Cell culture medium and its use, cell culture method, kit

Abstract

The application discloses a cell culture medium and application thereof, a cell culture method and a kit. The cell culture medium comprises toluhydroquinone, amino acids, vitamins, inorganic salts, polyamines, trace elements and a carbon source; the amino acids comprise L-methionine, L-proline, L-aspartic acid, L-tyrosine disodium salt, L-asparagine, L-cysteine, L-serine, L-tryptophan, L-lysine, L-alanine, L-histidine, L-threonine, L-valine, L-isoleucine, L-leucine, L-phenylalanine, L-glycine, L-arginine and L-glutamic acid; and the vitamins comprise vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid and alpha-lipoic acid. The application obtains a low-cost cell culture medium, and effectively meets the demand of expressing monoclonal antibodies, and realizes large-scale culture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a cell culture medium and application thereof, a cell culture method, and a kit. BACKGROUND

[0002] At present, the market demand for monoclonal antibody drugs expressed by CHO cells (Chinese Hamster Ovary cells) is large, and the supply cannot meet the demand. Therefore, large-scale animal cell suspension culture technology, which is one of the core technologies for commercial production of monoclonal antibodies, has become a new bottleneck restricting the development of antibody drugs.

[0003] One of the reasons why large-scale animal cell suspension culture technology has become a new bottleneck restricting the development of antibody drugs is that enterprises lack self-owned high-efficiency CHO cell culture medium, and international well-known culture media such as Gibco, Hyclone, and Merck cost up to thousands of yuan per liter, and the formula is secret, which is not conducive to subsequent culture process optimization.

[0004] There are various classification methods for cell culture medium, and the basic culture medium and the feed culture medium are a core classification method according to the function and action stage in the culture process, which is particularly important in modern industrialized cell culture (such as biopharmaceuticals).

[0005] The basic cell culture medium is a chemically defined or partially chemically defined liquid that provides basic nutrients necessary for in vitro cultured cells to survive, grow, and maintain basic functions. It is the starting liquid environment of the cell culture system, and its core function is to provide energy sources (mainly glucose), to provide building blocks (including amino acids (raw materials for synthesizing proteins) and nucleotides (raw materials for synthesizing nucleic acids)), to maintain a physiological environment (including inorganic salts (to maintain osmotic pressure and pH balance), buffer systems (such as sodium bicarbonate / CO2 system)), and to provide auxiliary factors (such as vitamins, which act as enzyme cofactors to participate in metabolic reactions).

[0006] The feed cell culture medium is a high-concentration nutrient concentrate that is added to the basic culture medium in batches or continuously during the cell culture process. Its core function is not to provide the initial environment, but to dynamically supplement the key nutrients consumed in large quantities by cells during high-speed metabolism and product synthesis, while controlling the accumulation of metabolic by-products. Specifically, it can supplement depleted nutrients (such as rapidly consumed glucose and essential amino acids), extend the culture period (to prevent cell apoptosis caused by nutrient depletion and extend the culture time from a few days to several weeks), improve yield and quality (through precise nutrient control to maximize the expression amount and consistency of target products), and regulate metabolism (by controlling the feeding strategy to change the cell metabolic pathway (such as reducing lactate accumulation)).

[0007] Therefore, how to develop a low-cost basic cell culture medium, a feed medium, and effectively meet the needs of expressing monoclonal antibodies has a very important significance for the commercial production of antibody drugs. SUMMARY

[0008] In order to overcome the defects that the CHO culture medium in the prior art is either too high in cost or poor in performance, the present application provides a cell culture medium and application thereof, a cell culture method and a kit. The basic cell culture medium (for example, Hengrui No. 1 BM03, which can also be referred to as HR No. 1 BM03) and the feed medium (for example, Hengrui No. 1 FeedB, which can also be referred to as HR No. 1 FeedB) in the present application can culture cell strains such as SHR-1314, SHR-1701 and SHR-1805, are low in cost, can effectively meet the needs of expressing monoclonal antibodies, and can realize large-scale culture.

[0009] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions.

[0010] In a first aspect of the present application, a basic cell culture medium is provided, which comprises a solid component and water;

[0011] The solid component comprises the following components: tophenone, amino acid, vitamin, inorganic salt, polyamine, trace element and carbon source;

[0012] The amino acid comprises: L-methionine, L-proline, L-aspartic acid, L-tyrosine disodium salt, L-asparagine, L-cysteine, L-serine, L-tryptophan, L-lysine, L-alanine, L-histidine, L-threonine, L-valine, L-isoleucine, L-leucine, L-phenylalanine, L-glycine, L-arginine and L-glutamic acid;

[0013] The vitamin comprises: vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid and alpha-lipoic acid;

[0014] The inorganic salt comprises: sodium phosphate, potassium chloride, zinc sulfate, sodium selenite, iron (II) sulfate, copper sulfate, calcium chloride, magnesium sulfate, sodium thiosulfate and sodium chloride;

[0015] The polyamine comprises: putrescine, spermidine and spermine;

[0016] The trace element comprises: manganese chloride, aluminum trichloride, cadmium sulfate and cobalt chloride;

[0017] The carbon source comprises: glucose and sodium pyruvate;

[0018] the concentration of the L-methionine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0019] the concentration of the L-methionine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0020] the concentration of the L-methionine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0021] the concentration of the L-methionine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0022] the concentration of the L-methionine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0023] the concentration of the L-methionine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0024] the concentration of the L-methionine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0025] In some embodiments of the application, the basal cell culture medium consists of the solid ingredients and the water.

[0026] In some embodiments of the application, the basal cell culture medium is used for cell culture and / or production of a protein of interest.

[0027] In some embodiments of the application, the cell is selected from a eukaryotic cell.

[0028] In some embodiments of the application, the eukaryotic cell is selected from a CHO cell or a 293 cell.

[0029] In some embodiments of the application, the protein of interest is selected from one or more of a PD-L1 / TGF-beta antibody, a Her2 antibody and an IL-17A antibody.

[0030] In some embodiments of the application, the concentration of the trophicin in the basal cell culture medium is 0.9-1.2 mg / L, for example 0.6 mg / L, 0.9 mg / L or 1.2 mg / L.

[0031] In some embodiments of the application, the concentration of the L-methionine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0032] In some embodiments of the application, the concentration of the L-methionine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0033] In some embodiments of the application, the L-aspartic acid is present in the basal cell culture medium at a concentration of 0.3 to 5 mM, for example 0.3 mM, 2.65 mM or 5 mM.

[0034] In some embodiments of the application, the L-tyrosine disodium salt is present in the basal cell culture medium at a concentration of 2.5 to 10 mM, for example 1 mM, 2.5 mM, 5.5 mM or 10 mM.

[0035] In some embodiments of the application, the L-asparagine is present in the basal cell culture medium at a concentration of 0.5 to 3 mM, for example 0.5 mM, 1.75 mM or 3 mM.

[0036] In some embodiments of the application, the L-cysteine is present in the basal cell culture medium at a concentration of 1 to 5 mM, for example 0.5 mM, 1 mM, 1.25 mM, 2.5 mM, 3 mM or 5 mM.

[0037] In some embodiments of the application, the L-serine is present in the basal cell culture medium at a concentration of 1 to 5 mM, for example 1 mM, 3 mM or 5 mM.

[0038] In some embodiments of the application, the L-tryptophan is present in the basal cell culture medium at a concentration of 2.5 to 10 mM, for example 1 mM, 2.5 mM, 4 mM, 5 mM, 7 mM or 10 mM.

[0039] In some embodiments of the application, the L-lysine is present in the basal cell culture medium at a concentration of 2 to 20 mM, for example 2 mM, 5 mM, 10 mM, 15 mM or 20 mM.

[0040] In some embodiments of the application, the L-alanine is present in the basal cell culture medium at a concentration of 1 to 5 mM, for example 1 mM, 3 mM or 5 mM.

[0041] In some embodiments of the application, the L-histidine is present in the basal cell culture medium at a concentration of 3 to 10 mM, also for example 3 mM, 6.5 mM or 10 mM.

[0042] In some embodiments of the application, the L-threonine is present in the basal cell culture medium at a concentration of 1 to 5 mM, for example 1 mM, 3 mM or 5 mM.

[0043] In some embodiments of the application, the L-valine is present in the basal cell culture medium at a concentration of 0.2 to 2 mM, for example 0.2 mM, 1.1 mM or 2 mM.

[0044] In some embodiments of the application, the concentration of L-isoleucine in the basal cell culture medium is 3-6 mM, for example 3 mM, 4.5 mM or 6 mM.

[0045] In some embodiments of the application, the concentration of L-leucine in the basal cell culture medium is 2-6 mM, for example 2 mM, 4 mM or 6 mM.

[0046] In some embodiments of the application, the concentration of L-phenylalanine in the basal cell culture medium is 5-10 mM, for example 5 mM, 7.5 mM or 10 mM.

[0047] In some embodiments of the application, the concentration of L-glycine in the basal cell culture medium is 0.1-2 mM, for example 0.1 mM, 1.05 mM or 2 mM.

[0048] In some embodiments of the application, the concentration of L-arginine in the basal cell culture medium is 2-6 mM, also for example 2 mM, 4 mM or 6 mM.

[0049] In some embodiments of the application, the concentration of L-glutamic acid in the basal cell culture medium is 5-20 mM, for example 5 mM, 12.5 mM or 20 mM.

[0050] In some embodiments of the application, the concentration of vitamin B1 in the basal cell culture medium is 0.005-0.1 mM, for example 0.005 mM, 0.05 mM or 0.1 mM.

