CN113088480A

CN113088480A - Culture medium for CHO cells and application thereof

Info

Publication number: CN113088480A
Application number: CN201911338826.8A
Authority: CN
Inventors: 秦婷; 钱宇辰
Original assignee: Innovent Biologics Suzhou Co Ltd
Current assignee: Sherpa Biotechnology Suzhou Co ltd
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2021-07-09
Anticipated expiration: 2039-12-23
Also published as: CN113088480B

Abstract

The invention provides a culture medium for CHO cells and application thereof. The culture medium is characterized by comprising a basal culture medium and a fed-batch culture medium with a final concentration of 120-fold milk-meal blood of 150g/L, wherein the culture medium is a culture medium which is clear in chemical components and does not contain serum, ornithine, hydroxyproline, p-aminobenzoic acid, thymine, thymidine, uracil, guanine and cytosine. The culture medium for the CHO cells can support long-term subculture growth of different subtype CHO cells; the growth of high cell density, the maintenance of later cell survival rate and the high expression of target products can be maintained in the batch culture of cells; the method is beneficial to the control of cell metabolism and product quality, and is convenient for the optimization of the culture process; the culture medium has low cost and convenient preparation, and is suitable for large-scale production of recombinant protein biological products.

Description

Culture medium for CHO cells and application thereof

Technical Field

The invention relates to the technical field of cell culture, in particular to a culture medium for CHO cells and application thereof.

Background

The animal cell culture technology is a key technology for industrially producing recombinant protein products at present, and with the expansion of the culture scale of mammalian cells and the increase of the requirements of biological medicines, the development of a serum-free culture medium based on the characteristics of cells and products becomes an important subject in the field of cell engineering. In the application fields of vaccine production, monoclonal antibodies, various bioactive proteins and other biological products, the components of a serum-free culture medium are optimized to ensure that engineering cells maintain higher viable cell density, higher cell viability and longer culture time, so that the method is an effective method for reducing the production cost.

Chinese hamster ovary cells (CHO cells) are currently the most widespread cell type in the large-scale production of recombinant proteins, and 70% of the biopharmaceuticals that have been marketed worldwide are CHO cell expression systems. The CHO cell has the advantages of high-efficiency gene expression, accurate protein posttranscriptional modification, flexible pathogen detection and the like, is easy for suspension culture, rarely secretes self protein, and is convenient for later-stage protein separation and purification. Compared with other cell types, CHO cells have the following advantages: the product is immortal and can be passaged for more than one hundred generations; belongs to fibroblast (fibroblast), is a non-secretory cell, rarely secretes CHO endogenous protein, and is very favorable for the separation and purification of target protein; in addition, the cell can also form active dimers, has glycosylation function and is an ideal host for expressing complex biological macromolecules.

The culture medium is the most direct and important environmental factor affecting the growth, metabolism and even survival of the cells. Traditional CHO cells can normally grow only by adding 5-10% of serum into a basic culture medium, but the components in the serum are complex and are not beneficial to downstream separation and purification, the risk of exogenous pollution is increased, the serum cost is high, and batch difference is inevitable. Therefore, the development of serum-free, chemically defined media is a hotspot in current CHO cell culture.

Compared with the traditional culture medium with serum, the serum-free culture medium has the following advantages: 1) no batch difference and no influence of unknown serum components on cells; 2) avoids exogenous pollution in serum and toxic action on cells; 3) the downstream separation and purification are convenient, and the recovery rate is improved; 4) the components are clear, the physiological state of cells can be researched, and culture media with different functional types can be designed and optimized according to different cell strains.

The culture medium disclosed in the prior art, which is suitable for CHO cells and has definite chemical components, has the following problems: when the cell culture medium is used for cell culture, the specific growth rate of cells in the logarithmic growth phase is slow, the highest viable cell density is low, the cell viability is poor, and the concentration of finally expressed products is low. For example, patent publication No. CN104328158B discloses a chemically defined medium suitable for mass production of animal cell-expressed products, which is chemically defined and free of any animal origin, but in each example of the invention, the specific growth rate in the logarithmic growth phase of cells in the batch culture process is only 0.41 to 0.55 days^-1The highest viable cell density is only 10.71 multiplied by 10⁶The integral value of the cells/ml and the living cells to the time is only 56.6 multiplied by 10 at most⁹Cell day/l, the final expressed product concentration is low (only 1618 mg/l), and the culture medium is difficult to meet the requirements of controlling the cost and improving the self-competitiveness in the market.

With the rapid development of recombinant protein preparation technology, the culture medium has low cost, simple preparation, convenient use, stable batch and high yield, and is increasingly the mainstream of industry selection. More and more enterprises tend to research and develop self-contained culture media, but the research aiming at different subtypes of CHO cells is few, and the independent development of a broad-spectrum culture medium is more difficult.

Disclosure of Invention

Problems to be solved by the invention

Aiming at the problems in the prior art, the application provides a culture medium which is used for culturing different subtype CHO cells and has definite chemical components and application thereof.

