CHO-K1 cell chemically limited basic culture medium

Abstract

The invention discloses a chemical defined culture medium formula for subculture of CHO-K1 host cells and high expression of an engineering cell strain product of a stable cell line constructed by taking CHO-K1 cells as hosts. The main components of the feed only comprise amino acids, inorganic salts, vitamins, trace elements and other components limited by chemical components. According to the invention, the growth density and motility of the engineering cell strain taking CHO-K1 as the host are improved, the tolerance of cells is increased, the maintenance period of the cells is prolonged, the product expression quantity of the CHO-K1 engineering cell strain is improved, and the cost of the culture medium is greatly reduced.

Description

Technical Field

[0001] This invention relates to the fields of medicine and biotechnology, and specifically to a chemically defined basal culture medium for CHO-K1 cells. Background Technology

[0002] Cell culture technology is one of the core foundations of modern biotechnology, playing a crucial role in numerous fields such as biopharmaceuticals, cell therapy, and basic medical research. Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovaries of Chinese hamsters. They possess a relatively stable genome and a high doubling time, allowing for large-scale culture and growth in serum-free or chemically defined media. Due to these unique advantages, CHO-K1 cells have become the preferred host cell for the production of recombinant proteins and other bioproducts in the biotechnology industry. The CHO-K1 cell culture medium, as a key environmental factor for cell growth and product expression, is crucial for improving cell culture efficiency, product quality and yield, and reducing contamination risks.

[0003] In a chemically defined culture medium, all components are known chemical substances whose concentrations and formulations can be precisely controlled. Suitable, personalized chemically defined culture media not only improve the stability and reproducibility of cell cultures but also facilitate further optimization of cell culture processes and research into cell metabolic mechanisms.

[0004] Currently, the price of CHO-K1 cell basal culture medium on the market is relatively expensive. It is difficult to find alternative products when the product is used on a large scale after it is launched, so the product's life cycle depends on the development of the culture medium company, which brings business risks. Some domestic culture media also add substances such as IGF, hormones, and hydrolysates, which not only makes the culture medium components contain unknown substances, but also brings certain risks to downstream purification. Patents CN118516299A and CN110894487 added hydrolysates, and patents CN115505561 and CN104073463 added hormones. Therefore, the invention of a low-cost, high-expression, stable, serum- and animal-derived protein-free basal culture medium is a real need in the current biopharmaceutical market. Summary of the Invention

[0005] To address the aforementioned limitations of existing technologies, this invention provides a chemically defined culture medium for CHO-K1 cells that supports high expression of the target product. This medium enables the culture of CHO-K1 cells and is free of serum and animal-derived proteins. This medium supports high-density cell growth and high expression of the target product, shortens the cell adaptation period to new culture media, prolongs the cell maintenance period, thereby increasing the accumulation of the target product and improving production efficiency.

[0006] This invention is achieved through the following technical solution:

[0007] A chemically defined basal culture medium for CHO-K1 cells, supporting host cell passage and high product expression, is composed of amino acids, vitamins, carbohydrates, and other additives. The amino acids are: L-arginine 0.32-0.6 g / L, L-histidine 0.15-0.35 g / L, L-isoleucine 0.25-0.68 g / L, L-leucine 0.26-0.55 g / L, L-lysine hydrochloride 0.28-0.7 g / L, L-methionine 0.05-0.25 g / L, L-phenylalanine 0.08-0.33 g / L, L-threonine 0.26-0.4 g / L, L-cysteine ​​hydrochloride 0.15-0.35 g / L, L... L-Tyrosine 0.18-0.68 g / L, L-Tryptophan 0.1-0.3 g / L, L-Valine 0.38-0.55 g / L, L-Alanine 0.04-0.25 g / L, L-Asparagine Monohydrate 0.32-0.55 g / L, L-Glutamic Acid 0.22-0.42 g / L, L-Glycine 0.035-0.06 g / L, L-Proline 0.3-0.55 g / L, L-Serine 0.7-1.0 g / L, L-Aspartic Acid 0.8-1.0 g / L.