[0051] In some embodiments of the application, the concentration of vitamin B2 in the basal cell culture medium is 0.005-0.05 mM, for example 0.005 mM, 0.025 mM or 0.05 mM.

[0052] In some embodiments of the application, the concentration of nicotinamide in the basal cell culture medium is 0.001-0.02 mM, for example 0.001 mM, 0.01 mM or 0.02 mM.

[0053] In some embodiments of the application, the concentration of vitamin B5 in the basal cell culture medium is 0.01-0.1 mM, for example 0.01 mM, 0.05 mM or 0.1 mM.

[0054] In some embodiments of the application, the concentration of vitamin B6 in the basal cell culture medium is 0.01-0.2 mM, for example 0.002 mM, 0.01 mM, 0.02 mM, 0.05 mM, 0.06 mM, 0.1 mM or 0.2 mM.

[0055] In some embodiments of the application, the concentration of vitamin B12 in the basal cell culture medium is 0.001-0.02 mM, for example 0.0002 mM, 0.001 mM, 0.002 mM, 0.005 mM, 0.006 mM, 0.01 mM or 0.02 mM.

[0056] In some embodiments of the application, the concentration of choline in the basal cell culture medium is 0.1-1 mM, for example 0.1 mM, 0.55 mM or 1 mM.

[0057] In some embodiments of the application, the concentration of folic acid in the basal cell culture medium is 0.005-0.5 mM, for example 0.005 mM, 0.25 mM or 0.5 mM.

[0058] In some embodiments of the application, the concentration of alpha-lipoic acid in the basal cell culture medium is 0.001-0.01 mM, for example 0.001 mM, 0.005 mM or 0.01 mM.

[0059] In some embodiments of the application, the concentration of sodium phosphate in the basal cell culture medium is 0.5-2 mM, for example 0.5 mM, 1.25 mM or 2 mM.

[0060] In some embodiments of the application, the concentration of potassium chloride in the basal cell culture medium is 0.5-2 mM, for example 0.5 mM, 1.25 mM or 2 mM.

[0061] In some embodiments of the application, the concentration of zinc sulfate in the basal cell culture medium is 0.0005-0.001 mM, for example 0.0005 mM, 0.00075 mM or 0.001 mM.

[0062] In some embodiments of the application, the concentration of sodium selenite in the basal cell culture medium is 0.00001-0.0001 mM, for example 0.00001 mM, 0.00005 mM or 0.0001 mM.

[0063] In some embodiments of the application, the concentration of iron (II) sulfate in the basal cell culture medium is 0.02-0.2 mM, for example 0.02 mM, 0.11 mM or 0.2 mM.

[0064] In some embodiments of the application, the concentration of copper sulfate in the basal cell culture medium is 0.0005-0.001 mM, for example 0.0005 mM, 0.00075 mM or 0.001 mM.

[0065] In some embodiments of the application, the concentration of calcium chloride in the basal cell culture medium is 0.05-0.5 mM, for example 0.05 mM, 0.25 mM or 0.5 mM.

[0066] In some embodiments of the application, the concentration of magnesium sulfate in the basal cell culture medium is 0.1-2 mM, for example 0.1 mM, 1.05 mM or 2 mM.

[0067] In some embodiments of the application, the concentration of sodium thiosulfate in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0068] In some embodiments of the application, the concentration of sodium chloride in the basal cell culture medium is 20-50 mM, for example 20 mM, 35 mM or 50 mM.

[0069] In some embodiments of the application, the concentration of putrescine in the basal cell culture medium is 0.01-0.1 mM, for example 0.01 mM, 0.055 mM or 0.1 mM.

[0070] In some embodiments of the application, the concentration of spermidine in the basal cell culture medium is 0.01-0.1 mM, for example 0.01 mM, 0.055 mM or 0.1 mM.

[0071] In some embodiments of the application, the concentration of spermine in the basal cell culture medium is 0.01-0.1 mM, also for example 0.01 mM, 0.055 mM or 0.1 mM.

[0072] In some embodiments of the application, the concentration of manganese chloride in the basal cell culture medium is 0.000001-0.0001 mM, for example 0.000001 mM, 0.00005 mM or 0.0001 mM.

[0073] In some embodiments of the application, the concentration of aluminum trichloride in the basal cell culture medium is 0.000001-0.0001 mM, for example 0.000001 mM, 0.00005 mM or 0.0001 mM.

[0074] In some embodiments of the application, the aluminum trichloride is aluminum trichloride hexahydrate.

[0075] In some embodiments of the application, the concentration of cadmium sulfate in the basal cell culture medium is 0.000001-0.0001 mM, for example 0.000001 mM, 0.00005 mM or 0.0001 mM.

[0076] In some embodiments of the present application, the concentration of the cobalt chloride in the basal cell culture medium is 0.000001-0.0001 mM, for example 0.000001 mM, 0.00005 mM or 0.0001 mM.

[0077] In some embodiments of the present application, the cobalt chloride is cobalt chloride hexahydrate.

[0078] In some embodiments of the present application, the concentration of the glucose in the basal cell culture medium is 20-40 mM, for example 20 mM, 30 mM or 40 mM.

[0079] In some embodiments of the present application, the concentration of the sodium pyruvate in the basal cell culture medium is 0.1-5 mM, for example 0.1 mM, 2.55 mM or 5 mM.

[0080] In a preferred embodiment of the present application, the concentrations of the trophic hormone, the amino acid, the vitamin, the inorganic salt, the polyamine, the trace element, the carbon source in the basal cell culture medium are as shown in the following table:

[0081]

[0082]

[0083] In some embodiments of the present application, the basal cell culture medium is formulated as shown in the following table:

[0084]

[0085]

[0086] In some embodiments of the present application, the basal cell culture medium is formulated as shown in the following table:

[0087]

[0088]

[0089] In some embodiments of the present application, the basal cell culture medium is formulated as shown in the following table:

[0090]

[0091]

[0092] In some embodiments of the present application, the basal cell culture medium is formulated as shown in the following table:

[0093]

[0094]

[0095] In a second aspect of the present application, a feeding medium is provided, the cell culture medium comprising a solid component and water;

[0096] The solid component comprises the following components: tophenone, amino acid, vitamin, inorganic salt, polyamine, trace element and carbon source;

[0097] The amino acid includes: L-methionine, L-proline, L-aspartic acid, L-tyrosine disodium salt, L-asparagine, L-cysteine, L-serine, L-tryptophan, L-lysine, L-alanine, L-histidine, L-threonine, L-valine, L-isoleucine, L-leucine, L-phenylalanine, L-glycine, L-arginine and L-glutamic acid;

[0098] The vitamin includes: vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid and α-thioctic acid;

[0099] The inorganic salt includes: sodium phosphate, potassium chloride, zinc sulfate, sodium selenite, iron (II) sulfate, copper sulfate, calcium chloride, magnesium sulfate, sodium thiosulfate and sodium chloride;

[0100] The polyamine includes: putrescine, spermidine and spermine;

[0101] The trace element includes: manganese chloride, aluminum trichloride, cadmium sulfate and cobalt chloride;

[0102] The carbon source includes: glucose and sodium pyruvate;

[0103] The concentration of tophenone in the feeding medium is 0.6-12 mg / L;

[0104] The concentration of L-tyrosine disodium salt in the feeding medium is 10-100 mM;

[0105] The concentration of L-cysteine in the feeding medium is 5-50 mM;

[0106] The concentration of L-tryptophan in the feeding medium is 10-100 mM;

[0107] The concentration of L-lysine in the feeding medium is 20-200 mM;

[0108] The concentration of vitamin B6 in the feeding medium is 0.1-2 mM;

[0109] The concentration of vitamin B12 in the feeding medium is 0.01-0.2 mM.

[0110] In some embodiments of the application, the feed medium consists of the solid components and the water.

[0111] In some embodiments of the application, the feed medium is used for cell culture and / or production of a protein of interest.

[0112] In some embodiments of the application, the cell is selected from a eukaryotic cell.

[0113] In some embodiments of the application, the eukaryotic cell is selected from a CHO cell or a 293 cell.

[0114] In some embodiments of the application, the protein of interest is selected from one or more of a PD-L1 / TGF-beta antibody, a Her2 antibody and an IL-17A antibody.

[0115] In some embodiments of the application, the concentration of the trophic factor in the feed medium is 6-12 mg / L, such as 6 mg / L, 9 mg / L or 12 mg / L.

[0116] In some embodiments of the application, the concentration of the L-methionine in the feed medium is 10-50 mM, such as 10 mM, 30 mM or 50 mM.

[0117] In some embodiments of the application, the concentration of the L-proline in the feed medium is 5-50 mM, such as 5 mM, 12.5 mM or 50 mM.

[0118] In some embodiments of the application, the concentration of the L-aspartic acid in the feed medium is 3-50 mM, such as 3 mM, 26.5 mM or 50 mM.

[0119] In some embodiments of the application, the concentration of the L-tyrosine disodium salt in the feed medium is 50-100 mM, such as 10 mM, 50 mM, 55 mM or 100 mM.

[0120] In some embodiments of the application, the concentration of the L-asparagine in the feed medium is 5-30 mM, such as 5 mM, 17.5 mM or 30 mM.

[0121] In some embodiments of the application, the concentration of the L-cysteine in the feed medium is 10-50 mM, such as 5 mM, 10 mM, 25 mM, 30 mM or 50 mM.

[0122] In some embodiments of the application, the concentration of the L-serine in the feed medium is 10-50 mM, also such as 10 mM, 30 mM or 50 mM.