Means for solving the problems

In view of the problems in the prior art, the present inventors have conducted intensive studies and repeated experiments, and have completed the present invention by screening a basic medium, screening a fed-batch medium, optimizing the content of amino acids in the medium, optimizing the content of tyrosine additionally added to the fed-batch medium, and screening and optimizing additives and trace elements based on an excellent medium development platform, and performing effect verification on CHO host cell platforms of different subtypes, such as CHO-S, CHO-K1, CHO-GS, and CHO-DG44, to finally screen a medium with definite chemical components suitable for the high-density growth of CHO cells, stably maintaining the survival rate of CHO cells, and highly expressing a target product. Namely, the present invention is as follows:

the first aspect of the present invention provides a culture medium for CHO cells, characterized in that the culture medium is a chemically defined and serum-, ornithine-, hydroxyproline-, p-aminobenzoic acid-, thymine-, thymidine-, uracil-, guanine-and cytosine-free medium, comprising a basal medium and a fed-batch medium at a final concentration of 120-150g/L,

the basic culture medium contains amino acid, vitamins, inorganic salt, trace elements and other components; wherein the amino acids include: alanine, arginine, asparagine, monohydrate, aspartic acid, cysteine, hydrochloride, monohydrate, cystine, dihydrochloride, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; wherein the vitamins include: biotin, calcium pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxine.hydrochloride, riboflavin, thiamine.hydrochloride, and vitamin B12; wherein the inorganic salt comprises: sodium dihydrogen phosphate monohydrate, magnesium sulfate, calcium chloride, potassium chloride, zinc sulfate heptahydrate, copper sulfate pentahydrate, ferric ammonium citrate, sodium selenite and sodium bicarbonate; wherein the other components comprise: one or more of dextran sulfate, putrescine, dihydrochloride, ethanolamine, and segmented polyether F68; the trace elements in the basic culture medium comprise: manganese sulfate monohydrate, sodium silicate nonahydrate, ammonium molybdate tetrahydrate, ammonium metavanadate, nickel sulfate hexahydrate, aluminum chloride hexahydrate, barium acetate, cobalt chloride hexahydrate, chromium chloride, sodium fluoride, germanium oxide, rubidium chloride and zirconium oxychloride octahydrate;

the fed-batch culture medium contains amino acids, vitamins, inorganic salts, trace elements and other components; wherein the amino acids include: arginine, asparagine, monohydrate, aspartic acid, cysteine, hydrochloride, monohydrate, cystine, dihydrochloride, glutamic acid, histidine, isoleucine, leucine, lysine, hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; wherein the vitamins include: calcium pantothenate, choline chloride, folic acid, inositol, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and vitamin B12; wherein the inorganic salt comprises: sodium pyruvate, sodium dihydrogen phosphate monohydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate, ferric ammonium citrate and sodium selenite; wherein the other ingredients comprise: one or more of the group consisting of putrescine, dihydrochloride, ethanolamine, segmented polyether F68, glucose and hydroxyethylpiperazine ethanethiosulfonic acid (HEPES); wherein the trace elements include: manganese sulfate monohydrate, sodium silicate nonahydrate, ammonium molybdate tetrahydrate, ammonium metavanadate, nickel sulfate hexahydrate, stannous chloride dihydrate, aluminum chloride hexahydrate, silver nitrate, barium acetate, potassium bromide, 5/2 cadmium chloride hydrate, cobalt chloride hexahydrate, chromium chloride, sodium fluoride, germanium oxide, potassium iodide, rubidium chloride and zirconium oxychloride octahydrate.

Further, other components in the basic culture medium also comprise glucose; the trace elements in the basic culture medium further comprise: one or more of the group consisting of stannous chloride dihydrate, silver nitrate, potassium bromide, 5/2 cadmium chloride hydrate and potassium iodide.

Further, the contents of sodium bicarbonate, glucose and dextran sulfate contained in the basic culture medium are respectively 1000-3000mg/L, 0-8000mg/L and 15-40 mg/L; preferably, the contents of the sodium bicarbonate, the glucose and the dextran sulfate are respectively 1500-2500mg/L, 2000-7000mg/L and 20-35 mg/L; more preferably, the contents of the sodium bicarbonate, the glucose and the dextran sulfate are 2000mg/L, 6000mg/L and 25mg/L respectively; the content of the asparagine-linked monohydrate contained in the fed-batch culture medium is 500-8000 mg/L; preferably, the content of the asparagine-monohydrate is 1000-5500 mg/L; more preferably, the content of asparagine-monohydrate is 4363 mg/L.

Furthermore, the contents of the 18 trace elements contained in the basal medium are respectively shown in the content ranges of column 2 in table 1; more preferably, as shown in the content range of column 3 in table 1; most preferably, as indicated in table 1 by the content ranges in column 4. The contents of the 18 trace elements contained in the fed-batch culture medium are respectively shown in the content range of the column 2 in the table 2; more preferably, as shown in the content range of column 3 in table 2; most preferably, as indicated in table 2 by the content ranges in column 4.

Further, the content of each of the amino acids, vitamins, inorganic salts and other components contained in the basal medium is shown in the content range of column 2 in table 1; more preferably, as shown in the content range of column 3 in table 1; most preferably, as indicated in table 1 by the content ranges in column 4. The contents of amino acids, vitamins, inorganic salts and other components contained in the fed-batch culture medium are respectively shown in the content range of column 2 in table 2; more preferably, as shown in the content range of column 3 in table 2; most preferably, as indicated in table 2 by the content ranges in column 4.