[0008] The vitamins are: biotin 0.00005-0.0002 g / L, inositol 0.01-0.06 g / L, riboflavin 0.00005-0.002 g / L, choline chloride 0.02-0.08 g / L, pyridoxine 0.0008-0.0026 g / L, magnesium phosphate of vitamin C 0.016-0.032 g / L, folic acid 0.005-0.006 g / L, cobalamin 0.0009-0.002 g / L, thiamine 0.003-0.01 g / L, nicotinamide 0.001-0.15 g / L, and calcium pantothenate 0.007-0.01 g / L.

[0009] The inorganic salts are: zinc sulfate 0.0006-0.0009 g / L, zinc chloride 0.0001-0.0008 g / L, sodium selenite 0.00001-0.0001 g / L, sulfuric acid hexahydrate 0.0000001-0.000001 g / L, ammonium molybdate 0.000005-0.00005 g / L, sodium metavanadate 0.0000006-0.000006 g / L, calcium chloride 0.1-0.6 g / L, sodium bicarbonate 1.8-3.0 g / L, potassium chloride 0.7-1.5 g / L, and magnesium sulfate 0. 0.06-0.09 g / L, magnesium chloride 0.03-0.05 g / L, sodium dihydrogen phosphate 1.0-1.5 g / L, disodium hydrogen phosphate 1.0-1.5 g / L, sodium iodide 0.00000003-0.0000075 g / L, potassium dihydrogen phosphate 1.0-1.5 g / L, copper sulfate 0.000006-0.0009 g / L, manganese sulfate monohydrate 0.00000002-0.0000002 g / L, ferric citrate 0.02-0.04 g / L, ferrous sulfate heptahydrate 0.0004-0.001 g / L.

[0010] The carbohydrates and other additives are: sodium acetate 0-0.2 g / L, linoleic acid 0.00001-0.0001 g / L, ethanolamine 0.01-0.1 g / L, putrescine 0.0005-0.005 g / L, 4-hydroxyethylpiperazine ethanesulfonic acid 2.0-4.5 g / L, D-glucose 6.0-8.0 g / L, sodium glycerophosphate 0.03-0.1 g / L, lipoic acid 0-0.002 g / L, and dextran sulfate 0.2-0 g / L. 0.5 g / L, trehalose 0-0.2 g / L, p-aminobenzoic acid 0-0.006 g / L, pyrroloquinoline quinone 0-0.001 g / L, glutathione 0.0008-0.001 g / L, poloxamer 1881-2 g / L, N-acetyl-L-cysteine ​​0-0.08 g / L, α-ketoglutarate 0-0.15 g / L, malic acid 0-0.67 g / L, oxaloacetic acid 0-0.2 g / L, ribose 0-0.2 g / L.

[0011] Further, the carbohydrates and other additives described in this invention are: 0.001-0.002 g / L thioctic acid, 0.1-0.2 g / L trehalose, 0.003-0.006 g / L p-aminobenzoic acid, 0.0005-0.001 g / L pyrroloquinoline quinone, 0.04-0.08 g / L N-acetyl-L-cysteine, 0.075-0.15 g / L α-ketoglutarate, 0.34-0.67 g / L malic acid, 0.1-0.2 g / L oxaloacetic acid, and 0.1-0.2 g / L ribose.

[0012] This invention provides a preferred chemically defined basal culture medium for CHO-K1 cells, wherein the amino acids are: L-arginine 0.46 g / L, L-histidine 0.25 g / L, L-isoleucine 0.47 g / L, L-leucine 0.41 g / L, L-lysine hydrochloride 0.49 g / L, L-methionine 0.15 g / L, L-phenylalanine 0.21 g / L, L-threonine 0.33 g / L, L- Cysteine ​​hydrochloride 0.25 g / L, L-tyrosine 0.43 g / L, L-tryptophan 0.2 g / L, L-valine 0.47 g / L, L-alanine 0.15 g / L, L-asparagine monohydrate 0.44 g / L, L-glutamic acid 0.32 g / L, L-glycine 0.048 g / L, L-proline 0.43 g / L, L-serine 0.9 g / L, L-aspartic acid 0.9 g / L;

