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CN-122012373-A - CHO-K1 cell chemistry limiting basic culture medium

CN122012373ACN 122012373 ACN122012373 ACN 122012373ACN-122012373-A

Abstract

The invention discloses a chemically defined culture medium formula for high expression of engineering cell strain products of a stable cell line constructed by taking CHO-K1 cells as hosts, which is used for subculturing CHO-K1 host cells. The main components of the food comprise amino acid, inorganic salt, vitamin, trace elements and other components limited by chemical components. The invention improves the growth density and the activity rate of the engineering cell strain taking CHO-K1 as a host, increases the tolerance of cells, prolongs the time of a cell maintenance period, improves the expression quantity of the product obtained by the CHO-K1 engineering cell strain, and greatly reduces the cost of a culture medium.

Inventors

  • JIA YU
  • LI ZIYI
  • Yun Linying
  • FENG JIRUI
  • ZHOU KUN
  • CAO HUI
  • ZHANG ZIJING
  • GENG YA
  • JIANG YIFAN
  • DONG JING
  • LI CHENXIN
  • YANG LIPING
  • Huo Huixin
  • An jiaxing
  • XUE WEN
  • XU FENGQIN

Assignees

  • 华北制药集团新药研究开发有限责任公司

Dates

Publication Date
20260512
Application Date
20241112

Claims (6)

