Cell culture medium and application thereof, cell culture method and kit

Abstract

The invention discloses a cell culture medium and application thereof, a cell culture method and a kit. The cell culture medium comprises tropolone, amino acid, vitamin, inorganic salt, polyamine, 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 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- The vitamins comprise vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid and alpha-lipoic acid. The low-cost cell culture medium is obtained, the requirement for expressing monoclonal antibodies can be effectively met, and large-scale culture can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a cell culture medium and its application, cell culture methods, and reagent kits. Background Technology

[0002] Currently, there is a large market demand for monoclonal antibody drugs expressed in CHO cells (Chinese Hamster Ovary cells), with supply falling short of demand. Therefore, large-scale animal cell suspension culture technology, one of the core technologies for the 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 companies lack their own high-efficiency CHO cell culture media. Meanwhile, internationally renowned culture media such as Gibco, Hyclone, and Merck cost thousands of yuan per liter, and their formulas are kept secret, which is not conducive to the subsequent optimization of culture processes.

[0004] There are many ways to classify cell culture media, but basal culture media and supplemented culture media are a core classification method based on their function and role in the culture process, which is especially important in modern industrialized cell culture (such as biopharmaceuticals).

[0005] A basal cell culture medium is a chemically defined or partially chemically defined liquid that provides the essential nutrients necessary for the survival, growth, and maintenance of basic functions of cells cultured in vitro. It serves as the initial liquid environment for cell culture systems, and its core functions are: providing an energy source (primarily glucose); providing building blocks (including amino acids, the raw materials for protein synthesis, and nucleotides, the raw materials for nucleic acid synthesis); maintaining the physiological environment (including inorganic salts, which maintain osmotic pressure and pH balance, and buffering systems such as the sodium bicarbonate / CO2 system); and providing cofactors (such as vitamins, which act as enzyme cofactors in metabolic reactions).

[0006] Fed-feed cell culture medium is a highly concentrated nutrient solution that is added to the basal medium in stages or continuously during cell culture. Its core function is not to provide the initial environment, but to dynamically replenish the key nutrients consumed in large quantities by cells during rapid metabolism and product synthesis, while controlling the accumulation of metabolic byproducts. Specifically: it can replenish depleted nutrients: replenishing rapidly consumed glucose, essential amino acids, etc.; it can extend the culture period: preventing cell apoptosis caused by nutrient depletion and extending the culture time from a few days to several weeks; it can improve yield and quality: maximizing the expression level and consistency of target products through precise nutrient control; and it can regulate metabolism: by controlling the feeding strategy, it can alter cellular metabolic pathways (such as reducing lactic acid accumulation).

[0007] Therefore, developing a low-cost basic cell culture medium and supplementary culture medium that can effectively meet the needs of monoclonal antibody expression is of great significance for the commercial production of antibody drugs. Summary of the Invention

[0008] To overcome the shortcomings of existing CHO culture media, which are either too costly or have poor efficiency, this invention provides a cell culture medium and its application, a cell culture method, and a kit. The basic cell culture medium (e.g., Hengrui No. 1 BM03, also known as HR1 BM03) and the supplemental culture medium (e.g., Hengrui No. 1 FeedB, also known as HR1 FeedB) of this invention can culture cell lines such as SHR-1314, SHR-1701, and SHR-1805 at a low cost, effectively meeting the needs for monoclonal antibody expression and enabling large-scale culture.

[0009] To achieve the above objectives, the present invention adopts the following technical solution.

[0010] In a first aspect, the present invention provides a basic cell culture medium comprising solid components and water;

[0011] The solid component comprises the following components: tyrosine, amino acids, vitamins, inorganic salts, polyamine, trace elements, and carbon source;

[0012] The amino acids include: 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 vitamins include: vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid, and alpha-lipoic acid;

[0014] The inorganic salts include: sodium phosphate, potassium chloride, zinc sulfate, sodium selenite, ferric sulfate (II), copper sulfate, calcium chloride, magnesium sulfate, sodium thiosulfate, and sodium chloride;

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

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

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

[0018] The concentration of tropenophenone in the basal cell culture medium is 0.6~1.2 mg / L;

[0019] The concentration of the L-tyrosine disodium salt in the basal cell culture medium is 1~10mM;

[0020] The concentration of L-cysteine ​​in the basal cell culture medium is 0.5~5mM;

[0021] The concentration of L-tryptophan in the basal cell culture medium is 1~10mM;

[0022] The concentration of L-lysine in the basal cell culture medium is 2-20 mM;

[0023] The concentration of vitamin B6 in the basal cell culture medium is 0.002~0.2mM;

[0024] The concentration of vitamin B12 in the basic cell culture medium is 0.0002~0.02mM.

[0025] In some embodiments of the present invention, the basic cell culture medium is composed of the solid components and the water.

[0026] In some embodiments of the present invention, the basic cell culture medium is used for cell culture and / or production of target proteins.

[0027] In some embodiments of the present invention, the cells are selected from eukaryotic cells.

[0028] In some embodiments of the present invention, the eukaryotic cells are selected from CHO cells or 293 cells.

[0029] In some embodiments of the present invention, the target protein is selected from one or more of PD-L1 / TGF-β antibody, Her2 antibody and IL-17A antibody.

[0030] In some embodiments of the present invention, the concentration of tropenophenone in the basal cell culture medium is 0.9 to 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 present invention, the concentration of 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 present invention, the concentration of L-proline in the basal cell culture medium is 0.5-5 mM, for example 0.5 mM, 1.25 mM or 5 mM.

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

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

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

[0036] In some embodiments of the present invention, the concentration of L-cysteine ​​in the basal cell culture medium is 1-5 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 present invention, the concentration of L-serine in the basal cell culture medium is 1-5 mM, for example 1 mM, 3 mM or 5 mM.

[0038] In some embodiments of the present invention, the concentration of L-tryptophan in the basal cell culture medium is 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 present invention, the concentration of L-lysine in the basal cell culture medium is 2 to 20 mM, for example 2 mM, 5 mM, 10 mM, 15 mM or 20 mM.

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

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

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

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

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

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

[0046] In some embodiments of the present invention, 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 present invention, the concentration of L-glycine in the basal cell culture medium is 0.1~2mM, for example 0.1mM, 1.05mM or 2mM.

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

[0049] In some embodiments of the present invention, 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 present invention, the concentration of vitamin B1 in the basal cell culture medium is 0.005~0.1mM, for example 0.005mM, 0.05mM or 0.1mM.

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

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

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

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

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

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

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

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

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

[0060] In some embodiments of the present invention, the concentration of potassium chloride in the basic cell culture medium is 0.5~2mM, for example 0.5mM, 1.25mM or 2mM.

[0061] In some embodiments of the present invention, the concentration of zinc sulfate in the basic cell culture medium is 0.0005~0.001mM, for example 0.0005mM, 0.00075mM or 0.001mM.

[0062] In some embodiments of the present invention, the concentration of sodium selenite in the basic 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 present invention, the concentration of ferric sulfate (II) in the basal cell culture medium is 0.02~0.2mM, for example 0.02mM, 0.11mM or 0.2mM.

[0064] In some embodiments of the present invention, the concentration of copper sulfate in the basic cell culture medium is 0.0005~0.001mM, for example 0.0005mM, 0.00075mM or 0.001mM.

[0065] In some embodiments of the present invention, the concentration of calcium chloride in the basic cell culture medium is 0.05~0.5mM, for example 0.05mM, 0.25mM or 0.5mM.

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

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

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

[0069] In some embodiments of the present invention, the concentration of putrescine in the basic cell culture medium is 0.01~0.1mM, for example 0.01mM, 0.055mM or 0.1mM.

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

[0071] In some embodiments of the present invention, the concentration of spermine in the basic cell culture medium is 0.01~0.1mM, or for example 0.01mM, 0.055mM or 0.1mM.