[0123] In some embodiments of the application, the concentration of L-tryptophan in the feed medium is 40-100 mM, also for example 10 mM, 40 mM, 50 mM, 70 mM or 100 mM.

[0124] In some embodiments of the application, the concentration of L-lysine in the feed medium is 100-200 mM, for example 20 mM, 100 mM, 150 mM or 200 mM.

[0125] In some embodiments of the application, the concentration of L-alanine in the feed medium is 10-50 mM, for example 10 mM, 30 mM or 50 mM.

[0126] In some embodiments of the application, the concentration of L-histidine in the feed medium is 30-100 mM, for example 30 mM, 65 mM or 100 mM.

[0127] In some embodiments of the application, the concentration of L-threonine in the feed medium is 10-50 mM, for example 10 mM, 30 mM or 50 mM.

[0128] In some embodiments of the application, the concentration of L-valine in the feed medium is 2-20 mM, also for example 2 mM, 11 mM or 20 mM.

[0129] In some embodiments of the application, the concentration of L-isoleucine in the feed medium is 30-60 mM, also for example 30 mM, 45 mM or 60 mM.

[0130] In some embodiments of the application, the concentration of L-leucine in the feed medium is 20-60 mM, also for example 20 mM, 40 mM or 60 mM.

[0131] In some embodiments of the application, the concentration of L-phenylalanine in the feed medium is 50-100 mM, for example 50 mM, 75 mM or 100 mM.

[0132] In some embodiments of the application, the concentration of L-glycine in the feed medium is 1-20 mM, for example 1 mM, 10.5 mM or 20 mM.

[0133] In some embodiments of the application, the concentration of L-arginine in the feed medium is 20-60 mM, for example 20 mM, 40 mM or 60 mM.

[0134] In some embodiments of the application, the concentration of L-glutamic acid in the feed medium is 50-200 mM, for example 50 mM, 125 mM or 200 mM.

[0135] In some embodiments of the application, the concentration of vitamin B1 in the feed medium is 0.05-1 mM, for example 0.05 mM, 0.5 mM or 1 mM.

[0136] In some embodiments of the application, the concentration of vitamin B2 in the feed medium is 0.05-0.5 mM, for example 0.05 mM, 0.25 mM or 0.5 mM.

[0137] In some embodiments of the application, the concentration of nicotinamide in the feed medium is 0.01-0.2 mM, for example 0.01 mM, 0.1 mM or 0.2 mM.

[0138] In some embodiments of the application, the concentration of vitamin B5 in the feed medium is 0.1-1 mM, for example 0.1 mM, 0.5 mM or 1 mM.

[0139] In some embodiments of the application, the concentration of vitamin B6 in the feed medium is 0.5-2 mM, for example 0.1 mM, 0.5 mM, 0.6 mM, 1 mM or 2 mM.

[0140] In some embodiments of the application, the concentration of vitamin B12 in the feed medium is 0.05-0.2 mM, for example 0.01 mM, 0.05 mM, 0.06 mM, 0.1 mM or 0.2 mM.

[0141] In some embodiments of the application, the concentration of choline in the feed medium is 1-10 mM, for example 1 mM, 5.5 mM or 10 mM.

[0142] In some embodiments of the application, the concentration of folic acid in the feed medium is 0.05-5 mM, for example 0.05 mM, 2.5 mM or 5 mM.

[0143] In some embodiments of the application, the concentration of alpha-lipoic acid in the feed medium is 0.01-0.1 mM, for example 0.01 mM, 0.05 mM or 0.1 mM.

[0144] In some embodiments of the application, the concentration of sodium phosphate in the feed medium is 5-20 mM, for example 5 mM, 12.5 mM or 20 mM.

[0145] In some embodiments of the application, the potassium chloride is at a concentration of 5 to 20 mM in the feed medium, for example 5 mM, 12.5 mM or 20 mM.

[0146] In some embodiments of the application, the zinc sulfate is at a concentration of 0.005 to 0.01 mM in the feed medium, for example 0.005 mM, 0.0075 mM or 0.01 mM.

[0147] In some embodiments of the application, the sodium selenite is at a concentration of 0.0001 to 0.001 mM in the feed medium, also for example 0.0001 mM, 0.0005 mM or 0.001 mM.

[0148] In some embodiments of the application, the iron (II) sulfate is at a concentration of 0.2 to 2 mM in the feed medium, for example 0.2 mM, 1.1 mM or 2 mM.

[0149] In some embodiments of the application, the copper sulfate is at a concentration of 0.005 to 0.01 mM in the feed medium, for example 0.005 mM, 0.0075 mM or 0.01 mM.

[0150] In some embodiments of the application, the calcium chloride is at a concentration of 0.5 to 5 mM in the feed medium, for example 0.5 mM, 2.5 mM or 5 mM.

[0151] In some embodiments of the application, the magnesium sulfate is at a concentration of 1 to 20 mM in the feed medium, for example 1 mM, 10.5 mM or 20 mM.

[0152] In some embodiments of the application, the sodium thiosulfate is at a concentration of 10 to 50 mM in the feed medium, for example 10 mM, 30 mM or 50 mM.

[0153] In some embodiments of the application, the sodium chloride is at a concentration of 200 to 500 mM in the feed medium, for example 200 mM, 350 mM or 500 mM.

[0154] In some embodiments of the application, the putrescine is at a concentration of 0.1 to 1 mM in the feed medium, also for example 0.1 mM, 0.55 mM or 1 mM.

[0155] In some embodiments of the application, the spermidine is at a concentration of 0.1 to 1 mM in the feed medium, for example 0.1 mM, 0.55 mM or 1 mM.

[0156] In some embodiments of the application, the concentration of spermine in the feed medium is 0.1-1 mM, also for example 0.1 mM, 0.55 mM or 1 mM.

[0157] In some embodiments of the application, the concentration of manganese chloride in the feed medium is 0.00001-0.001 mM, for example 0.00001 mM, 0.0005 mM or 0.001 mM.

[0158] In some embodiments of the application, the concentration of aluminum trichloride in the feed medium is 0.00001-0.001 mM, also for example 0.00001 mM, 0.0005 mM or 0.001 mM.

[0159] In some embodiments of the application, the aluminum trichloride is aluminum trichloride hexahydrate.

[0160] In some embodiments of the application, the concentration of cadmium sulfate in the feed medium is 0.00001-0.001 mM, for example 0.00001 mM, 0.0005 mM or 0.001 mM.

[0161] In some embodiments of the application, the concentration of cobalt chloride in the cell culture medium is 0.00001-0.001 mM, for example 0.00001 mM, 0.0005 mM or 0.001 mM.

[0162] In some embodiments of the application, the cobalt chloride is cobalt chloride hexahydrate.

[0163] In some embodiments of the application, the concentration of glucose in the feed medium is 200-400 mM, also for example 200 mM, 300 mM or 400 mM.

[0164] In some embodiments of the application, the concentration of sodium pyruvate in the feed medium is 1-50 mM, also for example 1 mM, 25.5 mM or 50 mM.

[0165] In a preferred embodiment of the application, the concentrations of the tocol, amino acids, vitamins, inorganic salts, polyamines, trace elements, carbon sources in the feed medium are as shown in the following table:

[0166]

[0167]

[0168] In some embodiments of the application, the feed medium is formulated as shown in the following table:

[0169]

[0170]

[0171] In some embodiments of the present application, the formulation of the feed medium is shown in the following table:

[0172]

[0173]

[0174] In some embodiments of the present application, the formulation of the feed medium is shown in the following table:

[0175]

[0176]

[0177] In a third aspect of the present application, there is provided a method for preparing the basal cell culture medium as described above, comprising the following steps:

[0178] mixing the ingredients in the basal cell culture medium to obtain the basal cell culture medium.

[0179] In a fourth aspect of the present application, there is provided a method for preparing the feed medium as described above, comprising the following steps:

[0180] mixing the ingredients in the feed medium to obtain the feed medium.

[0181] In a fifth aspect of the present application, there is provided a kit for culturing cells, comprising at least one first container and / or at least one second container, wherein:

[0182] the first container contains the basal cell culture medium as described above or the solid ingredients in the basal cell culture medium as described above, and the second container contains the feed medium as described above or the solid ingredients in the feed medium as described above.

[0183] In some embodiments of the present application, the first container contains the solid ingredients in the basal cell culture medium and the water.

[0184] In some embodiments of the present application, the solid ingredients in the basal cell culture medium and the water are separately contained in the first container.

[0185] In some embodiments of the present application, the first container contains the solid ingredients in the basal cell culture medium, which are mixed with water to prepare the corresponding concentration when used.

[0186] In some embodiments of the present application, the second container contains the solid ingredients in the feed medium and the water.

[0187] In some embodiments of the application, the second container contains the solid ingredients of the feed cell culture medium, and the water is added at the time of use.

[0188] In some embodiments of the application, the second container contains the solid ingredients of the feed cell culture medium, and the water is added at the time of use.

[0189] In some embodiments of the application, the first container contains Formula 1 and the second container contains Formula 16.

[0190] In some embodiments of the application, the first container contains Formula 2 and the second container contains Formula 17.

[0191] In some embodiments of the application, the first container contains Formula 3 and the second container contains Formula 18.

[0192] In some embodiments of the application, the first container contains Formula 8 and the second container contains Formula 19, Formula 20 or Formula 21.

[0193] In some embodiments of the application, the first container contains Formula 10 or Formula 11 and the second container contains Formula 22.

[0194] In some embodiments of the application, the first container contains Formula 12 and the second container contains Formula 23, Formula 24 or Formula 25.

