Culture medium for suspension culture of CHO (Chinese hamster ovary) cells as well as preparation method and application of culture medium

Abstract

The invention belongs to the technical field of biology, and particularly relates to a CHO cell suspension culture medium as well as a preparation method and application thereof. The culture medium comprises amino acids, vitamins, inorganic salts, trace elements, carbohydrates and other molecular compounds; the amino acids comprise glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. The culture medium provided by the invention can meet the high-density suspension culture of various CHO cells, can improve the production efficiency and reduce the production cost, and has wide application prospects and values. The culture medium provided by the invention overcomes the defects of the existing culture medium with determined chemical components, and provides more choices for the culture medium with determined chemical components in China.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, relates to a culture medium for CHO cell suspension culture and a preparation method and application thereof. Background Art

[0002] With the maturity of biomedical technology, more pathogen detection and diagnosis needs, coupled with the rapid development of biopharmaceuticals represented by targeted therapy and immunotherapy, have brought the recombinant protein industry into a period of vigorous development. The expression of recombinant proteins requires the support of high-expression hosts, vectors, and cell culture systems. Among the many expression systems, mammalian cell expression systems have attracted attention due to their unique advantages in processing, secretion, and glycosylation. Among them, Chinese hamster ovary cells (CHO) have the characteristics of efficient amplification and expression of recombinant genes, accurate post-transcriptional modification functions, and stable integration of exogenous proteins. They can be cultured in high-density suspensions, can withstand high shear forces and osmotic pressures, and rarely secrete their own endogenous proteins, which facilitates the separation and purification of downstream products. They have become the most widely used mainstream biological product expression system.

[0003] The steps for expressing recombinant protein biological products using the Chinese hamster ovary cell expression system are as follows: 1) Design and synthesize foreign genes to complete the construction of expression vectors; 2) Use appropriate transfection technology to deliver the vectors to CHO host cells; 3) Pressure screening to obtain qualified clones and expand the culture; 4) Express the target protein through CHO cell fermentation; 5) Separate and purify the target protein. In the application of the CHO cell expression system, the state of CHO cells is directly affected by the components of the culture medium, which is ultimately reflected in the expression of recombinant proteins.

[0004] Considering the cost, batch-to-batch variation, biohazard risk, and convenience of subsequent separation and purification of target proteins, serum-free and protein-free culture media are often preferred for CHO cell culture and recombinant protein expression. Among them, chemically defined culture media, because they do not contain any undefined or unclear supplements and purified growth factors, proteins, lipoproteins, and other substances that replace serum, are conducive to simplifying the product purification process, clarifying the role of specific components of the culture medium in cell physiology and protein expression, improving the controllability of protein expression, and reducing production costs, have broad prospects for commercial application. However, the commercial CHO cell chemically defined culture media with reliable and stable quality that are currently available are mostly imported products. They are expensive, have a long supply cycle, and have unclear formula ingredients. This not only increases the manufacturing cost of large-scale production of recombinant protein biological products using the CHO expression system, but also makes it impossible to optimize and match the culture medium formula for different cell lines, reducing the controllability of recombinant protein biological products. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a chemically defined culture medium that supports high-density suspension culture of CHO cells. The culture medium does not contain serum or animal-derived components, is suitable for the growth, passaging and protein expression of various CHO cell lines, supports high-density cell culture at high speeds and is low-cost, thus facilitating the large-scale production of biological products.

[0006] The details are as follows:

[0007] A culture medium for CHO cell suspension culture, the culture medium comprising amino acids, vitamins, inorganic salts, trace elements, carbohydrates and other molecular compounds;

[0008] The amino acids include glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

[0009] Preferably, the vitamins include thiamine hydrochloride, riboflavin, niacinamide, calcium D-pantothenate, pyridoxine, biotin, choline chloride, inositol, folic acid, cyanocobalamin and ascorbic acid;

[0010] Preferably, the inorganic salts include calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, dibasic sodium phosphate and monobasic sodium phosphate;

[0011] Preferably, the trace elements include ferric nitrate nonahydrate, ferrous sulfate heptahydrate, cupric sulfate pentahydrate, zinc sulfate heptahydrate, ferric citrate, sodium selenite, sodium metasilicate, barium acetate, potassium iodide, silver nitrate, manganese chloride tetrahydrate, sodium molybdate dihydrate, cobalt chloride, aluminum chloride, potassium bromide, sodium metavanadate, nickel chloride, stannous chloride, sodium fluoride, cadmium chloride, zirconium oxychloride, rubidium chloride, germanium tetroxide, chromium chloride and lithium chloride;

[0012] Preferably, the carbohydrates and other molecular compounds include glucose, linoleic acid, putrescine, spermine, lipoic acid, sodium pyruvate, ethanolamine, L-reduced glutathione, hydroxyethylpiperazineethanesulfonic acid and Pluronic F68.

[0013] Furthermore, the amino acids include: glycine 0.0210-0.0574 g / L, L-alanine 0.0060-0.0840 g / L, L-arginine 0.1246-1.5047 g / L, L-asparagine 0.582-2.030 g / L, L-aspartic acid 0.3550-1.2385 g / L, L-cysteine ​​0.0210-0.3878 g / L, L-cystine 0.0448-0.6226 g / L, L-glutamic acid 0.108-0.770 g / L, L-histidine 0.1500-0.3849 g / L, L-isoleucine 0.2905-1 .1900g / L, L-leucine 0.312-2.240g / L, L-lysine 0.3458-1.1900g / L, L-methionine 0.0630-0.2789g / L, L-phenylalanine 0.2017-0.6370g / L, L-proline 0.2412-1.7500g / L, L-serine 0.2721-1.4000g / L, L-threonine 0.25-0.77g / L, L-tryptophan 0.0702-0.3360g / L, L-tyrosine 0.1500-1.0579g / L and L-valine 0.264-0.630g / L;

[0014] The vitamins include: thiamine hydrochloride 0.0018-0.0091 g / L, riboflavin 0.0001-0.0109 g / L, niacinamide 0.0021-0.0112 g / L, D-calcium pantothenate 0.0015-0.0112 g / L, pyridoxine 0.0015-0.0077 g / L, biotin 0.0001-0.0026 g / L, choline chloride 0.0594-0.3500 g / L, inositol 0.0255-0.2940 g / L, folic acid 0.0027-0.0634 g / L, cyanocobalamin 0.0021-0.0154 g / L, and ascorbic acid 0.0000-0.0224 g / L;

[0015] The inorganic salts include: calcium chloride 0.021-0.175 g / L, magnesium chloride 0.011-0.147 g / L, magnesium sulfate 0.019-0.357 g / L, potassium chloride 0.33-0.77 g / L, sodium bicarbonate 1.20-3.36 g / L, sodium chloride 0.12-3.36 g / L, sodium phosphate dibasic 0.12-0.70 g / L, sodium phosphate monobasic 0.06-2.10 g / L;

