIENTIFIC: 25030081) to a medium designed based on Basal media (AJINOMOTO Co., Ltd.: BASAL10) at a final concentration of 4 mM.

[0304] CELLiST TM Feed medium A was prepared based on Feed media (AJINOMOTO Co., Ltd.: FEED2). Feed medium A containing 11 or 18 mM L-cysteine ​​and 60 g / L glucose at a final concentration was prepared according to the procedure described in the product manual of FEED2 so that the pH was 6.7 to 6.9.

[0305] A 110 mM L-cysteine ​​solution (pH 11.3) was prepared.

[0306] The prepared basal medium was used to adjust the concentration of 7 × 10 5 CHO cell suspensions with a concentration of 11 cells / mL were cultured for 14 days using an ambr15 cell culture apparatus (Sartorius). All experimental groups were performed with n=2. The experimental groups included: Feed medium A containing 11 or 18 mM L-cysteine, with 3.3% added daily from the second day of culture (11 mM zone, 18 mM zone), and Feed medium A containing 18 mM L-cysteine, with 3.3% added daily from the second day of culture, and 110 mM L-cysteine ​​solution, with 0.163% added daily from the second day of culture (equivalent to the 23-24 mM zone). The antibody production on the 14th day in these conditional zones was measured using Cedex Bio HT (ROCHE DIAGNOSTICS).

[0307] The results are as follows Figure 5 It was shown that the antibody production was improved by increasing the amount of L-cysteine ​​supplied during the culture process.

[0308] [Example 7]

[0309] Effect of N-acetylcysteine ​​on the liquid stability of L-cysteine ​​high concentration culture medium

[0310] CELLiST, a commercially available culture medium, was used. TMFeed media (AJINOMOTO Co., Ltd.: FEED2). FEED2 does not contain N-acetylcysteine. FEED2 was further added with L-cysteine ​​at a final concentration of 0, 4, 9 or 18 mM and N-acetylcysteine ​​at a final concentration of 0, 2, 10 or 20 mM. The prepared liquid medium was filtered using Stericup-GP, and 10 or 40 mL was placed in a 50 mL FALCON tube and stored at 4°C under light-shielding conditions for 29 days.

[0311] After storage, the precipitation of crystals was visually inspected and the results are shown in Table 5.

[0312] [Table 5]

[0313]

[0314] As shown in Table 5, in the FEED2 medium without L-cysteine ​​addition, no precipitation was detected after 29 days in either the condition of 10 mL of liquid volume or the condition of 40 mL of liquid volume.

[0315] In the medium to which 4 mM L-cysteine ​​was added to FEED2, precipitation was detected after 14 days under the condition of 10 mL of liquid volume and after 19 days under the condition of 40 mL of liquid volume. Under the condition of 10 mL of liquid volume, the detection date of precipitation was delayed to 19 days by adding 2 mM N-acetylcysteine, and precipitation was not confirmed even after 29 days in the medium to which 10 or 20 mM N-acetylcysteine ​​was added. Under the condition of 40 mL of liquid volume, precipitation was not confirmed even after 29 days in the medium to which 10 or 20 mM N-acetylcysteine ​​was added.

[0316] In addition, in the medium to which 9 mM L-cysteine ​​was added to FEED2, precipitation was detected after 6 days under the condition of 10 mL of liquid volume and after 10 days under the condition of 40 mL of liquid volume. Under the condition of 10 mL of liquid volume, the detection date of precipitation was delayed to 10 days and 29 days by adding 2 or 10 mM N-acetylcysteine, but precipitation was not confirmed even after 29 days in the medium to which 20 mM N-acetylcysteine ​​was added. Under the condition of 40 mL of liquid volume, the detection date of precipitation was delayed to 21 days by adding 10 mM N-acetylcysteine, but precipitation was not confirmed even after 29 days in the medium to which 20 mM N-acetylcysteine ​​was added.

[0317] In addition, in the medium to which 18 mM L-cysteine ​​was added to FEED2, precipitation was detected after 3 days in either the 10 mL liquid volume condition or the 40 mL liquid volume condition. In the 10 mL liquid volume condition, the detection date of precipitation was delayed to 11 days and 21 days by adding 10 or 20 mM N-acetylcysteine. In the 40 mL liquid volume condition, the detection date of precipitation was delayed to 11 days and 16 days by adding 10 or 20 mM N-acetylcysteine.

[0318] The above results show that the addition of N-acetylcysteine ​​suppresses the generation of precipitates in a liquid culture medium containing a high concentration of L-cysteine, thereby improving the stability of the liquid culture medium, compared with the case where N-acetylcysteine ​​is not added.