[0195] In some embodiments of the application, the first container contains Formula 13 and the second container contains Formula 23, Formula 24 or Formula 25.

[0196] In some embodiments of the application, the first container contains Formula 14 and the second container contains Formula 22 or Formula 24.

[0197] In some embodiments of the application, the first container contains Formula 15 and the second container contains Formula 22 or Formula 24.

[0198] In a sixth aspect of the application, there is provided the use of the basal cell culture medium as described above, the feed cell culture medium as described above or the kit for culturing cells as described above in cell culture and / or production of a protein of interest.

[0199] In some embodiments of the application, the cell is selected from a eukaryotic cell.

[0200] In some embodiments of the present application, the eukaryotic cell is selected from a CHO cell or a 293 cell.

[0201] In some embodiments of the present application, the protein of interest is selected from one or more of a PD-L1 / TGF-β antibody, a Her2 antibody, and an IL-17A antibody.

[0202] In a seventh aspect of the present application, a method for culturing cells is provided, the method comprising:

[0203] seeding cells into the basal cell culture medium for an expansion culture;

[0204] adding the feed medium to the culture system during the expansion culture, and continuing the expansion culture.

[0205] In some embodiments of the present application, the feed medium is added to the culture system from day 4 of the culture.

[0206] In some embodiments of the present application, the feed medium is added to the culture system at 0.5% to 5.0% of the initial culture volume on day 4, 6, 8, 10, 12 of the culture, for example, 2.8%, 3.8%, 4.4%, or 5.0%.

[0207] In some embodiments of the present application, the feed medium is added to the culture system at 2.8%, 3.8%, 5.0%, 5.0%, 4.4% of the initial culture volume on day 4, 6, 8, 10, 12 of the culture, respectively.

[0208] In some embodiments of the present application, the feed medium is added to the culture system at 5% of the initial culture volume on day 4, 6, 8, 10, 12 of the culture, respectively.

[0209] In some embodiments of the present application, the feed medium is added to the culture system at 5% of the initial culture volume on day 4, 6, 8, 10, 12, 14 of the culture, respectively.

[0210] In some embodiments of the present application, in the expansion culture, the seeding density is (0.4-1.0) x 10 6 cells / mL, and the initial pH of the culture system is controlled at 7.0±0.2, and the initial culture temperature is controlled at 36.0-37.0°C.

[0211] In some embodiments of the present application, in the expansion culture, the seeding density is (0.4-1.0) x 10 6 cells / mL, (0.4-0.6) x 10 6 cells / mL, or (0.6-0.8) x 106 0.5 x 10 6 cells / mL, 0.5 x 10 6 cells / mL, or 0.7 x 10 6 cells / mL.

[0212] In some embodiments of the present application, the culture temperature is reduced to 32.5-33.5°C when the culture system is cultured for 7 days.

[0213] In some embodiments of the present application, the pH value of the culture system is adjusted to 7.0±0.3 when the culture system is cultured for 10 days.

[0214] In some embodiments of the present application, the pH control method in the culture method is single CO2 mode control; the single CO2 mode control is as follows:

[0215] When the pH value of the culture system is higher than the upper limit, CO2 is introduced into the culture system;

[0216] When the pH value of the culture system is lower than the upper limit, Na2CO3 solution is added into the culture system.

[0217] In some embodiments of the present application, the stirring speed of the culture system is 250-300 rpm.

[0218] In some embodiments of the present application, the dissolved oxygen concentration (DO) of the culture system is 40-50%, for example, 45%, the percentage refers to the percentage of the dissolved oxygen concentration in the fermentation broth relative to the oxygen concentration in the air.

[0219] In some embodiments of the present application, when the glucose concentration in the culture system is lower than 4.0 g / L, glucose is supplemented to 6.0 g / L in the culture system.

[0220] In some embodiments of the present application, the cells are harvested when the culture is carried out for 14 days or the cell viability is lower than 80%.

[0221] In some embodiments of the present application, the cells are selected from eukaryotic cells.

[0222] In some embodiments of the present application, the eukaryotic cells are selected from CHO cells or 293 cells.

[0223] In a fifth aspect of the present application, a method for producing a target protein is provided, which comprises the following steps:

[0224] (i) culturing genetically engineered cells in a basal cell culture medium as described above under suitable conditions for expression, adding a feed medium to the basal cell culture medium, and expressing a protein of interest; and

[0225] (ii) isolating the protein of interest from the fermentation product.

[0226] In some embodiments of the present application, the cells are selected from eukaryotic cells.

[0227] In some embodiments of the present application, the eukaryotic cells are selected from CHO cells or 293 cells.

[0228] In some embodiments of the present application, the protein of interest is selected from one or more of a PD-L1 / TGF-β antibody, a Her2 antibody, and an IL-17A antibody.

[0229] The term "micronutrient" refers to inorganic salts that are essential but required in very low amounts in the process of cell growth and metabolism, with concentration ranges typically in the nM (nanomolar per liter) to μΜ (micromolar per liter) scale.

[0230] The term "mineral" refers to inorganic salts that are essential but required in higher amounts in the process of cell growth and metabolism, with concentration ranges typically in the mM (millimolar per liter) scale.

[0231] The term "CHO cell" refers to a transformed cell line established from Chinese hamster ovary tissue.

[0232] The term "basal medium" or "basal cell culture medium" is a cell culture medium used for culturing mammalian cells as defined below. It refers to the medium in which the cells are initially cultured from the start of a cell culture run and is not used as an additive for another medium, although various components can be added to the medium. The basal medium serves as a base to which optional further additives or a feed medium can be added during the culturing, i.e. cell culture run. The basal cell culture medium is provided from the start of the cell culture process. Generally, the basal cell culture medium provides nutrients such as a carbon source, amino acids, vitamins, major salts (e.g. sodium chloride or potassium chloride), various micronutrients (e.g. manganese sulfate), pH buffers, and glucose, etc.

[0233] The term "feed" or "feed medium" relates to a nutrient concentrate / concentrated nutrient composition used as a feed in a culture of mammalian cells, which can be added to the culture at some time after inoculation. The feed medium typically has higher concentrations of most, but not all, components of the basal cell culture medium. Generally, the feed medium replaces nutrients such as amino acids and carbohydrates that are consumed during the cell culture. The feed medium is typically added to the (basal) cell culture medium / fermentation broth in a fed-batch mode.

[0234] The above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present application.

[0235] The reagents and raw materials used in the present application are commercially available.

[0236] The positive progress effect of the present application is that:

[0237] The present application optimizes the cell culture medium to obtain a low-cost cell culture medium that can effectively meet the needs of expressing monoclonal antibodies and can realize large-scale culture. Specifically:

[0238] (1) For the cell culture medium (basic medium, feed medium) in the present application, the cells (such as SHR-1701 cells) can normally produce in low, medium and high concentration medium, and are safe and non-toxic.

[0239] (2) Compared with the commercially available classic medium, the highest cell density of the cell culture medium (basic medium, feed medium) in the present application is basically the same, but the cell maintenance is better in the later stage, and similar trends are shown in different cell strains.

[0240] (3) Compared with the commercially available classic medium, the antibody expression amount of the cell culture medium (basic medium, feed medium) in the present application can be increased by more than 30%, and the antibody quality is better (SEC-HPLC purity, reduced CE-SDS purity, non-reduced CE-SDS purity, IEC-HPLC main peak ratio is higher). BRIEF DESCRIPTION OF DRAWINGS

[0241] Figure 1 It is a cell growth chart of HR1 No. BM03 formula under different concentrations.

[0242] Figure 2 It is a batch feed cell growth chart under different concentrations of HR1 No. FeedB formula.

[0243] Figure 3 It is a cell growth comparison chart of expressing SHR-1701 cells using HR1 No. BM03 and HR1 No. FeedB medium, and commercially available medium, respectively.

[0244] Figure 4 It is a cell growth comparison chart of expressing SHR-1805 cells using HR1 No. BM03 and HR1 No. FeedB medium, and commercially available medium, respectively.

[0245] Figure 5 It is a cell growth comparison chart of expressing SHR-1314 cells using HR1 No. BM03 and HR1 No. FeedB medium, and commercially available medium, respectively. DETAILED DESCRIPTION

[0246] Before further description of the present application specific embodiments thereof, it is to be understood that the application is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description at least one embodiment. There are numerous specific details of construction and arrangements of components as described herein, and applicants depart therefrom in various specific embodiments disclosed herein. However, applicant has sought to provide an application which is as generic as possible, as well as to describe the application in sufficient detail that others skilled in the art can practice the application, without unnecessary restriction(s). Other embodiments, adaptations, and variations of such specific embodiments can be disclosed, and are within the scope of the present application. Also, it is to be understood that the specific methods, devices, materials, and the like which are described herein are exemplary and are not intended as limitations on the scope of the application.

[0247] When numerical ranges are given, understand that every numerical range is a range of values that includes any value between the two endpoints, unless otherwise specified in this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the application, unless otherwise indicated. Unless otherwise specified, all methods were carried out in accordance with conventional methods and procedures as is well known and as described in the literature, or as described herein.

[0248] The application is further described in the following examples, which do not limit the scope of the application described. Unless otherwise indicated, the experimental methods in the following examples were carried out according to conventional methods and conditions, or as described in the manufacturer's instructions.