Further, the sum of the concentrations of all the components in the fed-batch medium (i.e., the final concentration) was 140 g/L.

In particular, the present invention provides media comprising carbon sources, nitrogen sources, amino acids, vitamins, inorganic salts, lipids, buffers, trace elements and other nutrients to promote CHO cell growth, maintenance and product expression. The carbon source is used as the most important energy source substance, provides required energy for biosynthesis and a framework for product synthesis, exerts different effects in different carbon source forms, and is commonly used for glucose, galactose, mannose, fucose, glutamine, sodium pyruvate and the like. The nitrogen source is a major organization part of the synthesis of proteins in living organisms, and commonly used inorganic nitrogen sources include various ammonium salts, nitrates, and the like. Amino acid is the most key component in the culture medium with definite chemical components, and the amino acid with different component contents directly influences the growth and maintenance of cells and the expression of protein products, even influences the quality attributes of the products; the commonly used amino acids are divided into essential amino acids and non-essential amino acids, and the amino acids are required in different CHO cell types in different amounts. The vitamins provide a large amount of coenzyme factors for organisms and play an important role in the metabolic process; most vitamins are sensitive to strong light and heat and are easily oxidized. Lipid substances are important components of cell membranes, provide energy in cells, serve signal pathways, and are commonly used as fatty acids, phospholipids (e.g., ethanolamine), cholesterol, and the like. The trace elements have low content in the culture medium, but are favorable for maintaining the activity of in vivo enzymes, and the trace elements are combined with signal molecule groups to participate in metabolism, so that the stability of the culture medium can be greatly improved by the effective collocation of different elements. The inorganic salt and the buffer can regulate the permeability of cell membrane, maintain normal osmotic pressure and acid-base balance inside and outside the cell and promote the growth of the cell. Other nutrients, including some energy substances, hormones and serum substitutes, play an important role in maintaining normal cell growth and expression processes.

The culture medium which is suitable for CHO cells and has definite chemical components mainly comprises a Basal medium (Basal Media) and a fed-batch medium (Feed Media), and the components of the two Media comprise: carbon sources, nitrogen sources, amino acids, vitamins, inorganic salts, lipids, buffers, trace elements and other nutrients. The culture medium provided by the invention is suitable for a classic 'Fed-Batch' Batch culture process, the basic culture medium mainly supports the growth of prophase cells, and meanwhile, the culture is carried out by matching with a Fed-Batch culture medium; the fed-batch culture medium is designed based on the consumption rate of nutrient substances in a basic culture medium, is a highly concentrated culture medium of key nutrient substances, adopts modes of sectional fed-batch or continuous fed-batch and the like in the batch culture process, supplements the key nutrient substances in time, and ensures the maintenance of cell growth and the expression of target protein in the stable period of batch culture.

The components of the basic culture medium contained in the culture medium provided by the invention are detailed in the following table:

table 1: the basic culture medium contains the components and the content thereof

The basic culture medium provided by the invention contains 15-40mg/L of dextran sulfate and is used for preventing cells from agglomerating; 3000mg/L sodium bicarbonate 1000-; contains 0-8000mg/L glucose, and each trace element in the culture medium plays a critical role.

The components of the fed-batch culture medium contained in the culture medium provided by the invention are detailed in the following table:

table 2: the components and contents thereof contained in the fed-batch culture medium

The fed-batch culture medium provided by the invention is a high-concentration culture medium, the final concentration is within the range of 120-150g/L, and is preferably about 140g/L, so that the requirement of nutrient substances in the batch culture process is ensured, the volume of the fed-batch culture medium in the batch culture process is reduced, and the yield is greatly improved. Wherein "final concentration" means the sum of the concentrations of all ingredients in the fed-batch medium.

A second aspect of the invention provides a method of culturing recombinant CHO cells, the method comprising the steps of:

(1) resuscitating and shake-flask amplification culture are carried out on the recombinant CHO cells;

(2) according to the initial inoculation density (1.0 +/-0.3) multiplied by 10⁶Inoculating the recombinant CHO cells in the step (1) into the basic culture medium of any one of claims 1-6 at a DO of 20-80% and a pH of 6.80-7.20 per ml;

(3) culturing for 3, 5, 7, 9 and 11 days, and respectively feeding 4-5% (w/w) of the initial culture weight of the feeding culture medium as described in any one of claims 1-5.

Further, the specific operations of resuscitation and shake flask amplification culture in the step (1) are as follows: taking out the seed cells from liquid nitrogen or a refrigerator at minus 80 ℃ for resuscitation, inoculating the seed cells into a shake flask containing any one of the basal culture media provided by the first aspect of the invention, controlling the culture temperature to be 36.0-37.0 ℃ and 5-7% CO₂The shaking table speed is set to 120-140 r/min, the cultivation is carried out for 2-4 days according to (0.3-0.7) x 10⁶Carrying out shake flask gradual amplification passage at density of each/ml, wherein each stage of amplification culture lasts for 2-4 days, and when the cell density reaches (1.0-7.0) multiplied by 10⁶Inoculating to the next stage when the seed/ml is required; preferably, the cell viability rate is more than or equal to 85% in the seed recovery stage and more than or equal to 90% in the shake flask amplification stage;

in the step (2), the culture temperature is controlled to be 36.0-37.0 ℃ on the 1 st-4 th days of culture, and the culture temperature is reduced to be 32.0-34.0 ℃ from the 5 th day of culture;

in the step (3), the tyrosine concentrated solution with the final concentration of 0.8-1.6 g/L is additionally added while the fed-batch culture medium is fed.