[0016] The trace elements include: ferric nitrate nonahydrate 0.0001-0.0004 g / L, ferrous sulfate heptahydrate 0.0024-0.0111 g / L, copper sulfate pentahydrate 0.000002-0.005694 g / L, zinc sulfate heptahydrate 0.0024-0.0258 g / L, ferric citrate 0.021-0.105 g / L, sodium selenite 0.00000900-0.00004332 g / L, sodium metasilicate 0.000009-0.00002 1g / L, barium acetate 0.00000021-0.00002100g / L, potassium iodide 0.00000009-0.00000021g / L, silver nitrate 0.00000000-0.00000008g / L, manganese chloride tetrahydrate 0.00000105-0.00000341g / L, sodium molybdate dihydrate 0.00006828-0.00028773g / L, cobalt chloride 0.00000036-0.0002867 6g / L, Aluminum Chloride 0.00000000-0.00000210g / L, Potassium Bromide 0.00000004-0.00000070g / L, Sodium Metavanadate 0.00000090-0.00000601g / L, Nickel Chloride 0.00000180-0.00000923g / L, Stannous Chloride 0.00000000-0.00012621g / L, Sodium Fluoride 0.00000720-0.00001680 g / L, cadmium chloride 0.00000000-0.00005440g / L, zirconium oxychloride 0.00000581-0.00002426g / L, rubidium chloride 0.0000006-0.0000049g / L, germanium tetroxide 0.00000000-0.00000168g / L, chromium chloride 0.00000000-0.00000141g / L, lithium chloride 0.00000000-0.0000028g / L;

[0017] The carbohydrates and other molecular compounds include: glucose 3.60-9.38 g / L, linoleic acid 0.0001-0.0006 g / L, putrescine 0.0045-0.1540 g / L, spermine 0.000-0.007 g / L, lipoic acid 0.0000-0.0022 g / L, sodium pyruvate 0.0264-0.3080 g / L, ethanolamine 0.009-0.140 g / L, L-reduced glutathione 0.0002-0.0025 g / L, hydroxyethylpiperazine ethanesulfonic acid 0.000-3.332 g / L, and Pluronic F68 0.60-1.68 g / L.

[0018] Furthermore, the amino acids include: glycine 0.0245-0.0533 g / L, L-alanine 0.0070-0.0780 g / L, L-arginine 0.1453-1.3972 g / L, L-asparagine 0.6789-1.8850 g / L, L-aspartic acid 0.4142-1.1500 g / L, L-cysteine ​​0.0245-0.3601 g / L, L-cystine 0.0523-0.5781 g / L, L-glutamic acid 0.1260-0.7150 g / L, L-histidine 0.1750-0.3574 g / L, L-isoleucine 0.339- 1.105g / L, L-leucine 0.364-2.080g / L, L-lysine 0.4035-1.1050g / L, L-methionine 0.0735-0.2590g / L, L-phenylalanine 0.2353-0.5915g / L, L-proline 0.2814-1.6250g / L, L-serine 0.3175-1.3000g / L, L-threonine 0.2917-0.7150g / L, L-tryptophan 0.0819-0.3120g / L, L-tyrosine 0.1750-0.9823g / L, L-valine 0.308-0.585g / L;

[0019] The vitamins include: thiamine hydrochloride 0.0021-0.0085g / L, riboflavin 0.0001-0.0101g / L, niacinamide 0.0025-0.0104g / L, D-calcium pantothenate 0.0018-0.0104g / L, pyridoxine 0.0018-0.0072g / L, biotin 0.0001-0.0024g / L, choline chloride 0.0693-0.3250g / L, inositol 0.0298-0.2730g / L, folic acid 0.0032-0.0588g / L, cyanocobalamin 0.0025-0.0143g / L, ascorbic acid 0.0000-0.0208g / L;

[0020] The inorganic salts include: calcium chloride 0.025-0.163 g / L, magnesium chloride 0.013-0.137 g / L, magnesium sulfate 0.022-0.332 g / L, potassium chloride 0.39-0.72 g / L, sodium bicarbonate 1.40-3.12 g / L, sodium chloride 0.14-3.12 g / L, sodium phosphate dibasic 0.14-0.65 g / L, sodium phosphate monobasic 0.07-1.95 g / L;

[0021] The trace elements include: ferric nitrate nonahydrate 0.0001-0.0003 g / L, ferrous sulfate heptahydrate 0.0028-0.0103 g / L, copper sulfate pentahydrate 0.000003-0.005288 g / L, zinc sulfate heptahydrate 0.0028-0.0240 g / L, ferric citrate 0.0245-0.0975 g / L, sodium selenite 0.00001050-0.00004022 g / L, sodium metasilicate 0.0000105-0.0000 0195g / L, barium acetate 0.00000025-0.00001950g / L, potassium iodide 0.00000011-0.00000020g / L, silver nitrate 0.00000000-0.00000007g / L, manganese chloride tetrahydrate 0.00000123-0.00000317g / L, sodium molybdate dihydrate 0.00007966-0.00026717g / L, cobalt chloride 0.00000042-0.0002 6627g / L, Aluminum Chloride 0.00000000-0.00000195g / L, Potassium Bromide 0.00000004-0.00000065g / L, Sodium Metavanadate 0.00000105-0.00000558g / L, Nickel Chloride 0.00000210-0.00000857g / L, Stannous Chloride 0.0000000-0.0001172g / L, Sodium Fluoride 0.00000840-0.00001560 g / L, cadmium chloride 0.00000000-0.00005051g / L, zirconium oxychloride 0.00000678-0.00002253g / L, rubidium chloride 0.00000070-0.00000455g / L, germanium tetroxide 0.00000000-0.00000156g / L, chromium chloride 0.00000000-0.00000131g / L, lithium chloride 0.0000000-0.0000026g / L;

[0022] The carbohydrates and other molecular compounds include: glucose 4.20-8.71 g / L, linoleic acid 0.0001-0.0005 g / L, putrescine 0.0053-0.1430 g / L, spermine 0.0000-0.0065 g / L, lipoic acid 0.0000-0.0021 g / L, sodium pyruvate 0.0308-0.2860 g / L, ethanolamine 0.0105-0.1300 g / L, L-reduced glutathione 0.0003-0.0023 g / L, hydroxyethylpiperazine ethanesulfonic acid 0.000-3.094 g / L, and Pluronic F68 0.70-1.56 g / L.