[0072] In some embodiments of the present invention, the concentration of manganese chloride in the basic cell culture medium is 0.000001~0.0001mM, for example 0.000001mM, 0.00005mM or 0.0001mM.

[0073] In some embodiments of the present invention, the concentration of aluminum trichloride in the basic cell culture medium is 0.000001~0.0001mM, for example 0.000001mM, 0.00005mM or 0.0001mM.

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

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

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

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

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

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

[0080] In a preferred embodiment of the present invention, the concentrations of the tyrosine, amino acids, vitamins, inorganic salts, polyamine, trace elements, and carbon source in the basic cell culture medium are shown in the table below:

[0081]

[0082]

[0084] In some embodiments of the present invention, the formulation of the basic cell culture medium is shown in the table below:

[0085]

[0086]

[0088] In some embodiments of the present invention, the formulation of the basic cell culture medium is shown in the table below:

[0089]

[0090]

[0092] In some embodiments of the present invention, the formulation of the basic cell culture medium is shown in the table below:

[0093]

[0094]

[0096] In some embodiments of the present invention, the formulation of the basic cell culture medium is shown in the table below:

[0097]

[0098]

[0100] A second aspect of the present invention provides a fed culture medium, the cell culture medium comprising solid components and water;

[0101] The solid component comprises the following components: tyrosine, amino acids, vitamins, inorganic salts, polyamine, trace elements, and carbon source;

[0102] The amino acids include: 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;

[0103] The vitamins include: vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid, and alpha-lipoic acid;

[0104] The inorganic salts include: sodium phosphate, potassium chloride, zinc sulfate, sodium selenite, ferric sulfate (II), copper sulfate, calcium chloride, magnesium sulfate, sodium thiosulfate, and sodium chloride;

[0105] The polyamine includes: putrescine, spermidine, and spermine;

[0106] The trace elements include: manganese chloride, aluminum trichloride, cadmium sulfate, and cobalt chloride;

[0107] The carbon source includes: glucose and sodium pyruvate;

[0108] The concentration of tyrosine in the fed culture medium is 0.6~12 mg / L;

[0109] The concentration of the L-tyrosine disodium salt in the fed culture medium is 10~100mM;

[0110] The concentration of L-cysteine ​​in the fed culture medium is 5-50 mM.

[0111] The concentration of L-tryptophan in the supplemental culture medium is 10~100mM;

[0112] The concentration of L-lysine in the fed culture medium is 20-200 mM;

[0113] The concentration of vitamin B6 in the supplemental culture medium is 0.1~2mM;

[0114] The concentration of vitamin B12 in the supplemental culture medium is 0.01~0.2mM.

[0115] In some embodiments of the present invention, the feed culture medium consists of the solid components and the water.

[0116] In some embodiments of the present invention, the feed culture medium is used for cell culture and / or production of target proteins.

[0117] In some embodiments of the present invention, the cells are selected from eukaryotic cells.

[0118] In some embodiments of the present invention, the eukaryotic cells are selected from CHO cells or 293 cells.

[0119] In some embodiments of the present invention, the target protein is selected from one or more of PD-L1 / TGF-β antibody, Her2 antibody and IL-17A antibody.

[0120] In some embodiments of the present invention, the concentration of tyrosine in the supplemental culture medium is 6 to 12 mg / L, for example 6 mg / L, 9 mg / L or 12 mg / L.

[0121] In some embodiments of the present invention, the concentration of L-methionine in the supplemental culture medium is 10-50 mM, for example 10 mM, 30 mM or 50 mM.

[0122] In some embodiments of the present invention, the concentration of L-proline in the supplemental culture medium is 5 to 50 mM, for example 5 mM, 12.5 mM or 50 mM.

[0123] In some embodiments of the present invention, the concentration of L-aspartic acid in the fed culture medium is 3 to 50 mM, for example 3 mM, 26.5 mM or 50 mM.

[0124] In some embodiments of the present invention, the concentration of the L-tyrosine disodium salt in the supplemental culture medium is 50-100 mM, for example 10 mM, 50 mM, 55 mM or 100 mM.

[0125] In some embodiments of the present invention, the concentration of L-asparagine in the fed culture medium is 5 to 30 mM, for example 5 mM, 17.5 mM or 30 mM.

[0126] In some embodiments of the present invention, the concentration of L-cysteine ​​in the supplemental culture medium is 10-50 mM, for example 5 mM, 10 mM, 25 mM, 30 mM or 50 mM.

[0127] In some embodiments of the present invention, the concentration of L-serine in the supplemental culture medium is 10-50 mM, or for example 10 mM, 30 mM or 50 mM.

[0128] In some embodiments of the present invention, the concentration of L-tryptophan in the supplemental culture medium is 40-100 mM, or for example 10 mM, 40 mM, 50 mM, 70 mM or 100 mM.

[0129] In some embodiments of the present invention, the concentration of L-lysine in the supplemental culture medium is 100-200 mM, for example 20 mM, 100 mM, 150 mM or 200 mM.

[0130] In some embodiments of the present invention, the concentration of L-alanine in the supplemental culture medium is 10-50 mM, for example 10 mM, 30 mM or 50 mM.

[0131] In some embodiments of the present invention, the concentration of L-histidine in the supplemental culture medium is 30-100 mM, for example 30 mM, 65 mM or 100 mM.

[0132] In some embodiments of the present invention, the concentration of L-threonine in the supplemental culture medium is 10-50 mM, for example 10 mM, 30 mM or 50 mM.

[0133] In some embodiments of the present invention, the concentration of L-valine in the supplemental culture medium is 2 to 20 mM, for example 2 mM, 11 mM or 20 mM.

[0134] In some embodiments of the present invention, the concentration of L-isoleucine in the supplemental culture medium is 30-60 mM, for example 30 mM, 45 mM or 60 mM.

[0135] In some embodiments of the present invention, the concentration of L-leucine in the supplemental culture medium is 20-60 mM, for example 20 mM, 40 mM or 60 mM.

[0136] In some embodiments of the present invention, the concentration of L-phenylalanine in the supplemental culture medium is 50-100 mM, for example 50 mM, 75 mM or 100 mM.

[0137] In some embodiments of the present invention, the concentration of L-glycine in the supplemental culture medium is 1 to 20 mM, for example 1 mM, 10.5 mM or 20 mM.

[0138] In some embodiments of the present invention, the concentration of L-arginine in the supplemental culture medium is 20-60 mM, for example 20 mM, 40 mM or 60 mM.

[0139] In some embodiments of the present invention, the concentration of L-glutamic acid in the supplemental culture medium is 50-200 mM, for example 50 mM, 125 mM or 200 mM.

[0140] In some embodiments of the present invention, the concentration of vitamin B1 in the supplemental culture medium is 0.05~1mM, for example 0.05mM, 0.5mM or 1mM.

[0141] In some embodiments of the present invention, the concentration of vitamin B2 in the supplemental culture medium is 0.05~0.5mM, for example 0.05mM, 0.25mM or 0.5mM.

[0142] In some embodiments of the present invention, the concentration of nicotinamide in the supplemental culture medium is 0.01~0.2mM, for example 0.01mM, 0.1mM or 0.2mM.

[0143] In some embodiments of the present invention, the concentration of vitamin B5 in the supplemental culture medium is 0.1 to 1 mM, for example 0.1 mM, 0.5 mM or 1 mM.

[0144] In some embodiments of the present invention, the concentration of vitamin B6 in the supplemental culture medium is 0.5 to 2 mM, for example 0.1 mM, 0.5 mM, 0.6 mM, 1 mM or 2 mM.

[0145] In some embodiments of the present invention, the concentration of vitamin B12 in the supplemental culture medium is 0.05~0.2mM, for example 0.01mM, 0.05mM, 0.06mM, 0.1mM or 0.2mM.

[0146] In some embodiments of the present invention, the concentration of choline in the supplemental culture medium is 1 to 10 mM, for example 1 mM, 5.5 mM or 10 mM.