Further, the recombinant CHO cell is a CHO cell comprising a gene encoding a foreign protein; preferably, the CHO cell is CHO-S, CHO-K1, CHO-GS or CHO-DG44, and the foreign protein is an antibody; more preferably, the foreign protein is a monoclonal antibody or a bispecific antibody.

Specifically, the basic culture medium and the Fed-Batch culture medium provided by the invention adopt a Fed-Batch mode to carry out Batch culture on recombinant CHO cells containing the encoded foreign protein. The specific method for batch culture comprises the following steps: reviving and flask expanding the seed cells according to the initial inoculation densityDegree (1.0. + -. 0.3). times.10⁶The recombinant CHO cells containing the encoding foreign protein are inoculated into a basic culture medium per ml, the DO is controlled to be 20-80%, the pH is controlled to be 6.80-7.20, the early culture temperature is 36.0-37.0 ℃, and the temperature is reduced to 32.0-34.0 ℃ after the 5 th day of culture. Culturing to 3, 5, 7, 9 and 11 days, respectively feeding 4-5% (w/w) of feeding culture medium with initial culture weight and tyrosine concentrated solution with final concentration of 0.8-1.6 g/L (wherein the feeding culture medium and the tyrosine concentrated solution are fed at the same feeding time, but the tyrosine concentrated solution and the feeding culture medium are separately stored before feeding and are not mixed together, otherwise, nutrient components are separated out), detecting the concentration of glucose in the culture solution every day, and when the concentration of glucose is lower than 3.0g/L, adding the glucose concentrated solution to increase the concentration of glucose in the cell fluid to 5.0 g/L. The total amount of Antifoam (including but not limited to Antifoam (Hyclone, SH30897.01)) cannot exceed 100ppm, depending on the actual amount of foam manually added. Sampling and detecting the pH, viable cell density, cell viability and other biochemical indexes (such as glucose content, lactic acid content and ammonia content in the culture solution) of the cell sap every day in the culture process, and culturing till 12-16 days.

The specific operation of "resuscitating and shake flask expanding seed cells" described above is: taking out seed cells from a liquid nitrogen or a refrigerator at minus 80 ℃, recovering the seed cells, inoculating the recovered seed cells into 125ml of shake flasks containing a basic culture medium, supplementing the basic culture medium to 20-30 ml, controlling the culture temperature to be 36.0-37.0 ℃ and controlling the culture temperature to be 5-7% CO₂Under the condition (1), the shaking table speed is set to be 120-140 r/min, the cultivation is carried out for 2-4 days according to (0.3-0.7) multiplied by 10⁶Carrying out shake flask gradual amplification passage at density of each/ml, wherein each stage of amplification culture lasts for 2-4 days, and when the cell density reaches (1.0-7.0) multiplied by 10⁶When the number of seeds per ml is larger than or equal to 85%, the cell viability in the seed recovery stage is larger than or equal to 90%.

A third aspect of the present invention provides a use of the medium for CHO cells according to any one of the aspects provided in the first aspect of the present invention for expressing a foreign protein in CHO cells; preferably, the CHO cell is CHO-S, CHO-K1, CHO-GS or CHO-DG44, and the foreign protein is an antibody; more preferably, the foreign protein is a monoclonal antibody or a bispecific antibody.

ADVANTAGEOUS EFFECTS OF INVENTION

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1) the culture medium provided by the invention comprises a basic culture medium and a fed-batch culture medium, has definite chemical components, no animal source and no serum component, and is qualified in bacteria, heat source, pH and titer detection. The culture medium provided by the invention is beneficial to the production and passage maintenance of CHO cells, and is beneficial to the high-efficiency expression, separation and purification of protein products, thereby saving the cost of large-scale recombinant protein preparation.

2) The culture medium provided by the invention supports long-term subculture and in-vitro suspension culture of CHO cells of different subtypes (including CHO-S, CHO-K1, CHO-GS and CHO-DG 44). The culture medium is suitable for Fed-Batch culture, can be widely applied to various platforms of CHO cells (including CHO-S, CHO-K1, CHO-GS and CHO-DG44 platforms), is beneficial to growth and amplification of the CHO cells of various platforms, has excellent performance in Batch culture, can maintain high living cell density growth and maintenance of cell survival rate in the later stage of Batch culture, and simultaneously improves high expression of target products. Specifically, the basic culture medium contained in the culture medium is used for recovering CHO cells and amplifying the CHO cells in a shake flask, the Fed-batch culture medium contained in the culture medium is supplemented in a classical Fed-batch culture mode, the balanced supply of nutrient substances in the batch culture process is ensured, and the growth of high cell density (the highest viable cell density is about 15 multiplied by 10) in batch culture is favorably maintained (the highest viable cell density is about 15 multiplied by 10)⁶cells/ml-30X 10⁶Cell/ml), maintenance of later cell viability (cell viability is more than or equal to 90% at day 12-16) and high expression of target products (namely target proteins).