[0023] Furthermore, the amino acids include: glycine 0.0315-0.0451 g / L, L-alanine 0.009-0.066 g / L, L-arginine 0.1869-1.1823 g / L, L-asparagine 0.8729-1.5950 g / L, L-aspartic acid 0.5326-0.9731 g / L, L-cysteine ​​0.0315-0.3047 g / L, L-cystine 0.0672-0.4892 g / L, L-glutamic acid 0.162-0.605 g / L, L-histidine 0.2250-0.3024 g / L, L-isoleucine 0.4358-0. 9350g / L, L-leucine 0.468-1.760g / L, L-lysine 0.5188-0.9350g / L, L-methionine 0.0945-0.2192g / L, L-phenylalanine 0.3025-0.5005g / L, L-proline 0.3618-1.3750g / L, L-serine 0.4082-1.1000g / L, L-threonine 0.3751-0.6050g / L, L-tryptophan 0.1053-0.2640g / L, L-tyrosine 0.2250-0.8312g / L, L-valine 0.396-0.495g / L;

[0024] The vitamins include: thiamine hydrochloride 0.0027-0.0072 g / L, riboflavin 0.0002-0.0085 g / L, niacinamide 0.0032-0.0088 g / L, D-calcium pantothenate 0.0023-0.0088 g / L, pyridoxine 0.0023-0.0061 g / L, biotin 0.0001-0.0020 g / L, choline chloride 0.0891-0.2750 g / L, inositol 0.0383-0.2310 g / L, folic acid 0.0041-0.0498 g / L, cyanocobalamin 0.0032-0.0121 g / L, and ascorbic acid 0.0000-0.0176 g / L;

[0025] The inorganic salts include: calcium chloride 0.032-0.138 g / L, magnesium chloride 0.017-0.116 g / L, magnesium sulfate 0.029-0.281 g / L, potassium chloride 0.50-0.61 g / L, sodium bicarbonate 1.80-2.64 g / L, sodium chloride 0.18-2.64 g / L, sodium phosphate dibasic 0.18-0.55 g / L, sodium phosphate monobasic 0.09-1.65 g / L;

[0026] The trace elements include: ferric nitrate nonahydrate 0.0001-0.0003 g / L, ferrous sulfate heptahydrate 0.0036-0.0087 g / L, copper sulfate pentahydrate 0.000004-0.004474 g / L, zinc sulfate heptahydrate 0.0036-0.0203 g / L, ferric citrate 0.0315-0.0825 g / L, sodium selenite 0.00001350-0.00003403 g / L, sodium metasilicate 0.0000135-0.0000 0165g / L, barium acetate 0.00000032-0.00001650g / L, potassium iodide 0.00000014-0.00000017g / L, silver nitrate 0.00000000-0.00000006g / L, manganese chloride tetrahydrate 0.00000158-0.00000268g / L, sodium molybdate dihydrate 0.00010243-0.00022607g / L, cobalt chloride 0.00000054-0.0002 2531g / L, Aluminum Chloride 0.00000000-0.00000165g / L, Potassium Bromide 0.00000005-0.00000055g / L, Sodium Metavanadate 0.00000135-0.00000472g / L, Nickel Chloride 0.00000270-0.00000725g / L, Stannous Chloride 0.00000000-0.00009917g / L, Sodium Fluoride 0.0000108-0.0000132 g / L, cadmium chloride 0.00000000-0.00004274g / L, zirconium oxychloride 0.00000871-0.00001906g / L, rubidium chloride 0.00000090-0.00000385g / L, germanium tetroxide 0.00000000-0.00000132g / L, chromium chloride 0.00000000-0.00000111g / L, lithium chloride 0.0000000-0.0000022g / L;

[0027] The carbohydrates and other molecular compounds include: glucose 5.40-7.37 g / L, linoleic acid 0.0001-0.0005 g / L, putrescine 0.0068-0.1210 g / L, spermine 0.0000-0.0055 g / L, lipoic acid 0.0000-0.0018 g / L, sodium pyruvate 0.0396-0.2420 g / L, ethanolamine 0.0135-0.1100 g / L, L-reduced glutathione 0.0003-0.0020 g / L, hydroxyethylpiperazine ethanesulfonic acid 0.000-2.618 g / L, and Pluronic F68 0.90-1.32 g / L.

[0028] Preferably, the CHO cells include CHO-K1, expiCHO, CHO-S and CHO-K12F10.

[0029] Preferably, the culture medium further contains hydrochloric acid or sodium hydroxide. Preferably, the pH of the culture medium is 7.0 to 7.3.

[0030] On the other hand, the present invention discloses a method for preparing the culture medium for CHO cell suspension culture, comprising the following steps:

[0031] (1) mixing the amino acids, vitamins, inorganic salts, trace elements, carbohydrates and other molecular compounds with water;

[0032] (2) adding alkaline solution to aid dissolution;

[0033] (3) adding sodium bicarbonate;

[0034] (4) adjusting the pH of the solution;

[0035] (5) adding an osmotic pressure regulator to adjust the osmotic pressure to 250-350 Osm / L; preferably, the adjusted osmotic pressure is 290±10 Osm / L.

[0036] Preferably, the alkaline solution is NaOH.

[0037] The sodium bicarbonate can form a system with the carbon dioxide in the incubator to maintain the pH stability of the culture medium and ensure a neutral environment for cell growth.

[0038] Furthermore, the step (1) is added in the following order:

[0039] a. The amino acid dissolution according to any one of claims 1-7;

[0040] b. dissolving the inorganic salt according to any one of claims 1 to 7;

[0041] c. Add the vitamin according to any one of claims 1-7 into a concentrated solution;

[0042] d. Add the trace element according to any one of claims 1-7 into a concentrated solution;

[0043] e. dissolving any carbohydrate and other molecular compounds according to claim 1-7;

[0044] Preferably, the adding is dropwise addition.

[0045] The "dropping" is to add slowly to avoid precipitation.

[0046] On the other hand, the present invention discloses use of the medium for CHO cell suspension culture in culturing CHO cells.

[0047] Beneficial Effects

[0048] The chemically defined culture medium of the present application is obtained through a large number of experimental screenings. Its composition directly affects the culture effect, and there is no regularity to follow.

[0049] The chemically defined culture medium of the present invention can meet the needs of high-density suspension culture of CHO cells, improve production efficiency and reduce production costs; at the same time, because the ingredients are limited, it is conducive to the purification of the product, can improve the stability of the production batch, and is convenient for making adjustments to project problems, thereby ensuring the quality of the product and improving the controllability of the product. Ultimately, it can match cells to achieve large-scale high-density culture, and has broad application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is the growth curve of CHO-K1 2F10 cells;

[0051] Figure 2 is the growth curve of CHO-K1 2F10 cells after feeding;

[0052] Figure 3 Growth curves of CHO-K1 2F10, CHO-K1, expiCHO, and CHO-S cells;

[0053] Figure 4 A growth curve after feeding was performed for the single clone with the highest expression level;

[0054] Figure 5 This is the antibody expression determination curve. DETAILED DESCRIPTION

[0055] Specifically, in order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein. Unless otherwise indicated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0057] As used herein, the terms "including," "comprising," "having," "may," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0058] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (comprising recombinant technology), microbiology, biochemistry and immunology, which are within the capabilities of those skilled in the art. This technology is fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, 1984); "Animal Cell Culture" (RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (DM Weir and CC Black, 1989). ell, ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0059] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0060] The composition of the medium for the CHO cell suspension culture described in the following examples (including amino acids, vitamins, inorganic salts, trace elements, carbohydrates and other molecular compounds) was purchased from Shanghai Bioengineering; expiCHO and CHO-S cell lines were from Thermo; the cell line CHO-K12F10 was obtained by domestication and deposited in the General Microbiological Center of China Microbiological Culture Collection (CGMCC) with the deposit number CGMCC 45325.