[0319] It should be noted that the disclosures of the patents, patent applications, and publications cited in the present disclosure are incorporated herein by reference in their entirety.

[0320] Industrial Applicability

[0321] The culture medium of this embodiment can improve the productivity of products using recombinant proteins, and therefore can be suitably used in the production of antibody drugs using recombinant proteins, for example, and has industrial applicability.

Claims

1. A feed medium comprising 10-40 mM L-cysteine ​​and 1-40 mM N-acetylcysteine.

2. A feed medium comprising L-cysteine ​​and N-acetylcysteine, The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.

0.

3. The feed medium according to claim 1, comprising 15 to 30 mM L-cysteine.

4. The feed medium according to claim 1, comprising 2 to 10 mM N-acetylcysteine.

5. The feed medium according to claim 1, wherein The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.

0.

6. The feed medium according to claim 1, wherein The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.1 to 0.

4.

7. The feed medium according to claim 1, wherein The pH is 5.0~9.

0.

8. The feed medium according to claim 1, wherein The pH is 6.5~7.

6. The feed medium according to claim 1 , which is a serum-free medium and / or an animal protein-free medium.

10. The feed medium according to claim 1, which is a synthetic medium.

11. A powdered culture medium comprising L-cysteine ​​and N-acetylcysteine, wherein: The powder culture medium is used to prepare a feed medium containing 10-40 mM L-cysteine ​​and 1-40 mM N-acetylcysteine.

12. A powdered culture medium comprising L-cysteine ​​and N-acetylcysteine, wherein: The powder culture medium is used to prepare a feed culture medium in which the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04-1.

0.

13. The powder culture medium according to claim 11, wherein The feed medium contained 15-30 mM L-cysteine.

14. The powder culture medium according to claim 11, wherein The feed medium contained 2-10 mM N-acetylcysteine.

15. The powder culture medium according to claim 11, wherein The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the feed medium is 0.04 to 1.

0.

16. The powder culture medium according to claim 11, wherein The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the feed medium is 0.1 to 0.

4.

17. The powder culture medium according to claim 11, wherein The pH of the feed medium was 5.0~9.

0.

18. The powder culture medium according to claim 11, wherein The pH of the feed medium was 6.5~7.

6.

19. The powder culture medium according to claim 11, wherein The feed medium is serum-free and / or animal protein-free.

20. The powder culture medium according to claim 11, wherein The feed medium is a synthetic medium.

21. A method for culturing cells, comprising: The feed medium according to any one of claims 1 to 10 is added to a medium for culturing cells.

22. The method according to claim 21, wherein: The cell is an animal cell, a plant cell, a bacterial cell or a fungal cell.

23. The method according to claim 21, wherein: The cells are animal cells.

24. The method according to claim 21, wherein: The cells are mammalian cells.

25. The method according to claim 21, wherein: The cells are Chinese hamster ovary (CHO) cells.

26. The method according to claim 21, wherein: Cells express recombinant proteins.

27. The method according to claim 26, wherein: The recombinant protein comprises an antibody or an antigen-binding fragment thereof.

28. The method according to claim 27, wherein: The antibody or antigen-binding fragment thereof is derived from IgG.

29. The method according to claim 21, wherein: There is no precipitation of medium components due to heat load.

30. A method for producing a recombinant protein, comprising: The feed medium according to any one of claims 1 to 10 is added to a medium for culturing cells capable of producing a recombinant protein.

31. The method according to claim 30, wherein: The cell is an animal cell, a plant cell, a bacterial cell or a fungal cell.

32. The method of claim 30, wherein: The cells are animal cells.

33. The method of claim 30, wherein: The cells are mammalian cells.

34. The method of claim 30, wherein: The cells are Chinese hamster ovary (CHO) cells.

35. The method of claim 30, wherein: The recombinant protein comprises an antibody or an antigen-binding fragment thereof.

36. The method of claim 35, wherein: The antibody or antigen-binding fragment thereof is derived from IgG.

37. The method of claim 30, wherein: There is no precipitation of medium components due to heat load.

38. A method for improving the stability of a liquid culture medium, comprising: L-cysteine ​​and / or N-acetylcysteine ​​are added to the medium so that the concentration of L-cysteine ​​is 10 to 40 mM and the concentration of N-acetylcysteine ​​is 1 to 40 mM.

39. A method for improving the stability of a liquid culture medium, comprising: L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.

0.

40. A method for inhibiting the generation and / or precipitation of cystine in a liquid culture medium, comprising: L-cysteine ​​and / or N-acetylcysteine ​​are added to the medium so that the concentration of L-cysteine ​​is 10 to 40 mM and the concentration of N-acetylcysteine ​​is 1 to 40 mM.