  1. 1. A CHO-K1 cell chemistry limit basic culture medium consists of amino acid, vitamins, carbohydrate and other additives, and is characterized in that the amino acid is L-arginine 0.32-0.6g/L, L-histidine 0.15-0.35g/L, L-isoleucine 0.25-0.68g/L, L-leucine 0.26-0.55g/L, L-lysine hydrochloride 0.28-0.7g/L, L-methionine 0.05-0.25g/L, L-phenylalanine 0.08-0.33g/L, L-threonine 0.26-0.4g/L, L-cysteine hydrochloride 0.15-0.35g/L, L-tyrosine 0.18-0.68g/L, L-tryptophan 0.1-0.3g/L, L-valine 0.38-0.55g/L, L-alanine 0.04-0.25g/L, L-asparagine 0.32-0.55g/L, L-serine/glutamic acid 2-glutamic acid 0.35-35-35.26-0.35-35 g/glutamic acid 0.0350.55-0.35-35 g/35-serine; The vitamins are 0.00005-0.0002g/L of biotin, 0.01-0.06g/L of inositol, 0.00005-0.002g/L of riboflavin, 0.02-0.08g/L of choline chloride, 0.0008-0.0026g/L, vc g/032 g/L of magnesium phosphate, 0.005-0.006g/L of folic acid, 0.0009-0.002g/L of cobalamin, 0.003-0.01g/L of thiamine, 0.001-0.15g/L of nicotinamide and 0.007-0.01g/; The inorganic salt is zinc sulfate 0.0006-0.0009g/L, zinc chloride 0.0001-0.0008g/L, sodium selenite 0.00001-0.0001g/L, hexahydrate sulfuric acid 0.0000001-0.000001g/L, ammonium molybdate 0.000005-0.00005g/L, sodium metavanadate 0.0000006-0.000006g/L, calcium chloride 0.1-0.6g/L, sodium bicarbonate 1.8-3.0g/L, potassium chloride 0.7-1.5g/L, magnesium sulfate 0.06-0.09g/L, magnesium chloride 0.03-0.05g/L, sodium dihydrogen phosphate 1.0-1.5g/L, 1.0-1.5G/L of disodium hydrogen phosphate, 0.00000003-0.0000075g/L of sodium iodide, 1.0-1.5g/L of monopotassium phosphate, 0.000006-0.0009g/L of copper sulfate, 0.00000002-0.0000002g/L of manganese sulfate monohydrate, 0.02-0.04g/L of ferric citrate, and 0.0004-0.001g/L of ferrous sulfate heptahydrate; The carbohydrate and other additives are sodium acetate 0-0.2g/L, linoleic acid 0.00001-0.0001g/L, ethanolamine 0.01-0.1g/L, putrescine 0.0005-0.005g/L, 4-hydroxyethyl piperazine ethanesulfonic acid 2.0-4.5g/L, D-glucose 6.0-8.0g/L, sodium glycerophosphate 0.03-0.1g/L, lipoic acid 0-0.002g/L, dextran sulfate 0.2-0.5g/L, trehalose 0-0.2g/L, p-aminobenzoic acid 0-0.006g/L, pyrroloquinoline quinone 0-0.001g/L, glutathione 0.0008-0.001g/L, poloxamer 1881-2g/L, N-acetyl-L-cysteine 0-0.08g/L, alpha-ketoglutaric acid 0-0.15g/L, malic acid 0-0.67g/L, oxaloacetic acid 0.2 g-2 g/L, and ribose 2.67 g/L.
  2. 2. The CHO-K1 cell culture medium of claim 1, wherein the carbohydrate and other additives are lipoic acid 0.001-0.002g/L, trehalose 0.1-0.2g/L, p-aminobenzoic acid 0.003-0.006g/L, pyrroloquinoline quinone 0.0005-0.001g/L, N-acetyl-L-cysteine 0.04-0.08g/L, alpha-ketoglutaric acid 0.075-0.15g/L, malic acid 0.34-0.67g/L, oxaloacetic acid 0.1-0.2g/L, ribose 0.1-0.2g/L.
  3. 3. The CHO-K1 cell culture medium according to claim 2, wherein the amino acid is L-arginine 0.46g/L, L-histidine 0.25g/L, L-isoleucine 0.47g/L, L-leucine 0.41g/L, L-lysine hydrochloride 0.49g/L, L-methionine 0.15g/L, L-phenylalanine 0.21g/L, L-threonine 0.33g/L, L-cysteine hydrochloride 0.25g/L, L-tyrosine 0.43g/L, L-tryptophan 0.2g/L, L-valine 0.47g/L, L-alanine 0.15g/L, L-asparagine monohydrate 0.44g/L, L-glutamic acid 0.32g/L, L-glycine 0.048g/L, L-proline 0.43g/L, L-serine 0.9g/L, L-aspartic acid 0.9g/L; The vitamins are 0.00013g/L of biotin, 0.04g/L of inositol, 0.0013g/L of riboflavin, 0.05g/L of choline chloride, 0.0017g/L, vc g/L of magnesium phosphate salt of pyridoxine, 0.026g/L of folic acid, 0.006g/L of cobalamin, 0.0015g/L of thiamine, 0.008g/L of nicotinamide, and 0.009g/L of calcium pantothenate; The inorganic salt is zinc sulfate 0.0008g/L, zinc chloride 0.0005g/L, sodium selenite 0.00006g/L, nickel sulfate hexahydrate 0.0000006g/L, ammonium molybdate 0.000028g/L, sodium metavanadate 0.000003g/L, calcium chloride 0.4g/L, sodium bicarbonate 2.4g/L, potassium chloride 1.1g/L, magnesium sulfate 0.08g/L, magnesium chloride 0.04g/L, sodium dihydrogen phosphate 1.25g/L, disodium hydrogen phosphate 1.25g/L, potassium dihydrogen phosphate 1.25g/L, sodium iodide 0.0000038g/L, copper sulfate 0.000008g/L, manganese sulfate monohydrate 0.0000001g/L, ferric citrate 0.03g/L, and sodium sulfate heptahydrate 0.0007g/L; the carbohydrate and other additives are 0.1g/L of sodium acetate, 0.0005g/L of linoleic acid, 0.06g/L of ethanolamine, 0.0028g/L of putrescine, 3.25g/L, D g/L of 4-hydroxyethyl piperazine ethane sulfonic acid, 7g/L of glucose, 0.07g/L of sodium glycerophosphate, 0.001g/L of lipoic acid, 0.4g/L of dextran sulfate, 0.1g/L of trehalose, 0.003g/L of p-aminobenzoic acid, 0.0005g/L of pyrroloquinoline quinone, 0.0009g/L of glutathione, 1881.5g/L, N-acetyl-L-cysteine, 0.04g/L of alpha-ketoglutaric acid, 0.34g/L of malic acid, 0.1g/L of oxaloacetic acid and 0.1g/L of ribose.
  4. 4. A method for preparing a CHO-K1 cytochemically defined basal medium according to any one of claims 1 to 3, characterized in that: (1) Weighing all the components; (2) Dissolving in 0.8L pure water, and stirring until dissolving; (3) Regulating the pH value; (4) The volume is fixed to 1L by pure water; (5) Regulating osmotic pressure; (6) Sterile filtering to obtain the final product culture medium.
  5. 5. The method for preparing a CHO-K1 cytochemically defined basal medium according to claim 4, wherein the pH value is 7.0-7.4 and the osmotic pressure is 290-330mOsm/kg.
  6. 6. Use of a CHO-K1 cell chemolimiting basal medium according to any one of claims 1-3 for the cultivation of suspension CHO cells.