[0061] Preparation Example Method for Preparing Medium for CHO Cell Suspension Culture

[0062] The steps include:

[0063] (1) mixing the amino acids, vitamins, inorganic salts, trace elements, carbohydrates and other molecular compounds with water;

[0064] (2) adding alkaline solution to aid dissolution;

[0065] (3) adding sodium bicarbonate;

[0066] (4) Adjust the solution pH to 7.0-7.3;

[0067] (5) adding an osmotic pressure regulator to adjust the osmotic pressure to 250-350 Osm / L; preferably, the adjusted osmotic pressure is 290±10 Osm / L.

[0068] The details are as follows:

[0069] 1. Dissolution of amino acid components: Weigh sufficient amount of amino acids respectively and add them into 800 mL of water, stir at room temperature until dissolved, in some embodiments, a magnetic stirrer may be used for stirring. In some embodiments, an appropriate amount of sodium hydroxide may also be added to assist dissolution.

[0070] The sufficient amount of amino acids in this embodiment refers to the final concentration of each amino acid component after the final volume is set to 1L: glycine 0.0210-0.0574g / L, L-alanine 0.0060-0.0840g / L, L-arginine 0.1246-1.5047g / L, L-asparagine 0.582-2.030g / L, L-aspartic acid 0.3550-1.2385g / L, L-cysteine ​​0.0210-0.3878g / L, L-cystine 0.0448-0.6226g / L, L-glutamic acid 0.108-0.770g / L, L-histidine 0.1500-0.3849g / L, L -Isoleucine 0.2905-1.1900g / L, L-leucine 0.312-2.240g / L, L-lysine 0.3458-1.1900g / L, L-methionine 0.0630-0.2789g / L, L-phenylalanine 0.2017-0.6370g / L, L-proline 0.2412-1.7500g / L, L-serine 0.2721-1.4000g / L, L-threonine 0.25-0.77g / L, L-tryptophan 0.0702-0.3360g / L, L-tyrosine 0.1500-1.0579g / L, L-valine 0.264-0.630g / L.

[0071] 2. The inorganic salt can be directly weighed as solid powder. Weigh an appropriate amount and add it to step 1 until it is completely dissolved.

[0072] After dissolution and the volume is adjusted to 1L, the final concentrations of the inorganic salt components are: calcium chloride 0.021-0.175g / L, magnesium chloride 0.011-0.147g / L, magnesium sulfate 0.019-0.357g / L, potassium chloride 0.33-0.77g / L, sodium bicarbonate 1.20-3.36g / L, sodium chloride 0.12-3.36g / L, sodium phosphate dibasic 0.12-0.70g / L, sodium phosphate monobasic 0.06-2.10g / L.

[0073] 3. Prepare the vitamins to a certain 1000x mother liquor concentration and place at 4°C to avoid degradation. The shelf life should be measured and ensured to be within the shelf life range. Pipette a quantitative amount and add it to the solution after step 2, and observe carefully to avoid precipitation.

[0074] The vitamins are thiamine hydrochloride, riboflavin, nicotinamide, D-calcium pantothenate, pyridoxine, biotin, choline chloride, inositol, folic acid, cyanocobalamin, and ascorbic acid.

[0075] After the volume is fixed to 1L, the final concentrations of the vitamin components are: thiamine hydrochloride 0.0018-0.0091g / L, riboflavin 0.0001-0.0109g / L, niacinamide 0.0021-0.0112g / L, D-calcium pantothenate 0.0015-0.0112g / L, pyridoxine 0.0015-0.0077g / L, biotin 0.0001-0.0026g / L, choline chloride 0.0594-0.3500g / L, inositol 0.0255-0.2940g / L, folic acid 0.0027-0.0634g / L, cyanocobalamin 0.0021-0.0154g / L, ascorbic acid 0.0000-0.0224g / L.

[0076] 4. Add trace elements to the solution treated in step 3. Trace elements need to be prepared into a mother solution in advance, such as 1000x, and placed at 4°C away from light to avoid degradation. Measure the shelf life and make sure it is within the shelf life. Add metal elements slowly to avoid precipitation.

[0077] After the volume was fixed to 1L, the final concentrations of the trace elements were: ferric nitrate nonahydrate 0.0001-0.0004g / L, ferrous sulfate heptahydrate 0.0024-0.0111g / L, copper sulfate pentahydrate 0.000002-0.005694g / L, zinc sulfate heptahydrate 0.0024-0.0258g / L, ferric citrate 0.021-0.105g / L, sodium selenite 0.00000900-0.00004332g / L, sodium metasilicate 0.000009- 0.000021g / L, barium acetate 0.00000021-0.00002100g / L, potassium iodide 0.00000009-0.00000021g / L, silver nitrate 0.00000000-0.00000008g / L, manganese chloride tetrahydrate 0.00000105-0.00000341g / L, sodium molybdate dihydrate 0.00006828-0.00028773g / L, cobalt chloride 0.00000036-0.00 028676g / L, aluminum chloride 0.00000000-0.00000210g / L, potassium bromide 0.00000004-0.00000070g / L, sodium metavanadate 0.00000090-0.00000601g / L, nickel chloride 0.00000180-0.00000923g / L, stannous chloride 0.00000000-0.00012621g / L, sodium fluoride 0.00000720-0.000016 80g / L, cadmium chloride 0.00000000-0.00005440g / L, zirconium oxychloride 0.00000581-0.00002426g / L, rubidium chloride 0.0000006-0.0000049g / L, germanium tetroxide 0.00000000-0.00000168g / L, chromium chloride 0.00000000-0.00000141g / L, lithium chloride 0.00000000-0.0000028g / L.

[0078] 5. Dissolve carbohydrates and other molecular compounds in the solution after step 4. Among them, components such as glucose and putrescine have better solubility and can be added first.

[0079] After the volume was fixed to 1 L, the final concentrations of the carbohydrates and other molecular compounds were: glucose 3.60-9.38 g / L, linoleic acid 0.0001-0.0006 g / L, putrescine 0.0045-0.1540 g / L, spermine 0.000-0.007 g / L, lipoic acid 0.0000-0.0022 g / L, sodium pyruvate 0.0264-0.3080 g / L, ethanolamine 0.009-0.140 g / L, L-reduced glutathione 0.0002-0.0025 g / L, hydroxyethylpiperazine ethanesulfonic acid 0.000-3.332 g / L, and Pluronic F68 0.60-1.68 g / L.

[0080] 6. After complete dissolution, begin adjusting the pH to 7.0-7.3.

[0081] 7. Osmotic pressure was measured by adding a certain amount of sodium chloride to make the osmotic pressure at 290 ± 10 Osm / L.