3) The culture medium provided by the invention is beneficial to the control of cell metabolism and product quality, and is convenient for the optimization of a culture process.

4) The culture medium provided by the invention has low cost, is simple and convenient to prepare, and greatly reduces the cost; the process for culturing the CHO cells by using the culture medium provided by the invention is simple to operate, each process parameter is easy to control, the culture medium is suitable for large-scale production and preparation of recombinant protein biological products, protein medicines produced by the CHO cells can be greatly improved, the culture medium is used for treating diseases such as malignant tumors, autoimmune diseases, eyeground diseases, cardiovascular and cerebrovascular diseases and the like, and the market competitiveness of the products is improved.

In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail as follows:

drawings

FIG. 1 shows a graph of viable cell density over time in each test group and control group in the experiment screening basal medium.

FIG. 2 is a graph showing the change of cell viability with time in each test group and control group in the experiment for screening the basal medium

FIG. 3 shows a graph of product concentration over time in each test group and control group in the experiment screening basal medium.

FIG. 4 shows a graph of viable cell density over time in each test group and control group in screening fed-culture medium experiments.

FIG. 5 is a graph showing the change in cell viability over time in each test group and control group in the screening fed-batch medium test.

FIG. 6 shows a graph of product concentration over time for each test group and control group in screening fed-culture medium experiments.

FIG. 7 shows a statistical analysis predicted characterizer plot for 7 key amino acids analyzed using JMP statistical software.

FIG. 8 shows a graph of viable cell density over time in each experimental group with different concentrations of Asn added.

FIG. 9 shows a graph of cell viability over time in each experimental group with different concentrations of Asn added.

FIG. 10 shows a graph of the concentration of product over time in each test group with different concentrations of Asn added.

Fig. 11 shows a graph of viable cell density versus time in each test group and stand-up group in the additive screening test.

FIG. 12 shows a graph of the cell viability with time for each experimental group and the control group in the additive screening test.

Fig. 13 shows graphs of the product concentration with time in each test group and stand control group in the additive screening test.

FIG. 14 is a graph showing the change with time of the viable cell density in each test group and stand up against the group in the trace element content screening test.

FIG. 15 is a graph showing the cell motility with time in each test group and stand up group in the trace element content screening test.

FIG. 16 is a graph showing the change with time of the product concentrations in each test group and stand against group in the trace element content screening test.

Figure 17 shows a graph of viable cell density as a function of time with additional addition of different concentrations of tyrosine to the feed medium.

FIG. 18 shows a graph of cell viability over time with additional addition of different concentrations of tyrosine to the fed medium.

FIG. 19 shows a graph of product concentration as a function of time with additional addition of different concentrations of tyrosine to the feed medium.

FIG. 20 shows the doubling times of different generations for each cell line when different subtypes of CHO cells were cultured in the optimized medium of the present invention.

FIG. 21 is a graph showing viable cell density as a function of cell passage for different subtypes of CHO cells cultured in the optimized medium of the present invention.

FIG. 22 is a graph showing the change in cell viability with passage of cells when different subtypes of CHO cells were cultured in the optimized medium of the present invention.

FIG. 23 shows a graph of the change of viable cell density and cell viability over time of the CHO-K1 recombinant cell strain during fed culture.

FIG. 24 shows a graph of the product concentration of the CHO-K1 recombinant cell line as a function of time during the fed-batch culture.

FIG. 25 shows a graph of the viable cell density and cell viability of CHO-S recombinant cell lines over time during fed-batch culture.

FIG. 26 shows a graph of the product concentration of the CHO-S recombinant cell line as a function of time during the fed-batch culture.

FIG. 27 shows a graph of the viable cell density and cell viability of CHO-GS recombinant cell lines over time during fed-batch culture.

FIG. 28 shows a graph of the product concentration of the CHO-GS recombinant cell line as a function of time during fed-batch culture.

FIG. 29 shows a graph of the change in viable cell density and cell viability over time of the CHO-DG44 recombinant cell strain during fed culture.

FIG. 30 shows a graph of the change in product concentration over time during fed-culture of the CHO-DG44 recombinant cell strain.