[0249] SHR-1701 WCB (Working Cell Bank): Expressing PD-L1 / TGF-β antibody, the construction method is as follows: SHR-1701 light and heavy chain sequences (referring to patent CN 110050000A, heavy chain sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2, light chain sequence as shown in SEQ ID NO: 3) are respectively cloned into expression vectors pXC17.4 and pXC18.4 provided by Lonza company to form recombinant plasmids. The constructed recombinant plasmid is transfected into host cells (CHOK1SV GS-KO, from Lonza company) and further screened to obtain the original cell bank. The cells of the original cell bank are passaged and proliferated to prepare a batch of homogeneous cell suspension, which is quantitatively packaged in 1 mL per cell freezing tube and stored in liquid nitrogen for long-term preservation, which is the master cell bank. The cells of the master cell bank are passaged and proliferated to prepare a batch of homogeneous cell suspension, which is quantitatively packaged in 1 mL per cell freezing tube and stored in liquid nitrogen for long-term preservation, which is the working cell bank. 7 cells / mL, 1 mL per cell freezing tube, and stored in liquid nitrogen for long-term preservation, which is the master cell bank. The cells of the master cell bank are passaged and proliferated to prepare a batch of homogeneous cell suspension, which is quantitatively packaged in 1 mL per cell freezing tube and stored in liquid nitrogen for long-term preservation, which is the working cell bank. 7 cells / mL, 1 mL per cell freezing tube, and stored in liquid nitrogen for long-term preservation, which is the master cell bank. The cells of the master cell bank are passaged and proliferated to prepare a batch of homogeneous cell suspension, which is quantitatively packaged in 1 mL per cell freezing tube and stored in liquid nitrogen for long-term preservation, which is the working cell bank.

[0250] In the present application, SHR-1701 refers to PD-L1 (Programmed Death Ligand-1) / TGF-β (Transforming Growth Factor-β) antibody.

[0251] SHR-1805 WCB: expressing Her2 antibody, the construction method is that: the light and heavy chain sequences of SHR-1805 (trastuzumab light and heavy chain sequences) are respectively cloned into the expression vectors pXC17.4 and pXC18.4 provided by Lonza Company to form recombinant plasmids. The constructed recombinant plasmids are transfected into host cells (CHOK1SV GS-KO, from Lonza Company) and further screened to obtain the original cell bank. The cells in the original cell bank are passaged and proliferated to prepare a batch of homogeneous cell suspension, which is quantitatively packed in a cell freezing tube at 1x10 7 cells / mL, and stored in liquid nitrogen for long-term preservation, which is the master cell bank. The cells in the master cell bank are passaged and proliferated to prepare a batch of homogeneous cell suspension, which is quantitatively packed in a cell freezing tube at 1x10 7 cells / mL, and stored in liquid nitrogen for long-term preservation, which is the working cell bank.

[0252] In the present application, SHR-1805 refers to Her2 (human epidermal growth factor receptor 2) antibody.

[0253] SHR-1314 WCB: expressing IL-17A antibody, the construction method is that: the light and heavy chain sequences of SHR-1314 (the light chain shown in SEQ ID NO: 4 and the heavy chain shown in SEQ ID NO: 5, refer to the patent WO2021018191A1) are respectively cloned into the expression vectors pXC17.4 and pXC18.4 provided by Lonza Company to form recombinant plasmids. The constructed recombinant plasmids are transfected into host cells (CHOK1SV GS-KO, from Lonza Company) and further screened to obtain the original cell bank. The cells in the original cell bank are passaged and proliferated to prepare a batch of homogeneous cell suspension, which is quantitatively packed in a cell freezing tube at 1x10 7 cells / mL, and stored in liquid nitrogen for long-term preservation, which is the master cell bank. The cells in the master cell bank are passaged and proliferated to prepare a batch of homogeneous cell suspension, which is quantitatively packed in a cell freezing tube at 1x10 7 cells / mL, and stored in liquid nitrogen for long-term preservation, which is the working cell bank.

[0254] In the present application, SHR-1314 refers to IL-17A (interleukin-17A) antibody.

[0255] Tropolone is also called cycloheptatrienone, molecular formula C7H6O2, CAS number 533-75-5.

[0256] Iron (II) sulfate is an inorganic compound with the chemical formula FeSO4.

[0257] Putrescine refers to 1,4-butanediamine, an organic compound with the chemical formula C4H 12 N2, is a colorless crystal with the odor of hexahydropyridine.

[0258] Gibco Dynamis: purchased from Gibco, item number A2661503.

[0259] HyClone Cell Boost 7a, HyClone Cell Boost 7b: purchased from Hyclone, item numbers SH31026.01 and SH31027.07, respectively.

[0260] DO refers to the percentage of dissolved oxygen concentration in the fermentation broth relative to the oxygen concentration in the air.

[0261] The detection method of antibody expression amount is as follows: (1) solution preparation: mobile phase A (100 mM PBS + 150 mM NaCl, pH 7.4), mobile phase B (100 mM NaH2PO4, pH 2.5); (2) chromatographic conditions: flow rate 2.0 ml / min, injection volume 10 μL, column temperature 25 ℃, sample room temperature 2-8 ℃, detection wavelength 280 nm, running time 3 min; (3) elution conditions: 0-0.5 min isocratic 100% mobile phase A, 0.5-1.5 min isocratic 100% mobile phase B, 1.5-3 min isocratic 100% mobile phase A; (4) sample analysis: the sample to be tested is determined according to the chromatographic conditions and elution conditions, and the chromatogram is recorded; (5) data analysis: by comparing with the chromatogram of blank buffer, the chromatogram is integrated to calculate the antibody expression amount.

[0262] The detection method of SEC-HPLC (Size Exclusion Chromatography - High Performance Liquid Chromatography) purity is as follows: (1) solution preparation: mobile phase A (200 mM PBS+50 mM Na2SO4); (2) sample preparation: dilute the sample to a final concentration of about 10 mg / ml with mobile phase A; (3) chromatographic conditions: flow rate 0.5 ml / min, sample amount 100 μg, column temperature 30 ℃, sample room temperature 2-8 ℃, detection wavelength 280 nm, running time 40 min; (4) elution conditions: 100% mobile phase A isocratic elution for 40 min; (5) sample analysis: determine the prepared sample according to the chromatographic conditions and elution conditions, and record the chromatogram; (6) data analysis: compare the chromatogram with the blank buffer chromatogram, integrate the chromatogram, and calculate the peak area percentage of monomer, polymer and low molecular weight impurities by peak area normalization method.

[0263] The detection method of reduced CE-SDS (Reduced Capillary Electrophoresis - Sodium DodecylSulfate) purity is as follows: (1) reduced sample preparation: take 100 μg of sample and mix with SDS-MW Sample Buffer and 713 mM 2-mercaptoethanol to form a mixed solution, denature the mixed solution at 70 ℃ for 10 minutes, and cool for 5 minutes; (2) instrument parameters: capillary effective length 20 cm, temperature control 25 ℃, diode array detector wavelength 220 nm, sample room temperature 10 ℃; (3) sample analysis: take the prepared non-reduced sample in a 200 μL PCR tube, and determine according to the sample separation method, and record the chromatogram; (4) analyze and process data: compare the chromatogram with the blank control chromatogram, integrate the chromatogram of the detected sample, and calculate the corrected peak area percentage of the main peak, i.e. the reduced purity.

[0264] The detection method of non-reduced CE-SDS purity (Non-Reduced CE-SDS, non-reduced capillary electrophoresis-sodium dodecyl sulfate) is: (1) non-reduced sample preparation: take 100 μg sample and SDS-MW sample buffer, 250 mM iodoacetamide to make a mixed solution, the mixed solution is denatured at 70 ℃ for 10 minutes, and cooled for 5 minutes; (2) instrument parameters: capillary effective length 20 cm, temperature control 20 ℃, diode array detector wavelength 220 nm, sample chamber temperature 10 ℃; (3) sample analysis: take the prepared non-reduced sample in a 200 uL PCR tube, and determine according to the sample separation method, record the chromatogram; (4) analyze and process data: compare the chromatogram of the blank control with the chromatogram of the sample, integrate the chromatogram of the sample, and calculate the peak area percentage of the main peak, that is, the non-reduced purity.

[0265] The detection method of IEC-HPLC (Ion Exchange Chromatography - High Performance Liquid Chromatography, ion exchange chromatography-high performance liquid chromatography) is: (1) solution preparation: mobile phase A, 20 mM ACES (pH 7.0), mobile phase B, 20 mM ACES+300 mM NaCl (pH 7.0); (2) sample preparation: centrifuge at 12000 rpm for 1 min to take the supernatant, dilute to a final concentration of about 5.0 mg / mL; (3) chromatographic conditions: flow rate 0.8 ml / min, sample amount 50 μg, column temperature 40 ℃, sample chamber temperature 4 ℃, detection wavelength 280 nm, running time 60 min; (4) elution conditions: 0~5min isocratic 90% mobile phase A+10% mobile phase B, 5~50min gradient to 69% mobile phase A+31% mobile phase B, 50~55min isocratic 100% mobile phase B, 55~60min isocratic 90% mobile phase A+10% mobile phase B; (5) sample analysis: take the prepared sample to determine according to the chromatographic conditions and elution conditions, record the chromatogram; (6) data analysis: compare the chromatogram of the blank buffer with the chromatogram, integrate the chromatogram, and calculate the peak area percentage of the main peak, the basic zone peak area percentage and the acid zone peak area percentage by peak area normalization method.

[0266] Example 1 Determination of BM03 formulation of HR1

[0267] 1. Materials and methods

[0268] 1.1 Cell lines

[0269] Table 1

[0270]

[0271] 1.2 Concentrations of each component in HR1 BM03

[0272] Table 2

[0273]

[0274]

[0275] The method for preparing BM03 is as follows: Mix the components in the table above according to their concentrations and then dilute with water to the target volume.