[0082] 8. Final volume is 1L

[0083] 9. After filtering at 0.22um, package it into a special bottle.

[0084] 10. Take 20 ml for sterility test and 20 ml for precipitation test. After confirming sterility and no precipitation, store at 4°C and use within 6 months.

[0085] Example 2 Preparation of medium for CHO cell suspension culture 2

[0086] In this embodiment, the solution is prepared according to the method of embodiment 1 until each component is completely dissolved.

[0087] The final culture medium contains amino acids, vitamins, inorganic salts, trace elements, carbohydrates and other molecular compounds.

[0088] The final concentrations of the amino acid components are: glycine 0.0315-0.0451 g / L, L-alanine 0.009-0.066 g / L, L-arginine 0.1869-1.1823 g / L, L-asparagine 0.8729-1.5950 g / L, L-aspartic acid 0.5326-0.9731 g / L, L-cysteine ​​0.0315-0.3047 g / L, L-cystine 0.0672-0.4892 g / L, L-glutamic acid 0.162-0.605 g / L, L-histidine 0.2250-0.3024 g / L, L-isoleucine 0.4358-0. 9350g / L, L-leucine 0.468-1.760g / L, L-lysine 0.5188-0.9350g / L, L-methionine 0.0945-0.2192g / L, L-phenylalanine 0.3025-0.5005g / L, L-proline 0.3618-1.3750g / L, L-serine 0.4082-1.1000g / L, L-threonine 0.3751-0.6050g / L, L-tryptophan 0.1053-0.2640g / L, L-tyrosine 0.2250-0.8312g / L, L-valine 0.396-0.495g / L;

[0089] The final concentrations of the vitamin components are: thiamine hydrochloride 0.0027-0.0072 g / L, riboflavin 0.0002-0.0085 g / L, niacinamide 0.0032-0.0088 g / L, D-calcium pantothenate 0.0023-0.0088 g / L, pyridoxine 0.0023-0.0061 g / L, biotin 0.0001-0.0020 g / L, choline chloride 0.0891-0.2750 g / L, inositol 0.0383-0.2310 g / L, folic acid 0.0041-0.0498 g / L, cyanocobalamin 0.0032-0.0121 g / L, ascorbic acid 0.0000-0.0176 g / L;

[0090] The final concentrations of the inorganic salt components are: calcium chloride 0.032-0.138 g / L, magnesium chloride 0.017-0.116 g / L, magnesium sulfate 0.029-0.281 g / L, potassium chloride 0.50-0.61 g / L, sodium bicarbonate 1.80-2.64 g / L, sodium chloride 0.18-2.64 g / L, sodium phosphate dibasic 0.18-0.55 g / L, sodium phosphate monobasic 0.09-1.65 g / L;

[0091] The final concentrations of the trace elements are: ferric nitrate nonahydrate 0.0001-0.0003 g / L, ferrous sulfate heptahydrate 0.0036-0.0087 g / L, copper sulfate pentahydrate 0.000004-0.004474 g / L, zinc sulfate heptahydrate 0.0036-0.0203 g / L, ferric citrate 0.0315-0.0825 g / L, sodium selenite 0.00001350-0.00003403 g / L, sodium metasilicate 0.0000135-0.00003403 g / L. 0.0000165g / L, barium acetate 0.00000032-0.00001650g / L, potassium iodide 0.00000014-0.00000017g / L, silver nitrate 0.00000000-0.00000006g / L, manganese chloride tetrahydrate 0.00000158-0.00000268g / L, sodium molybdate dihydrate 0.00010243-0.00022607g / L, cobalt chloride 0.00000054-0.0 0022531g / L, aluminum chloride 0.00000000-0.00000165g / L, potassium bromide 0.00000005-0.00000055g / L, sodium metavanadate 0.00000135-0.00000472g / L, nickel chloride 0.00000270-0.00000725g / L, stannous chloride 0.00000000-0.00009917g / L, sodium fluoride 0.0000108-0.000013 2g / L, cadmium chloride 0.00000000-0.00004274g / L, zirconium oxychloride 0.00000871-0.00001906g / L, rubidium chloride 0.00000090-0.00000385g / L, germanium tetroxide 0.00000000-0.00000132g / L, chromium chloride 0.00000000-0.00000111g / L, lithium chloride 0.0000000-0.0000022g / L;

[0092] The final concentrations of the carbohydrates and other molecular compounds are: glucose 5.40-7.37 g / L, linoleic acid 0.0001-0.0005 g / L, putrescine 0.0068-0.1210 g / L, spermine 0.0000-0.0055 g / L, lipoic acid 0.0000-0.0018 g / L, sodium pyruvate 0.0396-0.2420 g / L, ethanolamine 0.0135-0.1100 g / L, L-reduced glutathione 0.0003-0.0020 g / L, hydroxyethylpiperazine ethanesulfonic acid 0.000-2.618 g / L, and Pluronic F68 0.90-1.32 g / L.

[0093] After the preparation is completed, carefully adjust the pH to 7.0 with hydrochloric acid, and then add sodium chloride to adjust the osmotic pressure to 290±10Osm / L. Finally, filter with a 0.22μm sterile membrane and store at 4℃ for long-term use.

[0094] Example 3 CHO cell fermentation test

[0095] 1. Take out a CHO-K12F10 frozen cell tube from liquid nitrogen, quickly dissolve the cells in a preheated water bath (within 1 min), wipe the outer wall with 75% ethanol and place it in a biosafety cabinet;

[0096] 2. Add 9 ml of preheated culture medium to a 15 ml centrifuge tube, slowly add the cell freezing solution, and centrifuge at room temperature, 1000 rpm, for 5 minutes;

[0097] 3. Discard the supernatant, add 8 ml of newly prepared culture medium to resuspend the cells (add glutamine), transfer to a culture dish, place in an incubator at 37°C, 180 rpm, 8% CO2, and observe the cell status the next day;

[0098] 4. Pipette 20ul of cell suspension, add typanblue dye, let stand for 1min, then observe cell viability and count under a microscope;

[0099] 5. After 72 hours, the cells can reach the passage state and be passaged to the P4 generation;

[0100] 6. After counting, 3x10 5 cells / ml cells for inoculation, total volume 30ml system (125ml shake flask), calculate the amount of cells to be added, and add directly to the new culture medium without centrifugation;

[0101] 7. Cultivate at 37°C, 180rpm, 8% CO2. During batch culture, take samples on the third, fifth, and seventh days to measure cell viability and density. Draw a growth curve based on the results, such as Figure 1 shown.

[0102] 8. During the batch culture process, samples were taken on the 3rd, 5th, 7th, 9th and 11th days to measure the cell density and viability, and the corresponding feed was added at the same time. After the feed was added on the 5th day, the temperature was reduced to 32°C. A growth curve was drawn based on the experimental results, such as Figure 2 shown.

[0103] 9. In the same way, CHO-K1, expiCHO, and CHO-S cells were revived and growth curves were drawn. Figure 3 shown.