[0147] In some embodiments of the present invention, the concentration of folic acid in the supplemental culture medium is 0.05~5mM, for example 0.05mM, 2.5mM or 5mM.

[0148] In some embodiments of the present invention, the concentration of α-lipoic acid in the fed culture medium is 0.01~0.1mM, for example 0.01mM, 0.05mM or 0.1mM.

[0149] In some embodiments of the present invention, the concentration of sodium phosphate in the supplemental culture medium is 5 to 20 mM, for example 5 mM, 12.5 mM or 20 mM.

[0150] In some embodiments of the present invention, the concentration of potassium chloride in the supplemental culture medium is 5-20 mM, for example 5 mM, 12.5 mM or 20 mM.

[0151] In some embodiments of the present invention, the concentration of zinc sulfate in the supplemental culture medium is 0.005~0.01mM, for example 0.005mM, 0.0075mM or 0.01mM.

[0152] In some embodiments of the present invention, the concentration of sodium selenite in the supplemental culture medium is 0.0001~0.001mM, or for example 0.0001mM, 0.0005mM or 0.001mM.

[0153] In some embodiments of the present invention, the concentration of ferric sulfate (II) in the supplemental culture medium is 0.2 to 2 mM, for example 0.2 mM, 1.1 mM or 2 mM.

[0154] In some embodiments of the present invention, the concentration of copper sulfate in the supplemental culture medium is 0.005~0.01mM, for example 0.005mM, 0.0075mM or 0.01mM.

[0155] In some embodiments of the present invention, the concentration of calcium chloride in the supplemental culture medium is 0.5 to 5 mM, for example 0.5 mM, 2.5 mM or 5 mM.

[0156] In some embodiments of the present invention, the concentration of magnesium sulfate in the supplemental culture medium is 1 to 20 mM, for example 1 mM, 10.5 mM or 20 mM.

[0157] In some embodiments of the present invention, the concentration of sodium thiosulfate in the supplemental culture medium is 10-50 mM, for example 10 mM, 30 mM or 50 mM.

[0158] In some embodiments of the present invention, the concentration of sodium chloride in the supplemental culture medium is 200-500 mM, for example 200 mM, 350 mM or 500 mM.

[0159] In some embodiments of the present invention, the concentration of putrescine in the feed culture medium is 0.1~1mM, or for example 0.1mM, 0.55mM or 1mM.

[0160] In some embodiments of the present invention, the concentration of spermidine in the supplemental culture medium is 0.1~1mM, for example 0.1mM, 0.55mM or 1mM.

[0161] In some embodiments of the present invention, the concentration of spermine in the supplemental culture medium is 0.1~1mM, or for example 0.1mM, 0.55mM or 1mM.

[0162] In some embodiments of the present invention, the concentration of manganese chloride in the supplemental culture medium is 0.00001~0.001mM, for example 0.00001mM, 0.0005mM or 0.001mM.

[0163] In some embodiments of the present invention, the concentration of aluminum trichloride in the supplemental culture medium is 0.00001~0.001mM, or for example 0.00001mM, 0.0005mM or 0.001mM.

[0164] In some embodiments of the present invention, the aluminum trichloride is aluminum trichloride hexahydrate.

[0165] In some embodiments of the present invention, the concentration of cadmium sulfate in the supplemental culture medium is 0.00001~0.001mM, for example 0.00001mM, 0.0005mM or 0.001mM.

[0166] In some embodiments of the present invention, the concentration of cobalt chloride in the cell culture medium is 0.00001~0.001mM, for example 0.00001mM, 0.0005mM or 0.001mM.

[0167] In some embodiments of the present invention, the cobalt chloride is cobalt chloride hexahydrate.

[0168] In some embodiments of the present invention, the concentration of glucose in the supplemental culture medium is 200-400 mM, or for example 200 mM, 300 mM or 400 mM.

[0169] In some embodiments of the present invention, the concentration of sodium pyruvate in the supplemental culture medium is 1 to 50 mM, for example 1 mM, 25.5 mM or 50 mM.

[0170] In a preferred embodiment of the present invention, the concentrations of the tyrosine, amino acids, vitamins, inorganic salts, polyamine, trace elements, and carbon source in the supplemental culture medium are shown in the table below:

[0171]

[0172]

[0174] In some embodiments of the present invention, the formulation of the supplemental culture medium is shown in the table below:

[0175]

[0176]

[0178] In some embodiments of the present invention, the formulation of the supplemental culture medium is shown in the table below:

[0179]

[0180]

[0182] In some embodiments of the present invention, the formulation of the supplemental culture medium is shown in the table below:

[0183]

[0184]

[0186] A third aspect of the present invention provides a method for preparing the aforementioned basic cell culture medium, comprising the following steps:

[0187] The components of the basic cell culture medium are mixed to obtain the basic cell culture medium.

[0188] A fourth aspect of the present invention provides a method for preparing the aforementioned fed culture medium, comprising the following steps:

[0189] The components of the feed medium are mixed to obtain the feed medium.

[0190] A fifth aspect of the invention provides a reagent kit for culturing cells, the reagent kit comprising at least one first container and / or at least one second container, wherein:

[0191] The first container contains the basal cell culture medium as described above or the solid components of the basal cell culture medium, and the second container contains the feed culture medium as described above or the solid components of the feed culture medium.

[0192] In some embodiments of the present invention, the first container contains the solid components of the basic cell culture medium and the water.

[0193] In some embodiments of the present invention, the first container contains the solid components of the basic cell culture medium and the water.

[0194] In some embodiments of the present invention, the first container contains the solid components of the basic cell culture medium, which are mixed with water to prepare a corresponding concentration during use.

[0195] In some embodiments of the present invention, the second container contains the solid components of the supplemental cell culture medium and the water.

[0196] In some embodiments of the present invention, the second container contains the solid components of the supplemental cell culture medium and the water.

[0197] In some embodiments of the present invention, the second container contains the solid components of the supplemental cell culture medium, which are mixed with water to prepare a corresponding concentration during use.

[0198] In some embodiments of the present invention, the first container contains formula 1 and the second container contains formula 16.

[0199] In some embodiments of the present invention, the first container contains formula 2 and the second container contains formula 17.

[0200] In some embodiments of the present invention, the first container contains formula 3 and the second container contains formula 18.

[0201] In some embodiments of the present invention, the first container contains the formula 8, and the second container contains the formula 19, the formula 20, or the formula 21.

[0202] In some embodiments of the present invention, the first container contains formula 10 or formula 11, and the second container contains formula 22.

[0203] In some embodiments of the present invention, the first container contains the formulation 12, and the second container contains the formulation 23, the formulation 24, or the formulation 25.

[0204] In some embodiments of the present invention, the first container contains the formula 13, and the second container contains the formula 23, the formula 24, or the formula 25.

[0205] In some embodiments of the present invention, the first container contains the formula 14, and the second container contains the formula 22 or the formula 24.

[0206] In some embodiments of the present invention, the first container contains the formulation 15, and the second container contains the formulation 22 or the formulation 24.

[0207] A sixth aspect of the invention provides the use of the aforementioned basic cell culture medium, the aforementioned supplemental culture medium, or the aforementioned kit for culturing cells in cell culture and / or production of target proteins.

[0208] In some embodiments of the present invention, the cells are selected from eukaryotic cells.

[0209] In some embodiments of the present invention, the eukaryotic cells are selected from CHO cells or 293 cells.

[0210] In some embodiments of the present invention, the target protein is selected from one or more of PD-L1 / TGF-β antibody, Her2 antibody and IL-17A antibody.

[0211] A seventh aspect of the present invention provides a method for culturing cells, the method comprising:

[0212] The cells were seeded into the basal cell culture medium for amplification culture;

[0213] In the amplification culture, the feed medium is added to the culture system, and the amplification culture continues.

[0214] In some embodiments of the present invention, the feed culture medium is added to the culture system starting from the fourth day of culture.