Description

CHO-K1 cell chemistry limiting basic culture medium Technical Field The invention relates to the fields of medicine, biotechnology and the like, in particular to a CHO-K1 cell chemistry limiting basal medium. Background Cell culture technology is one of the core foundations of modern biotechnology, and plays a vital role in various fields such as biopharmaceuticals, cell therapy, basic medical research and the like. Chinese Hamster Ovary (CHO) cells are an epithelial cell line derived from chinese hamster ovary, have a relatively stable genome and high doubling time, can be cultured on a large scale, and can be grown in serum-free or chemically defined media. Because of its unique advantages, CHO-K1 cells are the host cell of choice for the production of biological products such as recombinant proteins in the biotechnology industry. The CHO-K1 cell culture medium is taken as a key environmental factor of cell growth and product expression, and development and optimization of the medium have decisive significance for improving cell culture efficiency, product quality and yield, reducing pollution risk and the like. The chemical composition is clear that all the components in the culture medium are known chemical substances, and the concentration and the formula can be precisely controlled. The proper personalized chemical composition clear culture medium not only improves the stability and repeatability of cell culture, but also provides convenience for further optimizing the cell culture process and researching the cell metabolism mechanism. The CHO-K1 cell basic culture medium in the current market is relatively expensive, and the replacement product is difficult to find when the product is used on a large scale after being marketed, so that the life cycle of the product depends on the development of a culture medium company to bring about management risks, and substances such as IGF, hormone, hydrolysate and the like are added into part of the culture medium in China, so that not only are unknown substances contained in the culture medium components, but also a certain risk is brought to downstream purification, the hydrolysate is added into the culture medium, and the hormone is added into the culture medium in the patent CN118516299A, CN110894487, and the hormone is added into the culture medium in the patent CN115505561 and the patent CN104073463, so that the basic culture medium with low cost, high expression target product, stable performance and no serum and animal source protein is the actual demand of the current biomedical market. Disclosure of Invention Aiming at the prior art, the invention provides a CHO-K1 cell chemically defined medium supporting high expression of a product, which can realize the culture of CHO-K1 cells, and has no serum and animal source protein. The culture medium supports high-density growth of cells and high expression of target products, shortens the adaptability of the cells to a new culture medium, prolongs the maintenance period of the cells, further accumulates the target products and improves the production efficiency. The invention is realized by the following technical scheme: A CHO-K1 cell chemically defined basal medium supporting passage of host cells and high expression of products is composed of amino acids, vitamins, carbohydrates and other additives. The amino acid is L-arginine 0.32-0.6g/L, L-histidine 0.15-0.35g/L, L-isoleucine 0.25-0.68g/L, L-leucine 0.26-0.55g/L, L-lysine hydrochloride 0.28-0.7g/L, L-methionine 0.05-0.25g/L, L-phenylalanine 0.08-0.33g/L, L-threonine 0.26-0.4g/L, L-cysteine hydrochloride 0.15-0.35g/L, L-tyrosine 0.18-0.68g/L, L-tryptophan 0.1-0.3g/L, L-valine 0.38-0.55g/L, L-alanine 0.04-0.25g/L, L-asparagine monohydrate 0.32-0.55g/L, L-glutamic acid 0.22-0.42g/L, L-0.06 g/proline 0.35-35-tyrosine 0.18-0.68g/L, L-tryptophan 0.1-0.3-35-valine 0.38-0.55-valine/678-aspartic acid. The vitamins are 0.00005-0.0002g/L of biotin, 0.01-0.06g/L of inositol, 0.00005-0.002g/L of riboflavin, 0.02-0.08g/L of choline chloride, 0.0008-0.0026g/L, vc g/032 g/L of magnesium phosphate, 0.005-0.006g/L of folic acid, 0.0009-0.002g/L of cobalamin, 0.003-0.01g/L of thiamine, 0.001-0.15g/L of nicotinamide, and 0.007-0.01 g/L of calcium pantothenate. The inorganic salt is zinc sulfate 0.0006-0.0009g/L, zinc chloride 0.0001-0.0008g/L, sodium selenite 0.00001-0.0001g/L, hexahydrate sulfuric acid 0.0000001-0.000001g/L, ammonium molybdate 0.000005-0.00005g/L, sodium metavanadate 0.0000006-0.000006g/L, calcium chloride 0.1-0.6g/L, sodium bicarbonate 1.8-3.0g/L, potassium chloride 0.7-1.5g/L, magnesium sulfate 0.06-0.09g/L, magnesium chloride 0.03-0.05g/L, sodium dihydrogen phosphate 1.0-1.5g/L, disodium hydrogen phosphate 1.0-1.5g/L, sodium iodide 0.00000003-0.0000075g/L, potassium dihydrogen phosphate 1.0-1.5g/L, copper sulfate 0.000006-0.0009g/L, manganese sulfate 1.06-3.09 g/L, manganese sulfate 0.04-0.04 g/L, ferric sulfate 0.02-0.04 g/L. The carbohydrate and ot