[0104] According to the results, the self-developed formula culture medium can support the high-density growth of different CHO cells.

[0105] The "feed" medium of the present invention can be adjusted by a person skilled in the art according to the needs to adjust the concentration range of each component.

[0106] Example 4 Development of antibody expressing cell lines and fermentation production testing

[0107] 1. Take out a CHO-K12F10 cryotube from liquid nitrogen, quickly dissolve the cells in a preheated water bath (within 1 min), wipe the outer wall with 75% ethanol and place it in a biosafety cabinet;

[0108] 2. Add 9 ml of preheated self-developed culture medium to a 15 ml centrifuge tube, slowly add the cell freezing solution, and centrifuge at room temperature, 1000 rpm, for 5 minutes;

[0109] 3. Discard the supernatant, add 8 ml of self-developed culture medium to resuspend the cells (add glutamine), transfer to a culture dish, place in an incubator at 37°C, 120 rpm, 8% CO2, and observe the cell status the next day;

[0110] 4. Pipette 20ul of cell suspension, add typanblue dye, let stand for 1min, then observe cell viability and count under a microscope;

[0111] 5. Record the activity and number in the same way the next day;

[0112] 6. After 72 hours, cells can reach the passage state;

[0113] 7. After counting, 5x10 5 cells / ml cells for passaging, after determining the total volume, calculate the amount of cells to be added and directly introduce them into the new culture medium without centrifugation;

[0114] 8. Culture at 37°C, 180rpm, 8% CO2, cell density should not exceed 6x10 6 cells / ml;

[0115] 9. Centrifuge at 1000 rpm at room temperature for 5 min to collect cells;

[0116] 10. Wash cells twice with fresh culture medium or PBS;

[0117] 11. Adjust the cell density to 1x10 7 cells / ml, add 800ul to a 0.4cm electroporation cup, and add a total of 33ug of plasmid, 30ug of single vector, and 3ug of transposition vector, and gently pipette several times to mix the cells and plasmid;

[0118] 12. 0.3KV, 960uF electric shock 12-14ms;

[0119] 13. Aspirate the cells, add 7 ml of self-developed culture medium (with glutamine) and place in a culture dish, incubate at 37°C, 5% CO2 for 48 hours, and proceed to the next step after confirming the expression of the supernatant;

[0120] 14. According to the results of the 96h expression supernatant, the screening process is carried out after the expression is confirmed, and the cells are counted and viable;

[0121] 15. Determine the number of plates to be 3, the plate concentration to be 10,000 / well, no glutamine is added to the culture medium, and a final concentration of 50uM MSX is added;

[0122] 16. Place in an incubator for static culture at 37°C, 8% CO2 for 15 days;

[0123] 17. Determine the cell growth wells and perform ELISA on the supernatant to determine the top 5 minipool strains for expansion culture;

[0124] 18. After the 6-well plate is expanded, the supernatant is assayed by ELISA to determine the expression level of the cell pool;

[0125] 19. After 11 days of fermentation in 125ml shaker flask, determine the top three cell pools and proceed to the next step for single clone screening:

[0126] (1) Cell viability > 95%, culture medium was used to adjust the cell number to 0.5 / 200ul before plating;

[0127] (2) Five plates were plated in total and placed in an incubator for 12-14 days to detect the titer of the supernatant. Single clones with fast cell growth and high titer were selected for expansion. Six cell lines were determined for expansion. After expansion, 30 ml of the flask was shaken to verify the expression level. The single clone with the highest expression level was selected for fed-batch fermentation verification;

[0128] (3) Cultivate at 37°C, 180 rpm, 8% CO2. During batch culture, samples were taken on days 3, 5, 7, 9, 11, and 13 to measure cell density and viability, and corresponding feed was added. After adding feed on day 5, the temperature was lowered to 32°C. A growth curve was drawn based on the experimental results, such as Figure 4 shown.

[0129] (4) Antibody expression determination: The total amount of mouse antibody was determined by biochemical method. The expression of samples on days 3, 5, 7, 9, 11, and 13 was measured and the curve was drawn. Figure 5 shown.

[0130] The “feeding” culture medium of the present invention can be used by those skilled in the art to adjust the concentration range of each component according to needs.

[0131] The above experimental results show that the chemical composition-limited CHO cell culture medium of the present invention can support the growth and metabolism of various CHO cells in the current industry and scientific research community, can achieve higher cell density, cell density, and cell survival rate, and meet the needs of seed passage and expansion stages in the early stage of large-scale production, and has great application prospects. In particular, it has a great advantage on CHO-K12F10 cells, and has obvious advantages in both viable cell density and doubling rate, which is conducive to antibody expression and production.

[0132] The culture medium of the present invention is a stable and balanced whole, and the various components interact with each other to jointly promote the growth of Chinese hamster ovary cells, provide a rich nutritional environment for the growth of Chinese hamster ovary cells, and avoid various disadvantages of serum addition. The various components of the culture medium of the present invention work together to reduce the harmful metabolites produced by the growth and metabolism of Chinese hamster ovary cells. Therefore, the chemically defined culture medium of the present application can significantly improve the culture efficiency of CHO-K1 cells and shorten the doubling time. The culture medium of the present invention can promote the expression of functional proteins of Chinese hamster ovary cells and improve their performance.

[0133] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A medium for CHO cell suspension culture, characterized in that: The culture medium includes amino acids, vitamins, inorganic salts, trace elements, carbohydrates and other molecular compounds; The amino acids include glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

2. The culture medium according to claim 1, characterized in that The vitamins include thiamine hydrochloride, riboflavin, niacinamide, calcium D-pantothenate, pyridoxine, biotin, choline chloride, inositol, folic acid, cyanocobalamin and ascorbic acid; Preferably, the inorganic salts include calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, dibasic sodium phosphate and monobasic sodium phosphate; Preferably, the trace elements include ferric nitrate nonahydrate, ferrous sulfate heptahydrate, cupric sulfate pentahydrate, zinc sulfate heptahydrate, ferric citrate, sodium selenite, sodium metasilicate, barium acetate, potassium iodide, silver nitrate, manganese chloride tetrahydrate, sodium molybdate dihydrate, cobalt chloride, aluminum chloride, potassium bromide, sodium metavanadate, nickel chloride, stannous chloride, sodium fluoride, cadmium chloride, zirconium oxychloride, rubidium chloride, germanium tetroxide, chromium chloride and lithium chloride; Preferably, the carbohydrates and other molecular compounds include glucose, linoleic acid, putrescine, spermine, lipoic acid, sodium pyruvate, ethanolamine, L-reduced glutathione, hydroxyethylpiperazineethanesulfonic acid and Pluronic F68.