[0215] In some embodiments of the present invention, on days 4, 6, 8, 10, and 12 of the culture, 0.5% to 5.0% of the initial culture volume of the feed culture medium, for example 2.8%, 3.8%, 4.4%, or 5.0%, is added to the culture system.

[0216] In some embodiments of the present invention, on days 4, 6, 8, 10, and 12 of the culture, 2.8%, 3.8%, 5.0%, 5.0%, and 4.4% of the initial culture volume of the feed culture medium are added to the culture system, respectively.

[0217] In some embodiments of the present invention, on days 4, 6, 8, 10, and 12 of the culture, 5% of the initial culture volume of the feed culture medium is added to the culture system.

[0218] In some embodiments of the present invention, on days 4, 6, 8, 10, 12, and 14 of the culture, 5% of the initial culture volume of the feed culture medium is added to the culture system.

[0219] In some embodiments of the present invention, the inoculation density in the amplification culture is (0.4~1.0)×10⁻¹⁰. 6The cells / mL were controlled, and the initial pH of the culture system was controlled at 7.0±0.2, and the initial culture temperature was controlled at 36.0℃~37.0℃.

[0220] The seeding density can be selected according to the cell properties, for example, (0.8~1.0)×10⁻⁶. 6 cells / mL, (0.4~0.6)×10 6 cells / mL or (0.6~0.8)×10 6 cells / mL, or for example 0.9 × 10⁻⁶. 6 cells / mL, 0.5×10 6 cells / mL or 0.7×10 6 cells / mL.

[0221] In some embodiments of the present invention, when the culture system is cultured to day 7, the culture temperature is lowered to 32.5°C to 33.5°C.

[0222] In some embodiments of the present invention, when the culture system is cultured for 10 days, the pH value of the culture system is adjusted to 7.0 ± 0.3.

[0223] In some embodiments of the present invention, the pH control method in the cultivation method is a single CO2 mode control; the single CO2 mode control is as follows:

[0224] When the pH value in the culture system is higher than the upper limit, CO2 is introduced into the culture system;

[0225] When the pH value in the culture system is lower than the upper limit, Na2CO3 solution is added to the culture system.

[0226] In some embodiments of the present invention, the stirring speed in the culture system is 250-300 rpm.

[0227] In some embodiments of the present invention, the dissolved oxygen (DO) concentration in the culture system is 40-50%, for example 45%, where the percentage refers to the percentage of dissolved oxygen concentration in the fermentation broth relative to the oxygen concentration in the air.

[0228] In some embodiments of the present invention, when the glucose concentration in the culture system is lower than 4.0 g / L, glucose is added to the culture system to bring the concentration up to 6.0 g / L.

[0229] In some embodiments of the present invention, the cells are harvested when cultured to day 14 or when the cell viability is less than 80%.

[0230] In some embodiments of the present invention, the cells are selected from eukaryotic cells.

[0231] In some embodiments of the present invention, the eukaryotic cells are selected from CHO cells or 293 cells.

[0232] A fifth aspect of the present invention provides a method for producing a target protein, comprising the following steps:

[0233] (i) Under suitable expression conditions, genetically engineered cells are cultured in the basal cell culture medium as described above, the feed medium is added to the basal cell culture medium, and the target protein is expressed; and

[0234] (ii) Isolate the target protein from the fermentation product.

[0235] In some embodiments of the present invention, the cells are selected from eukaryotic cells.

[0236] In some embodiments of the present invention, the eukaryotic cells are selected from CHO cells or 293 cells.

[0237] In some embodiments of the present invention, the target protein is selected from one or more of PD-L1 / TGF-β antibody, Her2 antibody and IL-17A antibody.

[0238] The term "trace element" refers to inorganic salts that are essential for cell growth and metabolism but are required in very small amounts, with concentrations typically ranging from nM (nanomoles per liter) to μM (micromoles per liter).

[0239] The term "inorganic salt" refers to inorganic salts that are essential for cell growth and metabolism but are required in relatively high quantities, typically in the mM (millimoles per liter) range.

[0240] The term "CHO ​​cells" refers to a transformed cell line established from the ovarian tissue of Chinese hamsters.

[0241] 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 cells are cultured from the start of a cell culture run and is not used as an additive to another culture medium, although various components may be added to the medium. Basal medium acts as a basis for optional further additives or feed media that can be added during culture (i.e., cell culture runs). Basal cell culture medium is provided from the beginning of the cell culture process. Generally, basal cell culture medium provides nutrients such as carbon sources, amino acids, vitamins, main salts (e.g., sodium chloride or potassium chloride), various trace elements (e.g., manganese sulfate), pH buffers, and glucose, etc.

[0242] The term "feed" or "feed medium" refers to a nutrient concentrate / composition used as feed in mammalian cell cultures, which can be added to the culture at some point after inoculation. Feed medium typically contains a higher concentration of most, but not all, of the components of the basal cell culture medium. Generally, the feed medium replaces nutrients such as amino acids and carbohydrates consumed during cell culture. Feed medium is usually added to the (basal) cell culture medium / fermentation broth in a fed-batch manner.

[0243] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0244] The reagents and raw materials used in this invention are all commercially available.

[0245] The positive and progressive effects of this invention are as follows:

[0246] This invention optimizes cell culture media to obtain a low-cost cell culture medium that effectively meets the needs of monoclonal antibody expression and enables large-scale culture. Specifically:

[0247] (1) The cell culture media (basal culture medium, supplemental culture medium) in this invention can produce normally in low, medium and high concentration culture media, and are safe and non-toxic.

[0248] (2) Compared with commercially available classic culture media, the cell culture media (basal culture medium and supplemental culture medium) in this invention have the same maximum cell density, but the cell maintenance is better in the later stage, and similar trends are shown in different cell lines.

[0249] (3) Compared with commercially available classic culture media, the antibody expression level of the cell culture media (basal culture medium and supplemented culture medium) in this invention 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, and IEC-HPLC main peak ratio are higher). Attached Figure Description

[0250] Figure 1 The image shows cell growth at different concentrations of HR1 BM03 formulation.

[0251] Figure 2 This is a graph showing cell growth under different concentrations of FeedB formulation for HR1 in batches.

[0252] Figure 3 Comparison of cell growth in SHR-1701 cells cultured using HR1 BM03 and HR1 FeedB media, and commercially available media.

[0253] Figure 4 Comparison of cell growth in SHR-1805 expression cells cultured in HR1 BM03 and HR1 FeedB media, and commercially available media.

[0254] Figure 5 Comparison of cell growth in SHR-1314 expression cells cultured in HR1 BM03 and HR1 FeedB media, and commercially available media. Detailed Implementation

[0255] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0256] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, any value between the two endpoints of each range may be used. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement this invention.

[0257] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0258] SHR-1701 WCB (Working Cell Bank): Expresses PD-L1 / TGF-β antibody. Construction method: The SHR-1701 light and heavy chain sequences (refer 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) were cloned into expression vectors pXC17.4 and pXC18.4 provided by Lonza, respectively, to form recombinant plasmids. The constructed recombinant plasmids were transfected into host cells (CHOK1SV GS-KO, from Lonza) and further screened to obtain the original cell bank. Cells from the original cell bank were passaged and proliferated to prepare a batch of homogenized cell suspensions, which were then processed at 1×10⁻⁶. 7Cells / mL were quantitatively aliquoted into 1mL cell cryovials and stored in liquid nitrogen for long-term preservation, thus forming the master cell bank. Cells from the master cell bank were passaged and proliferated to prepare a batch of homogenized cell suspensions, which were then cultured at 1×10⁻⁶ cells / mL. 7 Cells / mL, each 1mL aliquot is quantitatively dispensed into cell cryopreservation tubes and stored in liquid nitrogen for long-term preservation, thus forming a working cell bank.

[0259] In this invention, SHR-1701 refers to a PD-L1 (programmed death ligand-1) / TGF-β (transforming growth factor-β) antibody.