Detailed Description

The embodiments of the present invention are described as examples of the present invention, and the present invention is not limited to the embodiments described below. Any equivalent modifications and substitutions to the embodiments described below are within the scope of the present invention for those skilled in the art. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

The components in the culture medium used in the embodiment of the invention can be purchased from the market, and the key instruments and experimental raw materials used in the cell culture process can be purchased from the market, and the specific information is as follows:

table 3: key instrument and equipment list for experiment

Device name	Origin and brand
		Cell counter	Beckman Coulter, USA
Cell counter	Beckman Coulter, USA
		Full-automatic biochemical analyzer	NOVA of America
Biochemical analyzer	Switzerland Roche
		FE20 laboratory pH meter	METTLER TOLEDO, switzerland
High performance liquid chromatograph	Agilent, USA
		Clean workbench	Suzhou Sujing Antai (AIRTECH)
Biological safety cabinet	Suzhou Sujing Antai (AIRTECH)
		Carbon dioxide shaking table	Kuhner, Switzerland
Centrifugal machine	Hunan Cence
		Centrifugal machine	Beckman Coulter, USA
Bioreactor	Startorius, germany
		Pipe connecting machine	Startorius, germany
Pipe sealing machine	Startorius, germany

Table 4: raw material list for experiment

Name of raw material	Purchasing company	Batch number
			Defoaming agent	Hyclone	SH30897.01
Sodium bicarbonate	Merck	1.06323.2500
			Anhydrous glucose	Merck	1.37048.5000
Sodium hydroxide	Sichuan Jinshan mountain	F20100001
			Dilute hydrochloric acid	Hunan Erkang	H43020202
Dynamis medium	Gibco	A26175
			Feed C + medium	Gibco	A25031

Example 1: screening of basal Medium

The screening of the basic culture medium adopts the classic mixed material design, and 5 groups of basic culture media for screening with different nutrient substance concentration ratios are selected: f1, F2, F3, F4 and F5 (the components of the screening basic medium are shown in Table 5), and the experiment design is carried out by using DOE software (specifically shown in Table 6); dynamis (Gbico, lot No.: A26175) was used as the medium for the Control group, and the Control group was set with repeated experiments (i.e., Control-1 and Control-2 shown in Table 6).

Test materials: CHO-S recombinant cells having the coding sequence of the anti-CTLA-4 monoclonal antibody incorporated therein were selected as seed cells.

The test method comprises the following steps: and (3) recovering the seed cells and performing shake flask amplification culture. Wherein, the recovery steps of the seed cells are as follows: taking out a cell strain from liquid nitrogen or-80 deg.C, water-bathing at 37.0 + -0.5 deg.C for 2min, thawing, transferring the cell suspension into a centrifuge tube containing 8.5ml Dynamis medium, centrifuging at 1000r/min for 5min, removing supernatant, resuspending the cell with Dynamis medium and transferring to 125mlIn shake flasks, make up to 30 ml. Placing the shake flask at 36.0-37.0 deg.C and 5-7% CO₂And culturing for 2-4 days under the condition of 120-140 r/min. The shake flask amplification step is as follows: the cells are cultured in Dynamis medium at a ratio of 0.3-0.7 × 10⁶Diluting the solution to a new shake flask with a density of 5-7% CO at 36.0-37.0 deg.C₂And culturing for 2-4 days under the condition of 120-140 r/min.

And (3) carrying out seed culture on the seed cells: the seed cells after the recovery and the shake flask amplification culture are subjected to adaptive culture (namely, the seed cells are subjected to adaptive amplification culture before batch culture) when being subcultured to N-1, specifically, the culture is carried out at 0.5 multiplied by 10⁶The seed cells are inoculated in each group of screening basal culture media in the table 5 for cell passage adaptive culture, the total culture volume is 25ml, and the cells are cultured for 3 days; after 3 days each group of cells was cultured in classical Batch at 0.8X 10⁶Cell density of cells/ml, culture volume 30ml, 6% CO at 36.5 ℃%₂200r/min, 80% humidity, sampling and counting on 3 rd, 5 th, 8 th and 11 th days, and keeping samples on 8 th and 11 th days to detect the product concentration.

As shown in fig. 1, 2, and 3, a graph of viable cell density, a graph of cell viability and a graph of product concentration over time in each set of basal media is shown, respectively. The dashed black lines in fig. 1-3 represent the control, the solid black lines represent the preferred test (i.e., group 10 in table 6), and the gray and white lines represent the remaining test groups.

From the results of FIGS. 1-3, it can be seen that: the viable cell density, cell viability and product concentration varied widely between the test groups. According to the comprehensive evaluation of indexes such as living cell density, cell survival rate, protein expression amount (namely product concentration) and the like, a better test group 10 is selected for the optimization of the subsequent fed-batch culture medium.

Table 5: composition of basal Medium for screening

Table 6: basal medium mixing design

Example 2: screening of feed Medium

And (3) experimental design: the 10 th group obtained by screening in example 1 was used as a basal medium, and 4 screening fed-batch media Feed1, Feed2, Feed3 and Feed4 with different compositions were screened by a mixed material design, wherein the 4 screening fed-batch media have the compositions shown in Table 7 and the mixed material design shown in Table 8. The Control medium was a combination of basal medium Dynamis and Feed medium Feed C +, and the Control group was set up for repeat experiments (i.e., Control-1 and Control-2 shown in Table 8).

Test materials: CHO-K1 recombinant cells incorporating the coding sequence of an anti-CD 20 monoclonal antibody.

The test method comprises the following steps: the CHO-K1 recombinant cells were rescued and shake flask expanded by the method described in example 1, followed by inoculating the cells into the preferred basal medium selected in example 1 for 3 days of subculture, and then subcultured at 0.8X 10⁶The cells/ml cell density was inoculated in 30ml of the feeding medium for selection, sampled and counted on

days

3, 5, 8, 11 and 14, biochemical markers were measured, and fed C + (Gibco, lot: A25031) at an initial volume of 6% (V/V) was added and cultured for 14 days for harvesting.