3. The culture medium according to claim 1 or 2, characterized in that The amino acids include: glycine 0.0210-0.0574 g / L, L-alanine 0.0060-0.0840 g / L, L-arginine 0.1246-1.5047 g / L, L-asparagine 0.582-2.030 g / L, L-aspartic acid 0.3550-1.2385 g / L, L-cysteine ​​0.0210-0.3878 g / L, L-cystine 0.0448-0.6226 g / L, L-glutamic acid 0.108-0.770 g / L, L-histidine 0.1500-0.3849 g / L, L-isoleucine 0.2905-1.19 00g / L, L-leucine 0.312-2.240g / L, L-lysine 0.3458-1.1900g / L, L-methionine 0.0630-0.2789g / L, L-phenylalanine 0.2017-0.6370g / L, L-proline 0.2412-1.7500g / L, L-serine 0.2721-1.4000g / L, L-threonine 0.25-0.77g / L, L-tryptophan 0.0702-0.3360g / L, L-tyrosine 0.1500-1.0579g / L and L-valine 0.264-0.630g / L; Preferably, the amino acids include: glycine 0.0245-0.0533 g / L, L-alanine 0.0070-0.0780 g / L, L-arginine 0.1453-1.3972 g / L, L-asparagine 0.6789-1.8850 g / L, L-aspartic acid 0.4142-1.1500 g / L, L-cysteine ​​0.0245-0.3601 g / L, L-cystine 0.0523-0.5781 g / L, L-glutamic acid 0.1260-0.7150 g / L, L-histidine 0.1750-0.3574 g / L, L-isoleucine 0.339-1 .105g / L, L-leucine 0.364-2.080g / L, L-lysine 0.4035-1.1050g / L, L-methionine 0.0735-0.2590g / L, L-phenylalanine 0.2353-0.5915g / L, L-proline 0.2814-1.6250g / L, L-serine 0.3175-1.3000g / L, L-threonine 0.2917-0.7150g / L, L-tryptophan 0.0819-0.3120g / L, L-tyrosine 0.1750-0.9823g / L, L-valine 0.308-0.585g / L; More preferably, the amino acids include: glycine 0.0315-0.0451 g / L, L-alanine 0.009-0.066 g / L, L-arginine 0.1869-1.1823 g / L, L-asparagine 0.8729-1.5950 g / L, L-aspartic acid 0.5326-0.9731 g / L, L-cysteine ​​0.0315-0.3047 g / L, L-cystine 0.0672-0.4892 g / L, L-glutamic acid 0.162-0.605 g / L, L-histidine 0.2250-0.3024 g / L, L-isoleucine 0.4358-0. 9350g / L, L-leucine 0.468-1.760g / L, L-lysine 0.5188-0.9350g / L, L-methionine 0.0945-0.2192g / L, L-phenylalanine 0.3025-0.5005g / L, L-proline 0.3618-1.3750g / L, L-serine 0.4082-1.1000g / L, L-threonine 0.3751-0.6050g / L, L-tryptophan 0.1053-0.2640g / L, L-tyrosine 0.2250-0.8312g / L, L-valine 0.396-0.495g / L.

4. The culture medium according to claim 3, characterized in that The vitamins include: thiamine hydrochloride 0.0018-0.0091 g / L, riboflavin 0.0001-0.0109 g / L, niacinamide 0.0021-0.0112 g / L, D-calcium pantothenate 0.0015-0.0112 g / L, pyridoxine 0.0015-0.0077 g / L, biotin 0.0001-0.0026 g / L, choline chloride 0.0594-0.3500 g / L, inositol 0.0255-0.2940 g / L, folic acid 0.0027-0.0634 g / L, cyanocobalamin 0.0021-0.0154 g / L, and ascorbic acid 0.0000-0.0224 g / L; Preferably, the inorganic salts include: calcium chloride 0.021-0.175 g / L, magnesium chloride 0.011-0.147 g / L, magnesium sulfate 0.019-0.357 g / L, potassium chloride 0.33-0.77 g / L, sodium bicarbonate 1.20-3.36 g / L, sodium chloride 0.12-3.36 g / L, sodium phosphate dibasic 0.12-0.70 g / L, sodium phosphate monobasic 0.06-2.10 g / L; Preferably, the trace elements include: ferric nitrate nonahydrate 0.0001-0.0004 g / L, ferrous sulfate heptahydrate 0.0024-0.0111 g / L, copper sulfate pentahydrate 0.000002-0.005694 g / L, zinc sulfate heptahydrate 0.0024-0.0258 g / L, ferric citrate 0.021-0.105 g / L, sodium selenite 0.00000900-0.00004332 g / L, sodium metasilicate 0.000009-0.00 0021g / L, barium acetate 0.00000021-0.00002100g / L, potassium iodide 0.00000009-0.00000021g / L, silver nitrate 0.00000000-0.00000008g / L, manganese chloride tetrahydrate 0.00000105-0.00000341g / L, sodium molybdate dihydrate 0.00006828-0.00028773g / L, cobalt chloride 0.00000036-0.00028 676g / L, Aluminum Chloride 0.00000000-0.00000210g / L, Potassium Bromide 0.00000004-0.00000070g / L, Sodium Metavanadate 0.00000090-0.00000601g / L, Nickel Chloride 0.00000180-0.00000923g / L, Stannous Chloride 0.00000000-0.00012621g / L, Sodium Fluoride 0.00000720-0.0000168 0g / L, cadmium chloride 0.00000000-0.00005440g / L, zirconium oxychloride 0.00000581-0.00002426g / L, rubidium chloride 0.0000006-0.0000049g / L, germanium tetroxide 0.00000000-0.00000168g / L, chromium chloride 0.00000000-0.00000141g / L, lithium chloride 0.00000000-0.0000028g / L; Preferably, the carbohydrates and other molecular compounds include: glucose 3.60-9.38 g / L, linoleic acid 0.0001-0.0006 g / L, putrescine 0.0045-0.1540 g / L, spermine 0.000-0.007 g / L, lipoic acid 0.0000-0.0022 g / L, sodium pyruvate 0.0264-0.3080 g / L, ethanolamine 0.009-0.140 g / L, L-reduced glutathione 0.0002-0.0025 g / L, hydroxyethylpiperazine ethanesulfonic acid 0.000-3.332 g / L, and Pluronic F68 0.60-1.68 g / L.