[0260] SHR-1805 WCB: Expressing Her2 antibody, constructed as follows: The SHR-1805 light and heavy chain sequences (trastuzumab light and heavy chain sequences) were cloned into expression vectors pXC17.4 and pXC18.4 provided by Lonza, respectively, to form recombinant plasmids. The constructed recombinant plasmids were transfected into host cells (CHOK1SV GS-KO, from Lonza) and further screened to obtain a primitive cell bank. Cells from the primitive cell bank were passaged and proliferated to prepare a batch of homogenized cell suspensions, which were then diluted at 1×10⁻⁶. 7 Cells / mL were quantitatively aliquoted into 1ml cryovials and stored in liquid nitrogen for long-term preservation, thus forming the master cell bank. Cells from the master cell bank were passaged and proliferated to prepare a batch of homogenized cell suspensions, which were then cultured at 1×10⁻⁶ cells / mL. 7 Cells / mL, each 1mL aliquot is quantitatively dispensed into cell cryopreservation tubes and stored in liquid nitrogen for long-term preservation, thus forming a working cell bank.

[0261] In this invention, SHR-1805 refers to Her2 (human epidermal growth factor receptor 2) antibody.

[0262] SHR-1314 WCB: Expressing IL-17A antibody, constructed as follows: The light and heavy chain sequences of SHR-1314 (referring to patent WO2021018191A1, SEQ ID NO:4 showing the light chain and SEQ ID NO:5 showing the heavy chain) were cloned into the expression vectors pXC17.4 and pXC18.4 provided by Lonza, respectively, to form recombinant plasmids. The constructed recombinant plasmids were transfected into host cells (CHOK1SV GS-KO, from Lonza) and further screened to obtain a primitive cell bank. Cells from the primitive cell bank were passaged and proliferated to prepare a batch of homogenized cell suspensions, which were then subjected to a 1×10⁻⁶ ppm reaction. 7 Cells / mL were quantitatively aliquoted into 1ml cryovials and stored in liquid nitrogen for long-term preservation, thus forming the master cell bank. Cells from the master cell bank were passaged and proliferated to prepare a batch of homogenized cell suspensions, which were then cultured at 1×10⁻⁶ cells / mL. 7Cells / mL, each 1mL aliquot is quantitatively dispensed into cell cryopreservation tubes and stored in liquid nitrogen for long-term preservation, thus forming a working cell bank.

[0263] In this invention, SHR-1314 refers to IL-17A (interleukin-17A) antibody.

[0264] Tolphenone, also known as cycloheptatrienolone, has the molecular formula C7H6O2 and CAS number 533-75-5.

[0265] Ferric sulfate (II) is an inorganic compound with the chemical formula FeSO4.

[0266] 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.

[0267] Gibco Dynamis: Purchased from Gibco, product number A2661503.

[0268] HyClone Cell Boost 7a and HyClone Cell Boost 7b: purchased from Hyclone, product numbers SH31026.01 and SH31027.07 respectively.

[0269] DO refers to the percentage of dissolved oxygen concentration in the fermentation broth relative to the oxygen concentration in the air.

[0270] The detection method for antibody expression level 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 chamber 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: take the sample to be tested and determine it according to the chromatographic conditions and elution conditions, and record the chromatogram; (5) Data analysis: by comparing with the chromatogram of the blank buffer, the antibody expression level is calculated by integrating the chromatogram.

[0271] The method for detecting the purity of SEC-HPLC (Size Exclusion Chromatography - High Performance Liquid Chromatography) is as follows: (1) Solution preparation: mobile phase A (200 mM PBS + 50 mM Na2SO4); (2) Sample preparation: dilute the sample with mobile phase A to a final concentration of about 10 mg / ml; (3) Chromatographic conditions: flow rate 0.5 ml / min, injection volume 100 μg, column temperature 30 ℃, sample chamber temperature 2~8 ℃, detection wavelength 280 nm, running time 40 min; (4) Elution conditions: elute isocratically with 100% mobile phase A for 40 min; (5) Sample analysis: take the prepared sample and determine it according to the chromatographic conditions and elution conditions, and record the chromatogram; (6) Data analysis: by comparing the chromatogram with the blank buffer, integrate the chromatogram, and calculate the peak area percentage of monomers, polymers and low molecular weight impurities by peak area normalization method.

[0272] The method for detecting the purity of reduced CE-SDS (Reduced Capillary Electrophoresis - Sodium Dodecyl Sulfate) is as follows: (1) Sample preparation: Take 100 μg of sample and mix it with SDS-MW Sample Buffer and 713 mM 2-mercaptoethanol to form a mixture. The mixture is denatured at 70 ℃ for 10 minutes and cooled for 5 minutes. (2) Instrument parameters: effective capillary length 20 cm, temperature control 25 ℃, diode array detector wavelength 220 nm, sample chamber temperature 10 ℃. (3) Sample analysis: Take the prepared non-reduced sample into a 200 μL PCR tube, and determine it according to the sample separation method. Record the chromatogram. (4) Data analysis: By comparing the chromatogram with the blank control, integrate the chromatogram of the detected sample and calculate the percentage of the corrected peak area of ​​the main peak, i.e., the reduction purity.

[0273] The detection method for non-reduced CE-SDS purity (non-reduced CE-SDS, non-reduced capillary electrophoresis-sodium dodecyl sulfate) is as follows: (1) Preparation of non-reduced sample: Take 100 μg of sample and mix it with SDS-MW Sample Buffer and 250 mM iodoacetamide to form a mixture. The mixture is denatured at 70 ℃ for 10 minutes and cooled for 5 minutes. (2) Instrument parameters: effective capillary 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 into a 200 uL PCR tube, and determine it according to the sample separation method. Record the chromatogram. (4) Data analysis and processing: By comparing the chromatogram with the blank control, integrate the chromatogram of the detected sample and calculate the percentage of the corrected peak area of ​​the main peak, i.e., the non-reduced purity.

[0274] The detection method of IEC-HPLC (Ion Exchange Chromatography - High Performance Liquid Chromatography) is as follows: (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, take the supernatant, and dilute to a final concentration of approximately 5.0 mg / mL; (3) Chromatographic conditions: Flow rate 0.8 ml / min, injection volume 50 μg, column temperature 40 ℃, sample chamber temperature 4 ℃, detection wavelength 280 nm, run time 60 minutes. 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 and determine it according to the chromatographic conditions and elution conditions, and record the chromatogram; (6) Data analysis: By comparing the chromatogram with the blank buffer, integrate the chromatogram, and use the peak area normalization method to calculate the peak area percentage of the main peak, the peak area percentage of the alkaline region and the peak area percentage of the acidic region respectively.

[0275] Example 1: Determination of the HR1 BM03 formulation

[0276] 1. Materials and Methods

[0277] 1.1 Cell lines

[0278] Table 1

[0279]

[0280] 1.2 Concentrations of each component in HR1 BM03

[0281] Table 2

[0282]

[0283]

[0284] 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.

[0285] 1.3 Cell passage culture

[0286] 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%.

[0287] 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.

[0288] 2. Experimental Results

[0289] 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.

[0290] Example 2: Determination of FeedB Formulation for HR1

[0291] 1. Materials and Methods

[0292] 1.1 Cell lines

[0293] Table 3

[0294]

[0295] 1.2 Concentration of each component in HR1 FeedB

[0296] Table 4

[0297]

[0298]

[0299] The method for preparing FeedB components is as follows: Mix the components in the table above according to their concentrations and then dilute with water to the target volume.

[0300] 1.3 Cell Culture

[0301] Take one cell line from the working cell bank and thaw it rapidly in a 37 ℃ water bath. In a biosafety cabinet, add the thawed cell solution to a 125 mL shake flask containing 40 mL of HR1 BM03 medium and place it in a CO2 shaker for culture. The culture conditions are: temperature 36 ℃, rotation speed 110 rpm, CO2 concentration 8.0%, and humidity 80%.