[0276] 1.3 Cell passage culture

[0277] Three cell lines were taken from the working cell bank and rapidly thawed in a 37 ℃ water bath. The thawed cell solutions were then added to 125 mL shake flasks containing 40 mL of the basal culture medium shown in Table 2 (one flask each for low, medium, and high concentrations) in a biosafety cabinet. The flasks were then placed in a CO2 shaker for incubation under the following conditions: temperature 36 ℃, rotation speed 110 rpm, CO2 concentration 8.0%, and humidity 80%.

[0278] When the cell density reaches (4-10)×10 6 Cells / mL: The low-concentration group was passaged once using the low-concentration culture medium shown in Table 2; the medium-concentration group was passaged once using the medium-concentration culture medium shown in Table 2; and the high-concentration group was passaged once using the high-concentration culture medium shown in Table 2. The viable cell density was controlled at 0.3 × 10⁻⁶ cells / mL after passage. 6 cells / mL or higher, until the cells grow to (4-10)×10⁻¹⁰. 6 When the number of cells / mL reaches a certain level, proceed with the next subculture, and continue subculturing for 30 days.

[0279] 2. Experimental Results

[0280] like Figure 1 As shown, SHR-1701 cells can be produced normally in low, medium, and high concentration culture media, and the doubling time is consistent, approximately 22 hours. This result fully demonstrates that low, medium, and high concentration culture media meet the growth requirements of SHR-1701 cells.

[0281] Example 2: Determination of FeedB Formulation for HR1

[0282] 1. Materials and Methods

[0283] 1.1 Cell lines

[0284] Table 3

[0285]

[0286] 1.2 Concentration of each component of Feed B No. 1

[0287] Table 4

[0288]

[0289]

[0290] The method for preparing the components of Feed B is as follows: mix the components in the above table according to the concentrations, and then add water to the target volume.

[0291] 1.3 Cell culture

[0292] Take one cell from the working cell bank and quickly thaw it in a 37 °C water bath. Then, in a biological safety cabinet, add the thawed cell solution to a 125 mL shake flask containing 40 mL of BM03 medium of No. 1 HR1, and place it in a CO2 shaker for culture. The culture conditions are as follows: temperature 36 °C, rotation speed 110 rpm, CO2 concentration 8.0%, and humidity 80%.

[0293] When the cell density reaches (4-10) x 10 6 cells / mL, perform shake flask fed-batch culture. A total of 3 groups are set, and the basic culture is HR1 No. 1 BM03 medium. The fed-batch medium uses low, medium, and high concentrations of HR1 No. 1 Feed B, respectively. The specific steps of shake flask fed-batch culture are as follows.

[0294] Inoculation: the inoculation density is controlled at (0.8-1.0) x 10 6 cells / mL, and the initial culture volume is 40 mL.

[0295] Set the shaker parameters: temperature 36.0 °C, rotation speed 110 rpm, carbon dioxide 8%, and humidity 80%.

[0296] Sugar supplementation: on the day of feeding, sample and detect the glucose concentration, which is less than 4 g / L. Then, supplement the sugar to a concentration of 6 g / L.

[0297] Feeding: on the 4th / 6th / 8th / 10th / 12th day, add HR1 No. 1 Feed B at 2.8%, 3.8%, 5.0%, 5.0%, and 4.4% of the initial volume, respectively (low, medium, and high concentrations for the three groups).

[0298] Harvesting: culture to the 14th day or when the viability is less than 80%.

[0299] 2. Experimental results

[0300] For example, Figure 2As shown, SHR-1701 cells were subjected to fed-batch culture with low, medium and high concentrations of HR No. 1 medium, and the cells could normally produce, and the antibody expression reached 4.45 g / L, 5.56 g / L, 4.03 g / L, respectively, all meeting the production requirements.

[0301] Example 3 Comparison of HR No. 1 BM03 and HR No. 1 FeedB with commercially available culture media in the culture of SHR-1701 cell strain that can express

[0302] 1. Materials and methods

[0303] 1.1 Cell strain

[0304] Table 5

[0305]

[0306] 1.2 Experimental methods

[0307] In this example, the concentrations of HR No. 1 BM03 in Example 1 and HR No. 1 FeedB in Example 2 were used for testing, and the commercially available culture media selected the classic combination Gibco Dynamis, HyClone Cell Boost 7a, HyClone Cell Boost 7b, and the scheme is shown in Table 6.

[0308] Table 6

[0309]

[0310] 2. Experimental results

[0311] SHR-1701 cell strain culture used HR No. 1 BM03 and HR No. 1 FeedB, commercially available culture medium (Gibco Dynamis, HyClone Cell Boost 7a, HyClone Cell Boost 7b), cell growth as Figure 3 As shown, compared with the commercially available classic medium, the highest cell density of HR No. 1 BM03 and HR No. 1 FeedB was basically the same, but the cell maintenance was better in the later period. Details are shown in Table 7.

[0312] Table 7

[0313]

[0314] The antibody expression and antibody quality are shown in Table 8 below: compared with the commercially available classic medium, the antibody expression of HR1 BM03 and HR1 FeedB after culture increased by more than 30%, and the antibody quality was better (SEC-HPLC purity, reduced CE-SDS purity, non-reduced CE-SDS purity, and IEC-HPLC main peak ratio were higher).

[0315] Table 8

[0316]

[0317] Example 4 Comparison of HR1 BM03 and HR1 FeedB with commercially available medium in the culture of SHR-1805 cell strain that can express

[0318] 1. Materials and methods

[0319] 1.1 Cell strain

[0320] Table 9

[0321]

[0322] 1.2 Experimental method

[0323] In this example, the concentrations of HR1 BM03 in Example 1 and HR1 FeedB in Example 2 were used for testing, and the commercially available medium selected Gibco Dynamis, HyClone Cell Boost 7a, and HyClone Cell Boost 7b. The scheme is shown in Table 10 below.

[0324] Table 10

[0325]

[0326] 2. Experimental results

[0327] The SHR-1805 cell strain was cultured using HR1 BM03 and HR1 FeedB, and the commercially available medium (Gibco Dynamis, HyClone Cell Boost 7a, and HyClone Cell Boost 7b). The cell growth is shown in Table 11 below: compared with the commercially available classic medium, the highest cell density of HR1 BM03 and HR1 FeedB was 10% higher, and the cell maintenance was better in the later stage. The details are shown in Table 11. Figure 4

[0328] Table 11

[0329]

[0330] ​The antibody expression and antibody quality are shown in Table 12 below: compared with the commercially available classic medium, the expression of antibody is increased by 45% after HR1 BM03 and HR1 FeedB are cultured, and the antibody quality is better (SEC-HPLC purity, reduced CE-SDS purity, non-reduced CE-SDS purity, and IEC-HPLC main peak ratio are higher).

[0331] Table 12

[0332]

[0333] Example 5 Comparison of HR1 BM03 and HR1 FeedB with commercially available medium in the culture of SHR-1314 cell strain that can express

[0334] 1. Materials and methods

[0335] 1.1 Cell strain

[0336] Table 13

[0337]

[0338] 1.2 Experimental method

[0339] In this example, the concentrations in HR1 BM03 in Example 1 and the concentrations in HR1 FeedB in Example 2 are used for testing, and the commercially available medium is selected as the classic combination Gibco Dynamis, HyClone Cell Boost 7a, and HyClone Cell Boost 7b. The scheme is shown in Table 14 below.

[0340] Table 14

[0341]

[0342] 2. Experimental results

[0343] The SHR-1314 cell strain culture uses HR1 BM03 and HR1 FeedB, and the commercially available medium (Gibco Dynamis, HyClone Cell Boost 7a, and HyClone Cell Boost 7b), and the cell growth is shown in Table 15 below: compared with the commercially available classic medium, the highest cell density of HR1 BM03 and HR1 FeedB is increased by 25%, and the cell maintenance is better in the later period. The details are shown in Table 15. Figure 5 Table 15

[0344]

[0345]

[0346] ​The antibody expression and antibody quality are shown in Table 16 below: compared with the commercially available classic medium, the expression of antibody of HR1 No. BM03 and HR1 No. FeedB after culture is increased by 50%, and the antibody quality is better (SEC-HPLC purity, reduced CE-SDS purity, non-reduced CE-SDS purity, and IEC-HPLC main peak ratio are higher).

[0347] Table 16

[0348]

[0349] Example 6 Effect of tropolone concentration on cell growth

[0350] On the basis of the concentration in the HR1 No. BM03 component in Example 1, the tropolone concentration in the HR1 No. BM03 basic medium is adjusted to 0, 0.6 mg / L, 1.2 mg / L, and 2.4 mg / L, respectively, and the initial inoculation density is controlled at 0.2 x 10 6 cells / ml, and the cell density is compared after 5 days of culture. The cultured cell strain is SHR-1701 WCB.

[0351] The experimental results show that the addition of no tropolone or too high concentration of tropolone in the basic medium will inhibit cell growth, and the data are shown in the table below.

[0352] Table 17

[0353]

[0354] Example 7 Effect of tyrosine disodium salt, cysteine, tryptophan, and lysine concentration on cell growth and expression

[0355] (1) Effect of tyrosine disodium salt, cysteine, tryptophan, and lysine concentration in the basic medium on cell growth and expression

[0356] A 50 mM L-tyrosine disodium salt + 25 mM L-cysteine + 50 mM L-tryptophan + 100 mM L-lysine stock solution (1x) is prepared, and 1x amino acid stock solution is mixed with the basic medium without these four amino acids (the ratio of other components of the basic medium without these four amino acids is the same as the concentration in the HR1 No. BM03 component in Example 1) to form basic media with different concentrations, and cell culture is performed. The cultured cell strain is SHR-1701 WCB. The initial inoculation density is consistent, all around 0.2 x 10 6 cells / ml, and the cell density is compared after 5 days of culture.