As shown in fig. 4-6, a graph of viable cell density, a graph of cell viability and a graph of product concentration over time are shown for each group, respectively. The dashed black lines in fig. 4-6 are controls, the solid black lines are the preferred test groups (i.e., group 10 in table 8), and the gray and white lines are the remaining test groups.

From the results of FIGS. 4-6, it can be seen that: there were significant differences in viable cell density, cell viability and product concentration between the different test groups. And comprehensively evaluating indexes such as viable cell density, cell viability rate and protein expression (namely product concentration) to determine the 10 th group of better fed-batch culture medium in the test group. Thus, the preferred combination of media (i.e., group 10 in basal media, group 10 in feed media) was selected for subsequent further optimization.

Table 7: components of feed media for screening

Table 8: feeding culture medium mixing design

Example 3: determination of key amino acid in culture medium and optimization of asparagine content in fed-batch culture medium

To further investigate the effect of the content of each amino acid (in which cystine and tyrosine are less soluble, which is not conducive to DOE design, and therefore the content of these two amino acids was not optimized) in the culture medium on CHO cell culture, the following experiments were performed:

and (3) experimental design: based on the studies of example 1 and example 2, 17 amino acids were optimally screened by the Deterministic Screening Design (DSD) (see table 9), Fed-Batch culture was performed using the Fed-Batch culture process described in example 2, and the predicted characterizer profiles (see in particular fig. 7) for 7 key amino acids (i.e., Arg, Asp, Cys, Gly, Glu, His, and Ser) were analyzed using JMP statistical software after the Batch culture was completed.

And (4) test conclusion: the results shown in FIG. 7 show that the content of asparagine (Asn) significantly affects the density and viability of cells, and is inversely related to the product concentration (i.e., the amount of protein expressed, Titer), so that it is confirmed that the content of Asn is appropriately reduced in the subsequent medium. Specifically, statistical analysis according to JMP DOE found: the content of Asn has obvious influence on the living cell density, the cell viability and the protein expression quantity, so that the Asn is the key amino acid in the amino acid of the fed-batch culture medium.

In the DOE screening experiment, two levels of screening are performed on Asn, namely, a low level (2252mg/L, referred to as Asn (-1) group), a high level (6475mg/L, referred to as Asn (1) group), and a central point (4363mg/L, referred to as Asn (0) group), as shown in fig. 7, increase in Asn concentration is beneficial to growth of cells at the early stage of batch culture, but as the concentration of Asn increases, cell density, viability and protein expression are significantly reduced at the later stage of batch culture, and Asn has a significant influence on batch culture, so that concentration confirmation experiments are performed on Asn, and the low level, the high level and the central point of the DOE screening experiment are respectively selected to perform batch culture one-factor confirmation again, and the results show that the results are consistent with DOE results. Specifically, according to the results of the DOE experiments, on the basis of the preferred fed-batch medium screened in example 2, the effect of different amounts of Asn on CHO cell culture was examined for 3 experimental groups, in which Asn concentrations were set to 2252mg/l (i.e., Asn (-1) group), 4363mg/l (i.e., Asn (0) group) and 6475mg/l (i.e., Asn (1) group), respectively.

And (4) test conclusion: as shown in fig. 8, 9, 10: the difference in the performance of different concentrations of Asn (Asn (-1) 2252mg/l, Asn (0) 4363mg/l, and Asn (1) 6475 mg/l) in the Fed-batch culture process was consistent with the results of the Deterministic Screening Design (DSD); the high concentration of Asn is not beneficial to maintaining the living cell density and the cell survival rate, and has a certain inhibiting effect on protein expression, while the addition of Asn is beneficial to the increase of the earlier-stage cell density, and the content of Asn in the finally selected fed-batch culture medium is 4363 mg/L.