[0302] When the cell density reaches (4-10)×10 6 Cells / mL were used for shake-flask fed-batch culture, with a total of 3 groups. The basal culture was carried out at a medium concentration of HR1 BM03, and the feeding medium was low, medium, and high concentrations of HR1 FeedB, respectively. The specific steps of shake-flask fed-batch culture are as follows.

[0303] Vaccination: The vaccination density should be controlled at (0.8~1.0)×10⁻⁶. 6 cells / mL, initial culture volume 40 mL.

[0304] Shaker parameters: Temperature: 36.0 ℃, Rotation speed: 110 rpm, Carbon dioxide: 8%, Humidity: 80%.

[0305] Sugar supplementation: If the glucose concentration is less than 4 g / L when sampled on the day of supplementation, supplement sugar to a concentration of 6 g / L.

[0306] Feeding: On days 4 / 6 / 8 / 10 / 12, add HR1 FeedB at 2.8%, 3.8%, 5.0%, 5.0%, and 4.4% of the initial volume (low, medium, and high concentrations were added to the three groups respectively).

[0307] Harvest: Cultivate to day 14 or when the survival rate is below 80%.

[0308] 2. Experimental Results

[0309] like Figure 2As shown, SHR-1701 cells were fed-batch cultured in low, medium, and high concentrations of HR1 medium. The cells produced normally in all cases, and the antibody expression levels reached 4.45 g / L, 5.56 g / L, and 4.03 g / L, respectively, all of which met the production requirements.

[0310] Example 3: Comparison of HR1 BM03 and HR1 FeedB in the culture of SHR-1701 cell lines with commercially available culture media.

[0311] 1. Materials and Methods

[0312] 1.1 Cell lines

[0313] Table 5

[0314]

[0315] 1.2 Experimental Methods

[0316] In this embodiment, the concentrations of HR1 BM03 in Example 1 and HR1 FeedB in Example 2 were used for the experiment. The commercially available culture media were selected from the classic combination of Gibco Dynamis, HyClone Cell Boost 7a, and HyClone Cell Boost 7b, as shown in Table 6 below.

[0317] Table 6

[0318]

[0319] 2. Experimental Results

[0320] SHR-1701 cell lines were cultured using HR1 BM03 and HR1 FeedB, respectively, and commercially available culture media (GibcoDynamis, HyClone Cell Boost 7a, HyClone Cell Boost 7b). Cell growth was as follows: Figure 3 As shown in Table 7, compared with commercially available classic culture media, the peak cell densities of HR1 BM03 and HR1 FeedB were basically the same, but the cell retention was better in the later stages.

[0321] Table 7

[0322]

[0323] The antibody expression levels and antibody quality are compared in Table 8 below: Compared with the commercially available classic culture medium, the antibody expression levels of HR1 BM03 and HR1 FeedB increased by more than 30% after culture, and the antibody quality was better (higher SEC-HPLC purity, reduced CE-SDS purity, non-reduced CE-SDS purity, and IEC-HPLC main peak ratio).

[0324] Table 8

[0325]

[0326] Example 4: Comparison of HR1 BM03 and HR1 FeedB in the culture of SHR-1805 cell lines with commercially available culture media.

[0327] 1. Materials and Methods

[0328] 1.1 Cell lines

[0329] Table 9

[0330]

[0331] 1.2 Experimental Methods

[0332] In this embodiment, the concentrations of HR1 BM03 in Example 1 and HR1 FeedB in Example 2 were used for the experiment. The commercially available culture media were selected from the classic combination of Gibco Dynamis, HyClone Cell Boost 7a, and HyClone Cell Boost 7b, as shown in Table 10 below.

[0333] Table 10

[0334]

[0335] 2. Experimental Results

[0336] SHR-1805 cell line was cultured using HR1 BM03 and HR1 FeedB, respectively, and commercially available culture media (GibcoDynamis, HyClone Cell Boost 7a, HyClone Cell Boost 7b). Cell growth was as follows: Figure 4 As shown in Table 11, compared with commercially available classic culture media, HR1 BM03 and HR1 FeedB showed a 10% higher peak cell density and better cell maintenance in the later stages.

[0337] Table 11

[0338]

[0339] The antibody expression levels and antibody quality are compared in Table 12 below: Compared with commercially available classic culture media, the antibody expression level increased by 45% after culturing with HR1 BM03 and HR1 FeedB, and the antibody quality was better (higher SEC-HPLC purity, reduced CE-SDS purity, non-reduced CE-SDS purity, and IEC-HPLC main peak ratio).

[0340] Table 12

[0341]

[0342] Example 5: Comparison of HR1 BM03 and HR1 FeedB in the culture of SHR-1314 cell lines with commercially available culture media.

[0343] 1. Materials and Methods

[0344] 1.1 Cell lines

[0345] Table 13

[0346]

[0347] 1.2 Experimental Methods

[0348] In this embodiment, the concentrations of HR1 BM03 in Example 1 and HR1 FeedB in Example 2 were used for the experiment. The commercially available culture media were selected from the classic combination of Gibco Dynamis, HyClone Cell Boost 7a, and HyClone Cell Boost 7b, as shown in Table 14 below.

[0349] Table 14

[0350]

[0351] 2. Experimental Results

[0352] SHR-1314 cell line was cultured using HR1 BM03 and HR1 FeedB, respectively, and commercially available culture media (GibcoDynamis, HyClone Cell Boost 7a, HyClone Cell Boost 7b). Cell growth was as follows: Figure 5 As shown in Table 15, compared with commercially available classic culture media, HR1 BM03 and HR1 FeedB showed a 25% higher peak cell density and better cell maintenance in the later stages.

[0353] Table 15

[0354]

[0355] The antibody expression levels and antibody quality are compared in Table 16 below: Compared with commercially available classic culture media, the antibody expression level increased by 50% after culturing in HR1 BM03 and HR1 FeedB, and the antibody quality was better (higher SEC-HPLC purity, reduced CE-SDS purity, non-reduced CE-SDS purity, and IEC-HPLC main peak ratio).

[0356] Table 16

[0357]

[0358] Example 6: Effect of tyrosine concentration on cell growth

[0359] Based on the concentration of HR1 BM03 component in Example 1, the concentration of tyrosine added to the HR1 BM03 basal medium was adjusted to 0, 0.6 mg / L, 1.2 mg / L, and 2.4 mg / L, respectively, with the initial inoculation density controlled at 0.2 × 10⁻⁶. 6 The cell density was compared after 5 days of culture. The cultured cell line was SHR-1701 WCB.

[0360] The experimental results show that the absence of tyrosine or excessive concentration in the basal culture medium inhibits cell growth, as shown in the table below.

[0361] Table 17

[0362]

[0363] Example 7: Effects of the concentrations of disodium tyrosine, cysteine, tryptophan, and lysine on cell growth and expression.

[0364] (1) Effects of the concentrations of disodium tyrosine, cysteine, tryptophan, and lysine in the basal culture medium on cell growth and expression.

[0365] A stock solution (1×) of 50 mM L-tyrosine disodium salt + 25 mM L-cysteine ​​+ 50 mM L-tryptophan + 100 mM L-lysine was prepared. This 1× amino acid stock solution was then mixed with basal medium containing none of these four amino acids (the other components of the basal medium without these four amino acids were in the same proportions as those in the HR1 BM03 component in Example 1) to create basal mediums of varying concentrations. Cell culture was then performed, and the cell line cultured was SHR-1701 WCB. The initial seeding density was kept consistent at 0.2 × 10⁻⁶. 6 The cell density was approximately 100 cells / ml, and the cells were compared after 5 days of culture.

[0366] Experimental results show that the absence of these four amino acids or their excessive concentration in the basal culture medium inhibits cell growth, as shown in the table below.

[0367] Table 18

[0368]

[0369] Table 19

[0370]

[0371] (2) Effects of the concentrations of disodium tyrosine, cysteine, tryptophan, and lysine in the fed culture medium on cell growth and expression.