[0357] The experimental results show that the addition of no these four amino acids or too high concentration of these four amino acids in the basic medium will inhibit cell growth, and the data are shown in the table below.

[0358] Table 18

[0359]

[0360] Table 19

[0361]

[0362] (2) Effect of the concentrations of tyrosine disodium salt, cysteine, tryptophan, lysine in the feed medium on cell growth and expression

[0363] The concentrations of the four amino acids, L-tyrosine disodium salt, L-cysteine, L-tryptophan, L-lysine in the fixed basal medium were 5 mM, 2.5 mM, 5 mM, 10 mM respectively, and the other components of the basal medium were in the same proportions as in the HR1 BM03 component in Example 1. The effect of the four amino acids in the feed medium on cell growth and expression was studied. A 500 mM L-tyrosine disodium salt + 250 mM L-cysteine + 500 mM L-tryptophan + 1000 mM L-lysine stock solution (1x) was prepared, and the 1x amino acid stock solution was mixed with a feed medium not containing the four amino acids (the other components of the feed medium not containing the four amino acids were in the same proportions as in the HR1 Feed B formula in Example 2) to prepare feed media of different concentrations, and cell culture was performed. The experimental method was the same as in Example 2.

[0364] Table 20

[0365]

[0366] Table 21

[0367]

[0368] Table 22

[0369]

[0370] Example 8 Effect of VB6 and VB12 on antibody expression

[0371] A 2 mM VB6 + 0.2 mM VB12 stock solution (1x) was prepared, and the 1x vitamin stock solution was mixed with a basal medium and a feed medium not containing the two vitamins (the other components of the basal medium not containing VB6 and VB12 were in the same proportions as in the HR1 BM03 component in Example 1, and the other components of the feed medium not containing VB6 and VB12 were in the same proportions as in the HR1 Feed B formula in Example 2) to prepare media of different concentrations, and the effect of the concentrations of VB6 and VB12 in the basal and feed media on antibody expression was studied. The experimental method was the same as in Example 2.

[0372] The results are shown in the following table: The antibody expression first increased with increasing VB6 and VB12 concentration, reached a peak and then decreased with increasing VB6 and VB12 concentration.

[0373] Table 23

[0374]

[0375] Table 24

[0376]

[0377] Although the specific embodiments of the present application are described above, it should be understood by those skilled in the art that the present application is only an example, and the protection scope of the present application is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present application, and such changes and modifications all fall within the protection scope of the present application.

Claims

1. A basal cell culture medium, characterized in that, The basic cell culture medium is composed of solid components and water; The solid components are composed of toluhydroquinone, amino acids, vitamins, inorganic salts, polyamines, trace elements and carbon sources; The amino acids are L-methionine, L-proline, L-aspartic acid, L-tyrosine disodium salt, L-asparagine, L-cysteine, L-serine, L-tryptophan, L-lysine, L-alanine, L-histidine, L-threonine, L-valine, L-isoleucine, L-leucine, L-phenylalanine, L-glycine, L-arginine and L-glutamic acid; The vitamins are vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid and alpha-lipoic acid; The inorganic salts are sodium phosphate, potassium chloride, zinc sulfate, sodium selenite, iron (II) sulfate, copper sulfate, calcium chloride, magnesium sulfate, sodium thiosulfate and sodium chloride; The polyamines are putrescine, spermidine and spermine; The trace elements are manganese chloride, aluminum trichloride, cadmium sulfate and cobalt chloride; The carbon sources are glucose and sodium pyruvate; The concentration of toluhydroquinone in the basic cell culture medium is 0.6-1.2 mg / L; The concentration of L-tyrosine disodium salt in the basic cell culture medium is 1-10 mM; The concentration of L-cysteine in the basic cell culture medium is 0.5-5 mM; The concentration of L-tryptophan in the basic cell culture medium is 1-10 mM; The concentration of L-lysine in the basic cell culture medium is 2-20 mM; The concentration of vitamin B6 in the basic cell culture medium is 0.002-0.2 mM; The concentration of vitamin B12 in the basic cell culture medium is 0.0002-0.02 mM; The concentration of L-methionine in the basic cell culture medium is 1-5 mM; The concentration of L-proline in the basic cell culture medium is 0.5-5 mM; The concentration of L-aspartic acid in the basic cell culture medium is 0.3-5 mM; The concentration of L-asparagine in the basic cell culture medium is 0.5-3 mM; The concentration of L-serine in the basic cell culture medium is 1-5 mM; The concentration of L-alanine in the basic cell culture medium is 1-5 mM; The concentration of L-histidine in the basic cell culture medium is 3-10 mM; The concentration of L-threonine in the basic cell culture medium is 1-5 mM; The concentration of L-valine in the basic cell culture medium is 0.2-2 mM; The concentration of L-isoleucine in the basic cell culture medium is 3-6 mM; The concentration of L-leucine in the basic cell culture medium is 2-6 mM; The concentration of L-phenylalanine in the basic cell culture medium is 5-10 mM; The concentration of L-glycine in the basic cell culture medium is 0.1-2 mM; The concentration of L-arginine in the basic cell culture medium is 2-6 mM; The concentration of the L-glutamic acid in the basic cell culture medium is 5-20 mM; The concentration of the vitamin B1 in the basic cell culture medium is 0.005-0.1 mM; The concentration of the vitamin B2 in the basic cell culture medium is 0.005-0.05 mM; The concentration of the nicotinamide in the basic cell culture medium is 0.001-0.02 mM; The concentration of the vitamin B5 in the basic cell culture medium is 0.01-0.1 mM; The concentration of the choline in the basic cell culture medium is 0.1-1 mM; The concentration of the folic acid in the basic cell culture medium is 0.005-0.5 mM; The concentration of the α-lipoic acid in the basic cell culture medium is 0.001-0.01 mM; The concentration of the sodium phosphate in the basic cell culture medium is 0.5-2 mM; The concentration of the potassium chloride in the basic cell culture medium is 0.5-2 mM; The concentration of the zinc sulfate in the basic cell culture medium is 0.0005-0.001 mM; The concentration of the sodium selenite in the basic cell culture medium is 0.00001-0.0001 mM; The concentration of the iron (II) sulfate in the basic cell culture medium is 0.02-0.2 mM; The concentration of the copper sulfate in the basic cell culture medium is 0.0005-0.001 mM; The concentration of the calcium chloride in the basic cell culture medium is 0.05-0.5 mM; The concentration of the magnesium sulfate in the basic cell culture medium is 0.1-2 mM; The concentration of the sodium thiosulfate in the basic cell culture medium is 1-5 mM; The concentration of the sodium chloride in the basic cell culture medium is 20-50 mM; The concentration of the putrescine in the basic cell culture medium is 0.01-0.1 mM; The concentration of the spermidine in the basic cell culture medium is 0.01-0.1 mM; The concentration of the spermine in the basic cell culture medium is 0.01-0.1 mM; The concentration of the manganese chloride in the basic cell culture medium is 0.000001-0.0001 mM; The concentration of the aluminum trichloride in the basic cell culture medium is 0.000001-0.0001 mM; The concentration of the cadmium sulfate in the basic cell culture medium is 0.000001-0.0001 mM; The concentration of the cobalt chloride in the basic cell culture medium is 0.000001-0.0001 mM; The concentration of the glucose in the basic cell culture medium is 20-40 mM; The concentration of the sodium pyruvate in the basic cell culture medium is 0.1-5 mM.

2. The basal cell culture medium of claim 1, wherein, The basic cell culture medium satisfies one or more of the following conditions: (1) The concentration of the L-tyrosine disodium salt in the basic cell culture medium is 2.5-10 mM; (2) The concentration of the L-cysteine in the basic cell culture medium is 1-5 mM; (3) The concentration of the L-tryptophan in the basic cell culture medium is 2.5-10 mM; (4) the concentration of the L-lysine in the basal cell culture medium is 2-20 mM; (5) the concentration of the vitamin B6 in the basal cell culture medium is 0.01-0.2 mM; (6) the concentration of the vitamin B12 in the basal cell culture medium is 0.001-0.02 mM; (7) the concentration of the tocofylliquin in the basal cell culture medium is 0.9-1.2 mg / L; and, (8) the basal cell culture medium is used for cell culture and / or production of a target protein.