[0013] The vitamins are: biotin 0.00013 g / L, inositol 0.04 g / L, riboflavin 0.0013 g / L, choline chloride 0.05 g / L, pyridoxine 0.0017 g / L, magnesium vitamin C phosphate 0.026 g / L, folic acid 0.006 g / L, cobalamin 0.0015 g / L, thiamine 0.008 g / L, nicotinamide 0.008 g / L, and calcium pantothenate 0.009 g / L.

[0014] The inorganic salts are: zinc sulfate 0.0008 g / L, zinc chloride 0.0005 g / L, sodium selenite 0.00006 g / L, nickel sulfate hexahydrate 0.0000006 g / L, ammonium molybdate 0.000028 g / L, sodium metavanadate 0.000003 g / L, calcium chloride 0.4 g / L, sodium bicarbonate 2.4 g / L, potassium chloride 1.1 g / L, and magnesium sulfate 0. 0.08 g / L, magnesium chloride 0.04 g / L, sodium dihydrogen phosphate 1.25 g / L, disodium hydrogen phosphate 1.25 g / L, potassium dihydrogen phosphate 1.25 g / L, sodium iodide 0.0000038 g / L, copper sulfate 0.000008 g / L, manganese sulfate monohydrate 0.0000001 g / L, ferric citrate 0.03 g / L, sulfuric acid heptahydrate 0.0007 g / L;

[0015] The carbohydrates and other additives are: sodium acetate 0.1 g / L, linoleic acid 0.0005 g / L, ethanolamine 0.06 g / L, putrescine 0.0028 g / L, 4-hydroxyethylpiperazine ethanesulfonic acid 3.25 g / L, D-glucose 7 g / L, sodium glycerophosphate 0.07 g / L, lipoic acid 0.001 g / L, dextran sulfate 0.4 g / L, trehalose 0.1 g / L, p-aminobenzoic acid 0.003 g / L, pyrrolidone 0.0005 g / L, glutathione 0.0009 g / L, poloxamer 188 1.5 g / L, N-acetyl-L-cysteine ​​0.04 g / L, α-ketoglutarate 0.075 g / L, malic acid 0.34 g / L, oxaloacetic acid 0.1 g / L, and ribose 0.1 g / L.

[0016] This invention provides a method for preparing a chemically defined basal culture medium for CHO-K1 cells:

[0017] (1) Weigh all the ingredients;

[0018] (2) Dissolve in 0.8L of pure water and stir until dissolved;

[0019] (3) Adjust the pH value;

[0020] (4) Make up to 1L with pure water;

[0021] (5) Adjust osmotic pressure;

[0022] (6) Aseptic filtration, which is the finished culture medium.

[0023] Furthermore, the pH value is 7.0-7.4, and the osmotic pressure is 290-330 mOsm / kg.

[0024] The application of the CHO-K1 cell chemically defined basal medium provided by this invention in culturing suspended CHO cells.

[0025] The beneficial effects of this invention are as follows:

[0026] The chemically defined culture medium for CHO-K1 cells provided by this invention has a pH of 7.0-7.4 and an osmotic pressure of 290-330 mOsm / kg, which can support the rapid growth of CHO-K1 cells under suspension conditions. It has a clearly defined chemical composition, is low in cost, and is easy to prepare and store. This invention does not contain serum or any components of serum, nor does it contain animal-derived components, hydrolysates, or growth factors. Compared with other culture media, its composition is simpler, product purification is easier, and cell acclimatization is unnecessary; cells can be directly inoculated into this medium for culture. This invention, without adding protein components or unknown ingredients, increases cell culture density and viability by adding antioxidants and metabolic intermediates, and also enhances the expression level of the target protein, reducing process development pressure, improving production efficiency, and expanding the application range of the culture medium. Attached Figure Description

[0027] Figure 1 Cell diagram of CHO-K1-031 host cells in suspension growth.