[0372] The concentrations of the four amino acids in the basal medium were fixed at 5 mM, 2.5 mM, 5 mM, and 10 mM, respectively. The other components of the basal medium were the same as those in the HR1 BMO3 component in Example 1. The effects of these four amino acids in the feed medium on cell growth and expression were investigated. A stock solution (1×) of 500 mM L-tyrosine disodium salt + 250 mM L-cysteine ​​+ 500 mM L-tryptophan + 1000 mM L-lysine was prepared. The 1× amino acid stock solution was mixed with feed medium without these four amino acids (the other components of the feed medium without these four amino acids were the same as those in the HR1 FeedB formula in Example 2) to prepare feed mediums of different concentrations, and cell cultures were performed. The experimental method was the same as in Example 2.

[0373] Table 20

[0374]

[0375] Table 21

[0376]

[0377] Table 22

[0378]

[0379] Example 8: Effects of VB6 and VB12 on antibody expression levels

[0380] A 2mM VB6 + 0.2mM VB12 stock solution (1×) was prepared. This 1× vitamin stock solution was then mixed with basal and feed media containing neither of these two vitamins to create different concentrations of culture media (the proportions of other components in the basal medium without VB6 and VB12 were the same as those in the HR1 BM03 component of Example 1, and the proportions of other components in the feed media without VB6 and VB12 were the same as those in the HR1 FeedB formulation of Example 2). The effects of VB6 and VB12 concentrations in the basal and feed media on antibody expression levels were investigated. The experimental methods were consistent with those in Example 2.

[0381] The results are shown in the table below: The antibody expression level first increased with the increase of VB6 and VB12 concentrations, reached a peak, and then decreased with the increase of VB6 and VB12 concentrations.

[0382] Table 23

[0383]

[0384] Table 24

[0385]

[0386] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention 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 invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A basic cell culture medium, characterized in that, The basic cell culture medium contains solid components and water; The solid component comprises the following components: tyrosine, amino acids, vitamins, inorganic salts, polyamine, trace elements, and carbon source; The amino acids include: 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 include: vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid, and alpha-lipoic acid; The inorganic salts include: sodium phosphate, potassium chloride, zinc sulfate, sodium selenite, ferric sulfate (II), copper sulfate, calcium chloride, magnesium sulfate, sodium thiosulfate, and sodium chloride; The polyamine includes: putrescine, spermidine, and spermine; The trace elements include: manganese chloride, aluminum trichloride, cadmium sulfate, and cobalt chloride; The carbon source includes: glucose and sodium pyruvate; The concentration of tropenophenone in the basal cell culture medium is 0.6~1.2 mg / L; The concentration of the L-tyrosine disodium salt in the basal cell culture medium is 1~10mM; The concentration of L-cysteine ​​in the basal cell culture medium is 0.5~5mM; The concentration of L-tryptophan in the basal cell culture medium is 1~10mM; The concentration of L-lysine in the basal cell culture medium is 2-20 mM; The concentration of vitamin B6 in the basal cell culture medium is 0.002~0.2mM; The concentration of vitamin B12 in the basic cell culture medium is 0.0002~0.02mM.

2. The basic cell culture medium as described in claim 1, characterized in that, The basic cell culture medium meets one or more of the following conditions: (1) The concentration of L-methionine in the basic cell culture medium is 1~5mM; (2) The concentration of L-proline in the basic cell culture medium is 0.5~5mM; (3) The concentration of L-aspartic acid in the basic cell culture medium is 0.3~5mM; (4) The concentration of the L-tyrosine disodium salt in the basic cell culture medium is 2.5~10mM; (5) The concentration of L-asparagine in the basic cell culture medium is 0.5~3mM; (6) The concentration of L-cysteine ​​in the basic cell culture medium is 1~5mM; (7) The concentration of L-serine in the basic cell culture medium is 1~5mM; (8) The concentration of L-tryptophan in the basic cell culture medium is 2.5~10mM; (9) The concentration of L-lysine in the basic cell culture medium is 2~20mM; (10) The concentration of L-alanine in the basic cell culture medium is 1~5mM; (11) The concentration of L-histidine in the basic cell culture medium is 3~10mM; (12) The concentration of L-threonine in the basic cell culture medium is 1~5mM; (13) The concentration of L-valine in the basic cell culture medium is 0.2~2mM; (14) The concentration of L-isoleucine in the basic cell culture medium is 3~6mM; (15) The concentration of L-leucine in the basic cell culture medium is 2~6mM; (16) The concentration of L-phenylalanine in the basic cell culture medium is 5~10mM; (17) The concentration of L-glycine in the basic cell culture medium is 0.1~2mM; (18) The concentration of L-arginine in the basic cell culture medium is 2-6 mM; (19) The concentration of L-glutamic acid in the basic cell culture medium is 5~20mM; (20) The concentration of vitamin B1 in the basic cell culture medium is 0.005~0.1mM; (21) The concentration of vitamin B2 in the basic cell culture medium is 0.005~0.05mM; (22) The concentration of nicotinamide in the basic cell culture medium is 0.001~0.02mM; (23) The concentration of vitamin B5 in the basic cell culture medium is 0.01~0.1mM; (24) The concentration of vitamin B6 in the basic cell culture medium is 0.01~0.2mM; (25) The concentration of vitamin B12 in the basic cell culture medium is 0.001~0.02mM; (26) The concentration of choline in the basal cell culture medium is 0.1~1mM; (27) The concentration of folic acid in the basal cell culture medium is 0.005~0.5mM; (28) The concentration of α-lipoic acid in the basic cell culture medium is 0.001~0.01mM; (29) The concentration of sodium phosphate in the basic cell culture medium is 0.5~2mM; (30) The concentration of potassium chloride in the basic cell culture medium is 0.5~2mM; (31) The concentration of zinc sulfate in the basic cell culture medium is 0.0005~0.001mM; (32) The concentration of sodium selenite in the basic cell culture medium is 0.00001~0.0001 mM; (33) The concentration of ferric sulfate (II) in the basic cell culture medium is 0.02~0.2mM; (34) The concentration of copper sulfate in the basic cell culture medium is 0.0005~0.001mM; (35) The concentration of calcium chloride in the basic cell culture medium is 0.05~0.5mM; (36) The concentration of magnesium sulfate in the basic cell culture medium is 0.1~2mM; (37) The concentration of sodium thiosulfate in the basic cell culture medium is 1~5mM; (38) The concentration of sodium chloride in the basic cell culture medium is 20~50mM; (39) The concentration of putrescine in the basic cell culture medium is 0.01~0.1mM; (40) The concentration of spermidine in the basic cell culture medium is 0.01~0.1mM; (41) The concentration of spermine in the basic cell culture medium is 0.01~0.1mM; (42) The concentration of manganese chloride in the basic cell culture medium is 0.000001~0.0001 mM; (43) The concentration of aluminum trichloride in the basic cell culture medium is 0.000001~0.0001 mM; (44) The concentration of cadmium sulfate in the basic cell culture medium is 0.000001~0.0001 mM; (45) The concentration of cobalt chloride in the basic cell culture medium is 0.000001~0.0001 mM; (46) The concentration of glucose in the basic cell culture medium is 20-40 mM; (47) The concentration of sodium pyruvate in the basic cell culture medium is 0.1~5mM; (48) The concentration of the tolphenidone in the basal cell culture medium is 0.9~1.2 mg / L; (49) The basic cell culture medium is composed of the solid components and the water; and (50) The basic cell culture medium is used for cell culture and / or production of target proteins.