41. A method for inhibiting the generation and / or precipitation of cystine in a liquid culture medium, comprising: L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.

0.

42. The method according to any one of claims 38 to 41, comprising: L-cysteine ​​and N-acetylcysteine ​​were added to the culture medium.

43. The method according to any one of claims 38 to 41, wherein The liquid culture medium contains 10~40mM L-cysteine. The method comprises: adding N-acetylcysteine ​​to a culture medium in such a manner that the concentration of N-acetylcysteine ​​is 1 to 40 mM.

44. The method according to any one of claims 38 to 41, wherein The liquid medium contains 1~40mM N-acetylcysteine. The method comprises: adding L-cysteine ​​to a culture medium so that the concentration of L-cysteine ​​is 10 to 40 mM.

45. The method according to any one of claims 38 to 41, comprising: L-cysteine ​​and / or N-acetylcysteine ​​are added to the medium so that the concentration of L-cysteine ​​becomes 15 to 30 mM.

46. ​​The method according to any one of claims 38 to 41, comprising: L-cysteine ​​and / or N-acetylcysteine ​​are added to the medium so that the concentration of N-acetylcysteine ​​is 2 to 10 mM.

47. The method of claim 38 or 40, comprising: L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.04 to 1.

0.

48. The method according to any one of claims 38 to 41, comprising: L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​is 0.1 to 0.

4.

49. The method according to any one of claims 38 to 41, comprising: L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the pH of the liquid medium is 5.0 to 9.

0.

50. The method according to any one of claims 38 to 41, comprising: L-cysteine ​​and / or N-acetylcysteine ​​is added to the medium so that the pH of the liquid medium is 6.5 to 7.

6.

51. A reagent for improving the stability of a liquid culture medium, comprising L-cysteine ​​and N-acetylcysteine, wherein: The liquid culture medium after adding the reagent contains 10-40 mM L-cysteine ​​and 1-40 mM N-acetylcysteine.

52. A reagent for improving the stability of a liquid culture medium, comprising L-cysteine ​​and N-acetylcysteine, wherein: The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.

0.

53. A reagent comprising N-acetylcysteine ​​for improving the stability of a liquid culture medium comprising 10 to 40 mM L-cysteine, wherein: The liquid culture medium after adding the reagent contains 1 to 40 mM N-acetylcysteine.

54. A reagent comprising N-acetylcysteine ​​for improving the stability of a liquid culture medium comprising L-cysteine, wherein: The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.

0.

55. A reagent for inhibiting the generation and / or precipitation of cystine in a liquid culture medium, comprising L-cysteine ​​and N-acetylcysteine, wherein: The liquid culture medium after adding the reagent contains 10-40 mM L-cysteine ​​and 1-40 mM N-acetylcysteine.

56. A reagent for inhibiting the generation and / or precipitation of cystine in a liquid culture medium, comprising L-cysteine ​​and N-acetylcysteine, wherein: The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.

0.

57. A reagent comprising N-acetylcysteine ​​for inhibiting the generation and / or precipitation of cystine in a liquid medium containing 10 to 40 mM L-cysteine, wherein: The liquid culture medium after adding the reagent contains 1 to 40 mM N-acetylcysteine.

58. A reagent comprising N-acetylcysteine ​​for inhibiting the production and / or precipitation of cystine in a liquid medium containing L-cysteine, wherein: The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.

0.

59. The reagent according to any one of claims 51 to 58, wherein The liquid culture medium after adding the reagent contains 15-30 mM L-cysteine.

60. The reagent according to any one of claims 51 to 58, wherein The liquid culture medium after adding the reagent contains 2-10 mM N-acetylcysteine.

61. The reagent according to any one of claims 51, 53, 55 and 57, wherein The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.04 to 1.

0.

62. The reagent according to any one of claims 51 to 58, wherein The molar concentration ratio of N-acetylcysteine ​​to L-cysteine ​​in the liquid culture medium after adding the reagent is 0.1 to 0.

4.

63. The reagent according to any one of claims 51 to 58, wherein The pH of the liquid culture medium after adding the reagent is 5.0-9.

0.

64. The reagent according to any one of claims 51 to 58, wherein The pH of the liquid culture medium after adding the reagent is 6.5-7.

6.

65. A culture medium supplement comprising N-acetylcysteine ​​and L-cysteine, wherein: The culture medium additive is used to prepare the feed medium according to any one of claims 1 to 10 or the powder culture medium according to any one of claims 11 to 20.

66. A culture medium supplement comprising N-acetylcysteine, wherein: The culture medium additive is used to prepare the feed medium according to any one of claims 1 to 10 or the powder culture medium according to any one of claims 11 to 20.

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