[0028] Figure 2 The viability density of CHO-K1-031 host cells in Experiment Example 1.

[0029] Figure 3 Cell viability density of CHO-K1-031 host cells in different culture media in Experiment Example 2.

[0030] Figure 4 The viability density of the CHO-K1 antibody engineered cell line in Experiment Example 3 under different culture media.

[0031] Figure 5 Antibody expression levels in CHO-K1 antibody-engineered cells under different culture media in Experiment Example 3.

[0032] Figure 6 The viability density of the CHO-K1 recombinant protein engineered cell line in Experiment Example 4 under different culture media.

[0033] Figure 7 Protein expression levels of CHO-K1 recombinant protein engineered cells in different culture media in Experiment Example 4. Detailed Implementation

[0034] The present invention will be further described in conjunction with the embodiments.

[0035] All raw materials used in this invention are cell culture medium materials and are stored according to relevant requirements. Unless otherwise specified, the experimental procedures described below are conventional experimental methods. The features of this invention are described below in conjunction with the accompanying drawings. It should be understood that the following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0036] Examples 1-3: Preparation of CHO-K1 cell chemically defined culture medium

[0037] The concentrations of amino acids, vitamins, inorganic salts, carbohydrates and other additives in Examples 1-3 are shown in Table 1.

[0038] Preparation method: Weigh all the dry powder ingredients for 1L and add them to 800ml of ultrapure water. Stir at room temperature for 20min until completely dissolved. Adjust the pH value to 6.9-7.4 with hydrochloric acid, then bring the volume to 1L with ultrapure water. Measure the osmotic pressure and adjust it to 290-330mOsm / kg with sodium chloride. Filter through a 0.22μm sterile membrane into a sterile bottle and store at 4℃.

[0039] Table 1. Composition of CHO-K1 chemically defined culture medium in Examples 1-3