3. The basic cell culture medium as described in claim 1, characterized in that, The formulation of the basic cell culture medium is selected from any one of formulations 1-15: ； ； ； 。 4. A fed culture medium, characterized in that, The feed culture medium contains solid components and water; The solid component comprises the following components: tyrosine, amino acids, vitamins, inorganic salts, polyamine, trace elements, and carbon source; The amino acids include: 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 include: vitamin B1, vitamin B2, nicotinamide, vitamin B5, vitamin B6, vitamin B12, choline, folic acid, and alpha-lipoic acid; The inorganic salts include: sodium phosphate, potassium chloride, zinc sulfate, sodium selenite, ferric sulfate (II), copper sulfate, calcium chloride, magnesium sulfate, sodium thiosulfate, and sodium chloride; The polyamine includes: putrescine, spermidine, and spermine; The trace elements include: manganese chloride, aluminum trichloride, cadmium sulfate, and cobalt chloride; The carbon source includes: glucose and sodium pyruvate; The concentration of tyrosine in the fed culture medium is 0.6~12 mg / L; The concentration of the L-tyrosine disodium salt in the fed culture medium is 10~100mM; The concentration of L-cysteine ​​in the fed culture medium is 5-50 mM. The concentration of L-tryptophan in the supplemental culture medium is 10~100mM; The concentration of L-lysine in the fed culture medium is 20-200 mM; The concentration of vitamin B6 in the supplemental culture medium is 0.1~2mM; The concentration of vitamin B12 in the supplemental culture medium is 0.01~0.2mM.

5. The fed culture medium as described in claim 4, characterized in that, The feed culture medium meets one or more of the following conditions: (1) The concentration of L-methionine in the supplemental culture medium is 10~50mM; (2) The concentration of L-proline in the supplemental culture medium is 5~50mM; (3) The concentration of L-aspartic acid in the fed culture medium is 3~50mM; (4) The concentration of the L-tyrosine disodium salt in the fed culture medium is 50~100mM; (5) The concentration of L-asparagine in the fed culture medium is 5~30mM; (6) The concentration of L-cysteine ​​in the supplemental culture medium is 10~50mM. (7) The concentration of L-serine in the supplemental culture medium is 10~50mM; (8) The concentration of L-tryptophan in the supplemental culture medium is 40~100mM; (9) The concentration of L-lysine in the supplemented culture medium is 100~200mM; (10) The concentration of L-alanine in the supplemented culture medium is 10~50mM; (11) The concentration of L-histidine in the supplemented culture medium is 30~100mM; (12) The concentration of L-threonine in the supplemented culture medium is 10~50mM; (13) The concentration of L-valine in the supplemented culture medium is 2~20mM; (14) The concentration of L-isoleucine in the supplemented culture medium is 30~60mM; (15) The concentration of L-leucine in the supplemented culture medium is 20~60mM; (16) The concentration of L-phenylalanine in the supplemented culture medium is 50~100mM; (17) The concentration of L-glycine in the supplemented culture medium is 1~20mM; (18) The concentration of L-arginine in the supplemented culture medium is 20~60mM; (19) The concentration of L-glutamic acid in the supplemental culture medium is 50~200mM; (20) The concentration of vitamin B1 in the supplemental culture medium is 0.05~1mM; (21) The concentration of vitamin B2 in the supplemental culture medium is 0.05~0.5mM; (22) The concentration of nicotinamide in the fed culture medium is 0.01~0.2mM; (23) The concentration of vitamin B5 in the supplemental culture medium is 0.1~1mM; (24) The concentration of vitamin B6 in the supplemental culture medium is 0.5~2mM; (25) The concentration of vitamin B12 in the supplemental culture medium is 0.05~0.2mM; (26) The concentration of choline in the supplemental culture medium is 1~10mM; (27) The concentration of folic acid in the supplemental culture medium is 0.05~5mM; (28) The concentration of α-lipoic acid in the fed culture medium is 0.01~0.1mM; (29) The concentration of sodium phosphate in the fed culture medium is 5-20 mM; (30) The concentration of potassium chloride in the supplemental culture medium is 5~20mM; (31) The concentration of zinc sulfate in the fed culture medium is 0.005~0.01mM; (32) The concentration of sodium selenite in the supplemented culture medium is 0.0001~0.001mM; (33) The concentration of ferric sulfate (II) in the supplemental culture medium is 0.2~2mM; (34) The concentration of copper sulfate in the supplemental culture medium is 0.005~0.01mM; (35) The concentration of calcium chloride in the supplemental culture medium is 0.5~5mM; (36) The concentration of magnesium sulfate in the fed culture medium is 1~20mM; (37) The concentration of sodium thiosulfate in the fed culture medium is 10~50mM; (38) The concentration of sodium chloride in the fed culture medium is 200~500mM; (39) The concentration of putrescine in the fed culture medium is 0.1~1mM; (40) The concentration of spermidine in the supplemental culture medium is 0.1~1mM; (41) The concentration of spermine in the supplemental culture medium is 0.1~1mM; (42) The concentration of manganese chloride in the supplemental culture medium is 0.00001~0.001 mM; (43) The concentration of aluminum trichloride in the supplemental culture medium is 0.00001~0.001 mM; (44) The concentration of cadmium sulfate in the supplemental culture medium is 0.00001~0.001 mM; (45) The concentration of cobalt chloride in the supplemental culture medium is 0.00001~0.001 mM; (46) The concentration of glucose in the supplemental culture medium is 200-400 mM; (47) The concentration of sodium pyruvate in the fed culture medium is 1~50mM; (48) The concentration of the tyrosine in the supplemental culture medium is 6-12 mg / L; (49) The basic cell culture medium is composed of the solid components and the water; and (50) The feed culture medium is used for cell culture and / or production of target protein.

6. The fed culture medium as described in claim 4, characterized in that, The formulation of the supplemental culture medium is selected from any one of formulations 16-25: ； ； 。 7. A reagent kit for culturing cells, characterized in that, The kit includes at least one first container and / or at least one second container, wherein: The first container contains the basic cell culture medium as described in any one of claims 1 to 3 or the solid components of the basic cell culture medium, and the second container contains the feed culture medium as described in any one of claims 4 to 6 or the solid components of the feed culture medium.

8. The reagent kit for culturing cells as described in claim 7, characterized in that, The first container contains formula 1, and the second container contains formula 16; Alternatively, the first container contains formula 2 and the second container contains formula 17; Alternatively, the first container contains formula 3 and the second container contains formula 18; Alternatively, the first container may contain formula 8, and the second container may contain formula 19, formula 20, or formula 21; Alternatively, the first container may contain formula 10 or formula 11, and the second container may contain formula 22; Alternatively, the first container may contain formula 12, and the second container may contain formula 23, formula 24, or formula 25; Alternatively, the first container may contain formula 13, and the second container may contain formula 23, formula 24, or formula 25; Alternatively, the first container may contain formula 14, and the second container may contain formula 22 or formula 24; Alternatively, the first container may contain formula 15, and the second container may contain either formula 22 or formula 24.

9. The use of a basic cell culture medium as described in any one of claims 1 to 3, a supplemental culture medium as described in any one of claims 4 to 6, or a kit for culturing cells as described in claim 7 or 8 in cell culture and / or production of target proteins.

10. The application as described in claim 9, characterized in that, The application 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 target protein 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 in that, The cultivation method includes: The cells are seeded into the basic cell culture medium as described in any one of claims 1 to 3 for amplification culture; In the amplification culture, the feed culture 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 cell culture method according to claim 11, characterized in that, The cells are selected from eukaryotic cells, and the eukaryotic cells are selected from CHO cells or 293 cells.

13. A method for producing a target protein, characterized in that, It includes the following steps: (i) Under suitable expression conditions, genetically engineered cells are cultured in the basic cell culture medium as described in any one of claims 1 to 3, the feed culture medium as described in any one of claims 4 to 6 is added to the basic cell culture medium, and the target protein is expressed; (ii) Isolate the target protein from the fermentation product.

14. The method for producing the target protein as described in claim 13, characterized in that, The method for producing the target protein 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 target protein is selected from one or more of PD-L1 / TGF-β antibody, Her2 antibody and IL-17A antibody.

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