3. The basal cell culture medium of claim 1, wherein, The basal cell culture medium is selected from any one of the following formulations: ； ； ； 。 4. A feed medium, characterized in that, The feed medium is composed of solid components and water; The solid components are composed of tocofylliquin, amino acids, vitamins, inorganic salts, polyamines, trace elements and carbon sources; The amino acids are L-methionine, L-proline, L-aspartic acid, L-tyrosine disodium salt, L-asparagine, L-cysteine, L-serine, L-tryptophan, L-lysine, L-alanine, L-histidine, L-threonine, L-valine, L-isoleucine, L-leucine, L-phenylalanine, L-glycine, L-arginine and L-glutamic acid; The vitamins are vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid and α-lipoic acid; The inorganic salts are sodium phosphate, potassium chloride, zinc sulfate, sodium selenite, iron (II) sulfate, copper sulfate, calcium chloride, magnesium sulfate, sodium thiosulfate and sodium chloride; The polyamines are putrescine, spermidine and spermine; The trace elements are manganese chloride, aluminum trichloride, cadmium sulfate and cobalt chloride; The carbon sources are glucose and sodium pyruvate; The concentration of the tocofylliquin in the feed medium is 6-12 mg / L; The concentration of the L-tyrosine disodium salt in the feed medium is 10-100 mM; The concentration of the L-cysteine in the feed medium is 5-50 mM; The concentration of the L-tryptophan in the feed medium is 10-100 mM; The concentration of the L-lysine in the feed medium is 20-200 mM; The concentration of the vitamin B6 in the feed medium is 0.1-2 mM; The concentration of the vitamin B12 in the feed medium is 0.01-0.2 mM; The concentration of the L-methionine in the feed medium is 10-50 mM; The concentration of the L-proline in the feed medium is 5-50 mM; The concentration of the L-aspartic acid in the feed medium is 3-50 mM; The concentration of the L-asparagine in the feed medium is 5-30 mM; The concentration of the L-serine in the feed medium is 10-50 mM; The concentration of the L-alanine in the feed medium is 10-50 mM; The concentration of the L-histidine in the feed medium is 30-100 mM; The concentration of the L-threonine in the feed medium is 10-50 mM; The concentration of the L-valine in the feed medium is 2-20 mM; The concentration of the L-isoleucine in the feed medium is 30-60 mM; The concentration of the L-leucine in the feed medium is 20-60 mM; The concentration of the L-phenylalanine in the feed medium is 50-100 mM; The concentration of the L-glycine in the feed medium is 1-20 mM; The concentration of the L-arginine in the feed medium is 20-60 mM; The concentration of the L-glutamic acid in the feed medium is 50-200 mM; The concentration of the vitamin B1 in the feed medium is 0.05-1 mM; The concentration of the vitamin B2 in the feed medium is 0.05-0.5 mM; The concentration of the nicotinamide in the feed medium is 0.01-0.2 mM; The concentration of the vitamin B5 in the feed medium is 0.1-1 mM; The concentration of the choline in the feed medium is 1-10 mM; The concentration of the folic acid in the feed medium is 0.05-5 mM; The concentration of the α-lipoic acid in the feed medium is 0.01-0.1 mM; The concentration of the sodium phosphate in the feed medium is 5-20 mM; The concentration of the potassium chloride in the feed medium is 5-20 mM; The concentration of the zinc sulfate in the feed medium is 0.005-0.01 mM; The concentration of the sodium selenite in the feed medium is 0.0001-0.001 mM; The concentration of the iron (II) sulfate in the feed medium is 0.2-2 mM; The concentration of the copper sulfate in the feed medium is 0.005-0.01 mM; The concentration of the calcium chloride in the feed medium is 0.5-5 mM; The concentration of the magnesium sulfate in the feed medium is 1-20 mM; The concentration of the sodium thiosulfate in the feed medium is 10-50 mM; The concentration of the sodium chloride in the feed medium is 200-500 mM; The concentration of the putrescine in the feed medium is 0.1-1 mM; The concentration of the spermidine in the feed medium is 0.1-1 mM; The concentration of the spermine in the feed medium is 0.1-1 mM; The concentration of the manganese chloride in the feed medium is 0.00001-0.001 mM; The concentration of the aluminum trichloride in the feed medium is 0.00001-0.001 mM; The concentration of the cadmium sulfate in the feed medium is 0.00001-0.001 mM; The concentration of the cobalt chloride in the feed medium is 0.00001-0.001 mM; The concentration of the glucose in the feed medium is 200-400 mM; The concentration of the sodium pyruvate in the feed medium is 1-50 mM.

5. The feed medium of claim 4, wherein, The feed medium satisfies one or more of the following conditions: The concentration of the L-valine in the feed medium is 2-20 mM; The concentration of the L-isoleucine in the feed medium is 30-60 mM; The concentration of the L-leucine in the feed medium is 20-60 mM; The concentration of the L-phenylalanine in the feed medium is 50-100 mM; The concentration of the L-glycine in the feed medium is 1-20 mM; The concentration of the L-arginine in the feed medium is 20-60 mM; The concentration of the L-glutamic acid in the feed medium is 50-200 mM; The concentration of the vitamin B1 in the feed medium is 0.05-1 mM; The concentration of the vitamin B2 in the feed medium is 0.05-0.5 mM; The concentration of the nicotinamide in the feed medium is 0.01-0.2 mM; The concentration of the vitamin B5 in the feed medium is 0.1-1 mM; The concentration of the choline in the feed medium is 1-10 mM; The concentration of the folic acid in the feed medium is 0.05-5 mM; The concentration of the α-lipoic acid in the feed medium is 0.01-0.1 mM; The concentration of the sodium phosphate in the feed medium is 5-20 mM; The concentration of the potassium chloride in the feed medium is 5-20 mM; The concentration of the zinc sulfate in the feed medium is 0.005-0.01 mM; The concentration of the sodium selenite in the feed medium is 0.0001-0.001 mM; The concentration of the iron (II) sulfate in the feed medium is 0.2-2 mM; The concentration of the copper sulfate in the feed medium is 0.005-0.01 mM; The concentration of the calcium chloride in the feed medium is 0.5-5 mM; The concentration of the magnesium sulfate in the feed medium is 1-20 mM; The concentration of the sodium thiosulfate in the feed medium is 10-50 mM; The concentration of the sodium chloride in the feed medium is 200-500 mM; The concentration of the putrescine in the feed medium is 0.1-1 mM; The concentration of the spermidine in the feed medium is 0.1-1 mM; The concentration of the spermine in the feed medium is 0.1-1 mM; The concentration of the manganese chloride in the feed medium is 0.00001-0.001 mM; The concentration of the aluminum trichloride in the feed medium is 0.00001-0.001 mM; The concentration of the cadmium sulfate in the feed medium is 0.00001-0.001 mM; The concentration of the cobalt chloride in the feed medium is 0.00001-0.001 mM; The concentration of the glucose in the feed medium is 200-400 mM; The concentration of the sodium pyruvate in the feed medium is 1-50 mM. The feed medium satisfies one or more of the following conditions: (1) the concentration of L-tyrosine disodium salt in the feed medium is 50-100 mM; (2) the concentration of L-cysteine in the feed medium is 10-50 mM; (3) the concentration of L-tryptophan in the feed medium is 40-100 mM; (4) the concentration of L-lysine in the feed medium is 100-200 mM; (5) the concentration of vitamin B6 in the feed medium is 0.5-2 mM; (6) the concentration of vitamin B12 in the feed medium is 0.05-0.2 mM; and, (7) the feed medium is used for cell culture and / or production of a protein of interest.

6. The feed medium of claim 4, wherein, The formulation of the feed medium is selected from any one of Formulation 16-Formulation 25: ； ； 。 7. A kit for culturing cells, characterized in that, The kit comprises at least one first container and / or at least one second container, wherein: the first container contains the basal cell culture medium as claimed in any one of claims 1-3 or the solid ingredients in the basal cell culture medium, and the second container contains the feed medium as claimed in any one of claims 4-6 or the solid ingredients in the feed medium.

8. The kit for culturing cells according to claim 7, wherein the first container comprises Formulation 1, and the second container comprises Formulation 16; alternatively, the first container comprises Formulation 2, and the second container comprises Formulation 17; alternatively, the first container comprises Formulation 3, and the second container comprises Formulation 18; alternatively, the first container comprises Formulation 8, and the second container comprises Formulation 19, Formulation 20 or Formulation 21; alternatively, the first container comprises Formulation 10 or Formulation 11, and the second container comprises Formulation 22; alternatively, the first container comprises Formulation 12, and the second container comprises Formulation 23, Formulation 24 or Formulation 25; alternatively, the first container comprises Formulation 13, and the second container comprises Formulation 23, Formulation 24 or Formulation 25; alternatively, the first container comprises Formulation 14, and the second container comprises Formulation 22 or Formulation 24; alternatively, the first container comprises Formulation 15, and the second container comprises Formulation 22 or Formulation 24.

9. Use of the basal cell culture medium as claimed in any one of claims 1-3, the feed medium as claimed in any one of claims 4-6 or the kit for culturing cells as claimed in claim 7 or 8 in cell culture and / or production of a protein of interest.

10. Use according to claim 9, wherein The use satisfies one or more of the following conditions: (1) the cells are selected from eukaryotic cells, and the eukaryotic cells are selected from CHO cells or 293 cells; and, (2) the protein of interest is selected from one or more of PD-L1 / TGF-β antibody, Her2 antibody and IL-17A antibody.

11. A method for culturing cells, characterized by, The culture method comprises: seeding cells into the basal cell culture medium as claimed in any one of claims 1-3 for expansion culture; In the amplification culture, a feed medium as described in any one of claims 4 to 6 is added to the culture system, and the amplification culture is continued.

12. The culturing method of cells according to claim 11, wherein The cell is selected from a eukaryotic cell selected from a CHO cell or a 293 cell.

13. A method of producing a protein of interest, characterized by, It comprises the following steps: (i) culturing genetically engineered cells in a basal cell culture medium as described in any one of claims 1 to 3 under suitable conditions for expression, adding a feed medium as described in any one of claims 4 to 6 to the basal cell culture medium, and expressing a protein of interest; and (ii) isolating the protein of interest from the fermentation product.

14. The method of producing a protein of interest according to claim 13, wherein The method for producing a protein of interest satisfies one or more of the following conditions: (1) the cell is selected from a eukaryotic cell selected from a CHO cell or a 293 cell; and (2) the protein of interest is selected from one or more of a PD-L1 / TGF-β antibody, a Her2 antibody, and an IL-17A antibody.

Images