[0040] Substance Name Example 1 Example 2 Example 3 amino acids L-arginine (g / L) 0.32 0.46 0.6 L-histidine (g / L) 0.15 0.25 0.35 L-Isoleucine (g / L) 0.25 0.47 0.68 L-Leucine (g / L) 0.26 0.41 0.55 L-Lysine hydrochloride (g / L) 0.28 0.49 0.7 L-methionine (g / L) 0.05 0.15 0.25 L-Phenylalanine (g / L) 0.08 0.21 0.33 L-Threonine (g / L) 0.26 0.33 0.4 L-cysteine ​​hydrochloride (g / L) 0.15 0.25 0.35 L-Tyrosine (g / L) 0.18 0.43 0.68 L-Tryptophan (g / L) 0.1 0.2 0.3 L-valine (g / L) 0.38 0.47 0.55 L-alanine (g / L) 0.04 0.15 0.25 L-Asparagine monohydrate (g / L) 0.32 0.44 0.55 L-Glutamic Acid (g / L) 0.22 0.32 0.42 L-glycine (g / L) 0.035 0.048 0.06 L-proline (g / L) 0.3 0.43 0.55 L-Serine (g / L) 0.7 0.9 1.0 L-Aspartic acid (g / L) 0.8 0.9 1.0 Vitamins (g / L) Biotin (g / L) 0.00005 0.00013 0.0002 Inositol (g / L) 0.01 0.04 0.06 Riboflavin (g / L) 0.0005 0.0013 0.002 Choline chloride (g / L) 0.02 0.05 0.08 Pyridoxine (g / L) 0.0008 0.0017 0.0026 Vitamin C magnesium phosphate (g / L) 0.016 0.026 0.035 Folic acid (g / L) 0.005 0.006 0.006 Cobalamin (g / L) 0.0009 0.0015 0.002 Thiamine (g / L) 0.003 0.008 0.01 Nicotinamide (g / L) 0.001 0.008 0.015 Calcium pantothenate (g / L) 0.007 0.009 0.01 Inorganic salts Zinc sulfate (g / L) 0.0006 0.0008 0.0009 Zinc chloride (g / L) 0.0001 0.0005 0.0008 Sodium selenite (g / L) 0.00001 0.00006 0.0001 Nickel sulfate hexahydrate (g / L) 0.0000001 0.0000006 0.000001 Ammonium molybdate (g / L) 0.000005 0.000028 0.00005 Sodium metavanadate (g / L) 0.0000006 0.000003 0.000006 Calcium chloride (g / L) 0.1 0.4 0.6 Sodium bicarbonate (g / L) 1.8 2.4 3.0 Potassium chloride (g / L) 0.7 1.1 1.5 Magnesium sulfate (g / L) 0.06 0.08 0.09 Magnesium chloride (g / L) 0.03 0.04 0.05 Sodium dihydrogen phosphate (g / L) 1.0 1.25 1.5 Disodium hydrogen phosphate (g / L) 1.0 1.25 1.5 Potassium dihydrogen phosphate (g / L) 1.0 1.25 1.5 Sodium iodide (g / L) 0.00000003 0.0000038 0.0000075 Copper sulfate (g / L) 0.000006 0.000008 0.000009 Manganese sulfate monohydrate (g / L) 0.00000002 0.0000001 0.0000002 Ferric citrate (g / L) 0.02 0.03 0.04 Ferrous sulfate heptahydrate (g / L) 0.0004 0.0007 0.001 Carbohydrates and others Sodium acetate (g / L) 0 0.1 0.2 Linoleic acid (g / L) 0.00001 0.0005 0.001 Ethanolamine (g / L) 0.01 0.06 0.1 Putrescine (g / L) 0.0005 0.0028 0.005 4-Hydroxyethylpiperazine ethanesulfonate (g / L) 2.0 3.25 4.5 D-glucose (g / L) 6 7 8 Sodium glycerophosphate (g / L) 0.03 0.07 0.1 Alpha-lipoic acid (g / L) 0 0.001 0.002 Dextran sulfate (g / L) 0.2 0.4 0.5 Trehalose (g / L) 0 0.1 0.2 p-Aminobenzoic acid (g / L) 0 0.003 0.006 Pyrroloquinoline quinone (g / L) 0 0.0005 0.001 Glutathione (g / L) 0.0008 0.0009 0.001 Poloxamer 188 (g / L) 1.0 1.5 2.0 N-acetyl-L-cysteine ​​(g / L) 0 0.04 0.08 α-Ketoglutarate (g / L) 0 0.075 0.15 Malic acid (g / L) 0 0.34 0.67 Oxaloacetic acid (g / L) 0 0.1 0.2 Ribose (g / L) 0 0.1 0.2

[0041] Experimental Example 1:

[0042] Prepare the culture medium as described in Example 1. CHO-K1-031 host cells were seeded at a density of 5E5 cells / mL into 30ml Erlenmeyer flasks containing 30ml of medium. The flasks were then placed in a shaker at 37±2℃, 5%±1% CO2 concentration, 110-120rpm, and 75%±5% humidity. When the cell density reached 2.0E6-4.0E6 cells / mL (usually in 2-4 days), the above procedure was repeated for cell passage. After more than 10 passages, the cells were observed under a microscope to have clear edges, plump morphology, no clumping, and good adaptation. Figure 1 As shown.

[0043] During the passage culture process, cell samples were taken before each passage, and cell viability was calculated and cell density was monitored using trypan blue staining. All results showed normal host cell growth rate, viability above 95%, and pre-passage cell density exceeding 2.0 E6 cells / mL. Figure 2 .