5. The culture medium according to claim 4, characterized in that The vitamins include: thiamine hydrochloride 0.0021-0.0085g / L, riboflavin 0.0001-0.0101g / L, niacinamide 0.0025-0.0104g / L, D-calcium pantothenate 0.0018-0.0104g / L, pyridoxine 0.0018-0.0072g / L, biotin 0.0001-0.0024g / L, choline chloride 0.0693-0.3250g / L, inositol 0.0298-0.2730g / L, folic acid 0.0032-0.0588g / L, cyanocobalamin 0.0025-0.0143g / L, ascorbic acid 0.0000-0.0208g / L; The inorganic salts include: calcium chloride 0.025-0.163 g / L, magnesium chloride 0.013-0.137 g / L, magnesium sulfate 0.022-0.332 g / L, potassium chloride 0.39-0.72 g / L, sodium bicarbonate 1.40-3.12 g / L, sodium chloride 0.14-3.12 g / L, sodium phosphate dibasic 0.14-0.65 g / L, sodium phosphate monobasic 0.07-1.95 g / L; The trace elements include: ferric nitrate nonahydrate 0.0001-0.0003 g / L, ferrous sulfate heptahydrate 0.0028-0.0103 g / L, copper sulfate pentahydrate 0.000003-0.005288 g / L, zinc sulfate heptahydrate 0.0028-0.0240 g / L, ferric citrate 0.0245-0.0975 g / L, sodium selenite 0.00001050-0.00004022 g / L, sodium metasilicate 0.0000105-0.0000 0195g / L, barium acetate 0.00000025-0.00001950g / L, potassium iodide 0.00000011-0.00000020g / L, silver nitrate 0.00000000-0.00000007g / L, manganese chloride tetrahydrate 0.00000123-0.00000317g / L, sodium molybdate dihydrate 0.00007966-0.00026717g / L, cobalt chloride 0.00000042-0.0002 6627g / L, Aluminum Chloride 0.00000000-0.00000195g / L, Potassium Bromide 0.00000004-0.00000065g / L, Sodium Metavanadate 0.00000105-0.00000558g / L, Nickel Chloride 0.00000210-0.00000857g / L, Stannous Chloride 0.0000000-0.0001172g / L, Sodium Fluoride 0.00000840-0.00001560 g / L, cadmium chloride 0.00000000-0.00005051g / L, zirconium oxychloride 0.00000678-0.00002253g / L, rubidium chloride 0.00000070-0.00000455g / L, germanium tetroxide 0.00000000-0.00000156g / L, chromium chloride 0.00000000-0.00000131g / L, lithium chloride 0.0000000-0.0000026g / L; The carbohydrates and other molecular compounds include: glucose 4.20-8.71 g / L, linoleic acid 0.0001-0.0005 g / L, putrescine 0.0053-0.1430 g / L, spermine 0.0000-0.0065 g / L, lipoic acid 0.0000-0.0021 g / L, sodium pyruvate 0.0308-0.2860 g / L, ethanolamine 0.0105-0.1300 g / L, L-reduced glutathione 0.0003-0.0023 g / L, hydroxyethylpiperazine ethanesulfonic acid 0.000-3.094 g / L, Pluronic F68 0.70-1.56 g / L; Preferably, the vitamins include: thiamine hydrochloride 0.0027-0.0072 g / L, riboflavin 0.0002-0.0085 g / L, niacinamide 0.0032-0.0088 g / L, D-calcium pantothenate 0.0023-0.0088 g / L, pyridoxine 0.0023-0.0061 g / L, biotin 0.0001-0.0020 g / L, choline chloride 0.0891-0.2750 g / L, inositol 0.0383-0.2310 g / L, folic acid 0.0041-0.0498 g / L, cyanocobalamin 0.0032-0.0121 g / L, ascorbic acid 0.0000-0.0176 g / L; The inorganic salts include: calcium chloride 0.032-0.138 g / L, magnesium chloride 0.017-0.116 g / L, magnesium sulfate 0.029-0.281 g / L, potassium chloride 0.50-0.61 g / L, sodium bicarbonate 1.80-2.64 g / L, sodium chloride 0.18-2.64 g / L, sodium phosphate dibasic 0.18-0.55 g / L, sodium phosphate monobasic 0.09-1.65 g / L; The trace elements include: ferric nitrate nonahydrate 0.0001-0.0003 g / L, ferrous sulfate heptahydrate 0.0036-0.0087 g / L, copper sulfate pentahydrate 0.000004-0.004474 g / L, zinc sulfate heptahydrate 0.0036-0.0203 g / L, ferric citrate 0.0315-0.0825 g / L, sodium selenite 0.00001350-0.00003403 g / L, sodium metasilicate 0.0000135-0.0000 0165g / L, barium acetate 0.00000032-0.00001650g / L, potassium iodide 0.00000014-0.00000017g / L, silver nitrate 0.00000000-0.00000006g / L, manganese chloride tetrahydrate 0.00000158-0.00000268g / L, sodium molybdate dihydrate 0.00010243-0.00022607g / L, cobalt chloride 0.00000054-0.0002 2531g / L, Aluminum Chloride 0.00000000-0.00000165g / L, Potassium Bromide 0.00000005-0.00000055g / L, Sodium Metavanadate 0.00000135-0.00000472g / L, Nickel Chloride 0.00000270-0.00000725g / L, Stannous Chloride 0.00000000-0.00009917g / L, Sodium Fluoride 0.0000108-0.0000132 g / L, cadmium chloride 0.00000000-0.00004274g / L, zirconium oxychloride 0.00000871-0.00001906g / L, rubidium chloride 0.00000090-0.00000385g / L, germanium tetroxide 0.00000000-0.00000132g / L, chromium chloride 0.00000000-0.00000111g / L, lithium chloride 0.0000000-0.0000022g / L; The carbohydrates and other molecular compounds include: glucose 5.40-7.37 g / L, linoleic acid 0.0001-0.0005 g / L, putrescine 0.0068-0.1210 g / L, spermine 0.0000-0.0055 g / L, lipoic acid 0.0000-0.0018 g / L, sodium pyruvate 0.0396-0.2420 g / L, ethanolamine 0.0135-0.1100 g / L, L-reduced glutathione 0.0003-0.0020 g / L, hydroxyethylpiperazine ethanesulfonic acid 0.000-2.618 g / L, and Pluronic F68 0.90-1.32 g / L.

6. The culture medium according to any one of claims 1 to 5, characterized in that The CHO cells include CHO-K1, expiCHO, CHO-S and CHO-K12F10.

7. The culture medium according to any one of claims 1 to 5, characterized in that The culture medium further contains hydrochloric acid or sodium hydroxide. Preferably, the pH of the culture medium is 7.0-7.

3.

8. The method for preparing a culture medium according to any one of claims 1 to 7, characterized in that: The steps include: (1) mixing the amino acids, vitamins, inorganic salts, trace elements, carbohydrates and other molecular compounds described in any one of claims 1 to 7 with water; (2) adding alkaline solution to aid dissolution; (3) adding sodium bicarbonate; (4) adjusting the pH of the solution; (5) adding an osmotic pressure regulator to adjust the osmotic pressure to 250-350 Osm / L; preferably, the adjusted osmotic pressure is 290±10 Osm / L.

9. The method according to claim 8, characterized in that The step (1) is added in the following order: a. The amino acid dissolution according to any one of claims 1 to 7; b. dissolving the inorganic salt according to any one of claims 1 to 7; c. Add the vitamin according to any one of claims 1-7 into a concentrated solution; d. Add the trace element according to any one of claims 1-7 into a concentrated solution; e. dissolving any carbohydrate and other molecular compounds according to claim 1-7; Preferably, the adding is dropwise addition.

10. Use of the culture medium according to any one of claims 1 to 7 in culturing CHO cells.

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