[0044] Experiment Example 2

[0045] The culture medium described in Example 1 was prepared, and CHO-K1-031 host cells were seeded at a density of 5E5 cells / mL into 125mL Erlenmeyer flasks containing 30mL of medium. The flasks were then cultured in a shaker at 37±2℃, 5%±1% CO2 concentration, 110-120rpm, and 75%±5% humidity. Cell viability was calculated and cell density was monitored by sampling and counting cells every 48-72 hours using trypan blue staining. Culture was terminated when cell viability fell below 80%, constituting a batch culture experiment. Compared with a batch culture experiment conducted simultaneously using the commercial culture medium (CD CHO031, purchased from Gansu Jianshun Biotechnology Co., Ltd., catalog number 88031-585), the self-made medium showed good adaptation, with a viability exceeding 99% at day 3, higher than the commercially available medium previously screened for these cells. The maximum density was significantly increased, nearly 8% higher than the commercial control medium. At day 10 (the end of culture), both viability and density remained higher than the commercial medium control group. Figure 3 .

[0046] Experimental Example 3

[0047] An engineered cell line constructed using CHO-K1 cells as the host was cultured in a self-made culture medium. This cell line was used to express a monoclonal antibody. A commercially available culture medium, pre-screened for this cell line, was used as a control for comparison. The commercially available culture medium was formulated as a 1:1 mixture of CHO MaxA1 and CHO MaxC (purchased from Maibang Pharmaceuticals, catalog numbers MB1105.102 and 1103.102, respectively).

[0048] Prepare the culture media used in Examples 1, 2, and 3, and seed them at a density of 5E5 cells / mL into 125 mL Erlenmeyer flasks containing 30 mL of culture medium. Place the Erlenmeyer flasks in a shaker at a temperature of 37±2℃, a carbon dioxide concentration of 5%±1%, a rotation speed of 110-120 rpm, and a humidity of 75%±5%. Every 48-72 hours, take samples, count cells, and calculate cell viability using trypan blue staining. Monitor cell density and detect antibody expression levels using a biochemical analyzer. End the culture when the cell viability is below 80%, which constitutes a batch culture experiment.

[0049] Comparing the cell culture results with those of commercially available culture media and Examples 1, 2, and 3, it was found that throughout the entire culture period, the cell density in all experimental groups using the self-made culture media was higher than that in the control group using the commercial culture media. Specifically, the cell density in the culture media experimental group of Example 2 was nearly 30% higher than that of the control group. At the end of the culture, antibody expression was significantly increased by more than 60%. Figure 4 , Figure 5 .

[0050] Experiment Example 4

[0051] An engineered cell line constructed using CHO-K1 cells as the host was cultured in a self-made culture medium. This cell line was used to express a recombinant protein, and a commercially available culture medium for this cell line, which had been pre-screened, was used as a control for comparison. The commercially available culture medium was CD optiCHO (purchased from Gibco, catalog number 12681-011).

[0052] Prepare the culture media as described in Examples 1, 2, and 3. Seed cells at a density of 5E5 cells / mL into 125mL Erlenmeyer flasks containing 30mL of culture medium. Place the flasks in a shaker at a temperature of 37±2℃, a carbon dioxide concentration of 5%±1%, a rotation speed of 110-120rpm, and a humidity of 75%±5%. Every 48-72 hours, take samples, count cells, and calculate cell viability using trypan blue staining. Monitor cell density and detect protein content using a specific protein detection kit. End culture when cell viability is below 80%, which constitutes a batch culture experiment.

[0053] Comparing the batch culture experiments conducted simultaneously with commercially available culture media, it was found that after cell inoculation, the growth rate in the self-made culture medium was significantly higher than that in the control group, and the viability was also higher throughout the entire culture cycle. In particular, the data from D5 and D7 showed that the self-made culture medium in Example 2 significantly increased the peak cell density by approximately 45%, and maintained a viability of over 98% at D7, thus extending the time for sample accumulation. The expression level of recombinant proteins was also significantly increased, with yields more than double that of the control group. Figure 6 , Figure 7 .

Claims

1. A chemically defined basal culture medium for CHO-K1 cells, composed of amino acids, vitamins, carbohydrates, and other additives, characterized in that, The amino acids are: L-arginine 0.32-0.6 g / L, L-histidine 0.15-0.35 g / L, L-isoleucine 0.25-0.68 g / L, L-leucine 0.26-0.55 g / L, L-lysine hydrochloride 0.28-0.7 g / L, L-methionine 0.05-0.25 g / L, L-phenylalanine 0.08-0.33 g / L, L-threonine 0.26-0.4 g / L, L-cysteine ​​hydrochloride 0.15-0.35 g / L, L... L-Tyrosine 0.18-0.68 g / L, L-Tryptophan 0.1-0.3 g / L, L-Valine 0.38-0.55 g / L, L-Alanine 0.04-0.25 g / L, L-Asparagine Monohydrate 0.32-0.55 g / L, L-Glutamic Acid 0.22-0.42 g / L, L-Glycine 0.035-0.06 g / L, L-Proline 0.3-0.55 g / L, L-Serine 0.7-1.0 g / L, L-Aspartic Acid 0.8-1.0 g / L; The vitamins are: biotin 0.00005-0.0002 g / L, inositol 0.01-0.06 g / L, riboflavin 0.00005-0.002 g / L, choline chloride 0.02-0.08 g / L, pyridoxine 0.0008-0.0026 g / L, magnesium phosphate of vitamin C 0.016-0.032 g / L, folic acid 0.005-0.006 g / L, cobalamin 0.0009-0.002 g / L, thiamine 0.003-0.01 g / L, nicotinamide 0.001-0.15 g / L, and calcium pantothenate 0.007-0.01 g / L. The inorganic salts are: zinc sulfate 0.0006-0.0009 g / L, zinc chloride 0.0001-0.0008 g / L, sodium selenite 0.00001-0.0001 g / L, sulfuric acid hexahydrate 0.0000001-0.000001 g / L, ammonium molybdate 0.000005-0.00005 g / L, sodium metavanadate 0.0000006-0.000006 g / L, calcium chloride 0.1-0.6 g / L, sodium bicarbonate 1.8-3.0 g / L, potassium chloride 0.7-1.5 g / L, magnesium sulfate 0.06-0.09 g / L, magnesium chloride 0.03-0.05 g / L, and sodium dihydrogen phosphate 1.0-1.5 g / L. Disodium hydrogen phosphate 1.0-1.5 g / L, sodium iodide 0.00000003-0.0000075 g / L, potassium dihydrogen phosphate 1.0-1.5 g / L, copper sulfate 0.000006-0.0009 g / L, manganese sulfate monohydrate 0.00000002-0.0000002 g / L, ferric citrate 0.02-0.04 g / L, ferrous sulfate heptahydrate 0.0004-0.001 g / L; The carbohydrates and other additives are: sodium acetate 0-0.2 g / L, linoleic acid 0.00001-0.0001 g / L, ethanolamine 0.01-0.1 g / L, putrescine 0.0005-0.005 g / L, 4-hydroxyethylpiperazine ethanesulfonic acid 2.0-4.5 g / L, D-glucose 6.0-8.0 g / L, sodium glycerophosphate 0.03-0.1 g / L, lipoic acid 0-0.002 g / L, and dextran sulfate 0.2-0 g / L. 0.5 g / L, trehalose 0-0.2 g / L, p-aminobenzoic acid 0-0.006 g / L, pyrroloquinoline quinone 0-0.001 g / L, glutathione 0.0008-0.001 g / L, poloxamer 1881-2 g / L, N-acetyl-L-cysteine ​​0-0.08 g / L, α-ketoglutarate 0-0.15 g / L, malic acid 0-0.67 g / L, oxaloacetic acid 0-0.2 g / L, ribose 0-0.2 g / L.

2. The CHO-K1 cell culture medium according to claim 1, characterized in that, The carbohydrates and other additives are 0.001-0.002 g / L lipoic acid, 0.1-0.2 g / L trehalose, 0.003-0.006 g / L p-aminobenzoic acid, 0.0005-0.001 g / L pyrroloquinoline quinone, 0.04-0.08 g / L N-acetyl-L-cysteine, 0.075-0.15 g / L α-ketoglutarate, 0.34-0.67 g / L malic acid, 0.1-0.2 g / L oxaloacetic acid, and 0.1-0.2 g / L ribose.

3. The CHO-K1 cell culture medium according to claim 2, characterized in that, The amino acids are: L-arginine 0.46 g / L, L-histidine 0.25 g / L, L-isoleucine 0.47 g / L, L-leucine 0.41 g / L, L-lysine hydrochloride 0.49 g / L, L-methionine 0.15 g / L, L-phenylalanine 0.21 g / L, L-threonine 0.33 g / L, L-cysteine ​​hydrochloride 0.25 g / L, L-tyrosine 0.43 g / L, L-tryptophan 0.2 g / L, L-valine 0.47 g / L, L-alanine 0.15 g / L, L-asparagine monohydrate 0.44 g / L, L-glutamic acid 0.32 g / L, L-glycine 0.048 g / L, L-proline 0.43 g / L, L-serine 0.9 g / L, and L-aspartic acid 0.9 g / L. The vitamins are: biotin 0.00013 g / L, inositol 0.04 g / L, riboflavin 0.0013 g / L, choline chloride 0.05 g / L, pyridoxine 0.0017 g / L, magnesium vitamin C phosphate 0.026 g / L, folic acid 0.006 g / L, cobalamin 0.0015 g / L, thiamine 0.008 g / L, nicotinamide 0.008 g / L, and calcium pantothenate 0.009 g / L. The inorganic salts are: zinc sulfate 0.0008 g / L, zinc chloride 0.0005 g / L, sodium selenite 0.00006 g / L, nickel sulfate hexahydrate 0.0000006 g / L, ammonium molybdate 0.000028 g / L, sodium metavanadate 0.000003 g / L, calcium chloride 0.4 g / L, sodium bicarbonate 2.4 g / L, potassium chloride 1.1 g / L, and magnesium sulfate 0. 0.08 g / L, magnesium chloride 0.04 g / L, sodium dihydrogen phosphate 1.25 g / L, disodium hydrogen phosphate 1.25 g / L, potassium dihydrogen phosphate 1.25 g / L, sodium iodide 0.0000038 g / L, copper sulfate 0.000008 g / L, manganese sulfate monohydrate 0.0000001 g / L, ferric citrate 0.03 g / L, sulfuric acid heptahydrate 0.0007 g / L; The carbohydrates and other additives are: sodium acetate 0.1 g / L, linoleic acid 0.0005 g / L, ethanolamine 0.06 g / L, putrescine 0.0028 g / L, 4-hydroxyethylpiperazine ethanesulfonic acid 3.25 g / L, D-glucose 7 g / L, sodium glycerophosphate 0.07 g / L, lipoic acid 0.001 g / L, dextran sulfate 0.4 g / L, trehalose 0.1 g / L, p-aminobenzoic acid 0.003 g / L, pyrrolidone 0.0005 g / L, glutathione 0.0009 g / L, poloxamer 188 1.5 g / L, N-acetyl-L-cysteine ​​0.04 g / L, α-ketoglutarate 0.075 g / L, malic acid 0.34 g / L, oxaloacetic acid 0.1 g / L, and ribose 0.1 g / L.

4. The method for preparing the CHO-K1 cell chemically defined basal culture medium according to any one of claims 1-3, characterized in that: (1) Weigh all the ingredients; (2) Dissolve in 0.8L of pure water and stir until dissolved; (3) Adjust the pH value; (4) Make up to 1L with pure water; (5) Adjust osmotic pressure; (6) Aseptic filtration, which is the finished culture medium.

5. The method for preparing the CHO-K1 cell chemically defined basal culture medium according to claim 4, characterized in that: The pH value is 7.0-7.4, and the osmotic pressure is 290-330 mOsm / kg.

6. The use of the CHO-K1 cytochemically defined basal medium according to any one of claims 1-3 in culturing suspension CHO cells.

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