Table 9: DSD deterministic screening design

Group of	Arg	Asn	Asp	Cys	Gly	Glu	His	Ile	Leu	Lys	Met	Phe	Pro	Ser	Thr	Trp	Val
																		1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
2	0	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1
																		3	1	0	1	-1	-1	1	1	1	1	-1	1	-1	1	-1	-1	-1	-1
4	-1	0	-1	1	1	-1	-1	-1	-1	1	-1	1	-1	1	1	1	1
																		5	1	-1	0	1	-1	-1	1	1	1	1	-1	1	-1	1	-1	-1	-1
6	-1	1	0	-1	1	1	-1	-1	-1	-1	1	-1	1	-1	1	1	1
																		7	1	1	-1	0	1	-1	-1	1	1	1	1	-1	1	-1	1	-1	-1
8	-1	-1	1	0	-1	1	1	-1	-1	-1	-1	1	-1	1	-1	1	1
																		9	1	1	1	-1	0	1	-1	-1	1	1	1	1	-1	1	-1	1	-1
10	-1	-1	-1	1	0	-1	1	1	-1	-1	-1	-1	1	-1	1	-1	1
																		11	1	-1	1	1	-1	0	1	-1	-1	1	1	1	1	-1	1	-1	1
12	-1	1	-1	-1	1	0	-1	1	1	-1	-1	-1	-1	1	-1	1	-1
																		13	1	-1	-1	1	1	-1	0	1	-1	-1	1	1	1	1	-1	1	-1
14	-1	1	1	-1	-1	1	0	-1	1	1	-1	-1	-1	-1	1	-1	1
																		15	1	-1	-1	-1	1	1	-1	0	1	-1	-1	1	1	1	1	-1	1
16	-1	1	1	1	-1	-1	1	0	-1	1	1	-1	-1	-1	-1	1	-1
																		17	1	-1	-1	-1	-1	1	1	-1	0	1	-1	-1	1	1	1	1	-1
18	-1	1	1	1	1	-1	-1	1	0	-1	1	1	-1	-1	-1	-1	1
																		19	1	1	-1	-1	-1	-1	1	1	-1	0	1	-1	-1	1	1	1	1
20	-1	-1	1	1	1	1	-1	-1	1	0	-1	1	1	-1	-1	-1	-1
																		21	1	-1	1	-1	-1	-1	-1	1	1	-1	0	1	-1	-1	1	1	1
22	-1	1	-1	1	1	1	1	-1	-1	1	0	-1	1	1	-1	-1	-1
																		23	1	1	-1	1	-1	-1	-1	-1	1	1	-1	0	1	-1	-1	1	1
24	-1	-1	1	-1	1	1	1	1	-1	-1	1	0	-1	1	1	-1	-1
																		25	1	-1	1	-1	1	-1	-1	-1	-1	1	1	-1	0	1	-1	-1	1
26	-1	1	-1	1	-1	1	1	1	1	-1	-1	1	0	-1	1	1	-1
																		27	1	1	-1	1	-1	1	-1	-1	-1	-1	1	1	-1	0	1	-1	-1
28	-1	-1	1	-1	1	-1	1	1	1	1	-1	-1	1	0	-1	1	1
																		29	1	1	1	-1	1	-1	1	-1	-1	-1	-1	1	1	-1	0	1	-1
30	-1	-1	-1	1	-1	1	-1	1	1	1	1	-1	-1	1	0	-1	1
																		31	1	1	1	1	-1	1	-1	1	-1	-1	-1	-1	1	1	-1	0	1
32	-1	-1	-1	-1	1	-1	1	-1	1	1	1	1	-1	-1	1	0	-1
																		33	1	1	1	1	1	-1	1	-1	1	-1	-1	-1	-1	1	1	-1	0
34	-1	-1	-1	-1	-1	1	-1	1	-1	1	1	1	1	-1	-1	1	0
																		35	1	-1	1	1	1	1	-1	1	-1	1	-1	-1	-1	-1	1	1	-1
36	-1	1	-1	-1	-1	-1	1	-1	1	-1	1	1	1	1	-1	-1	1
																		37	1	-1	-1	1	1	1	1	-1	1	-1	1	-1	-1	-1	-1	1	1
38	-1	1	1	-1	-1	-1	-1	1	-1	1	-1	1	1	1	1	-1	-1
																		39	1	1	-1	-1	1	1	1	1	-1	1	-1	1	-1	-1	-1	-1	1
40	-1	-1	1	1	-1	-1	-1	-1	1	-1	1	-1	1	1	1	1	-1
																		41	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
42	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Example 4: effect of other additives on CHO cell culture

From the prior art references, the person skilled in the art knows: hydroxyproline, a non-essential amino acid, is a degradation product of collagen, participates in the synthesis of glycine, and simultaneously regulates the reduction-oxidation state of cells; in organisms, ornithine mainly participates in uric acid circulation and plays an important role in discharging ammonia nitrogen in the organisms; the p-aminobenzoic acid can capture free radicals and resist oxidation; thymine, thymidine, uracil, guanine, cytosine, and the like are nucleic acid precursors and are necessary for synthesizing DNA. However, the addition of the above substances inevitably increases the production cost of the medium, and this example examines the effect of the above substances on CHO cell culture.

Based on the optimization results of the culture media in examples 1-3, the following additives were screened simultaneously: ornithine (Ornithine), hydroxyproline (Hy-proline), p-aminobenzoic acid (PABA), Thymine (Thymine), Thymidine (Thymidine), uracil (Uridine), guanine (Guanosine), cytosine (Cytidine).

And (3) experimental design: the above additives were tested for single factor effect based on media platform experience and references known in the art, and the effect of each additive on cell culture was assessed by monitoring the course of the Fed-Batch culture of cells.

The test method comprises the following steps: at 0.8X 10⁶Cells/ml were seeded in 250ml baffled shake flasks of 80ml volume. The parameters of shaking table culture are as follows: 6% CO₂Sampling at 36.5 deg.C, 130r/min, 3 rd, 5 th, 7 th, 9 th, 11 th and 14 th days to detect viable cell density and cell viability, and monitoring glucose content with biochemical analyzer when glucose concentration is reached<Increasing the final concentration of glucose to 6g/L at the time of 2g/L, simultaneously respectively supplementing fed-batch culture media with the initial volume of 4% (V/V) of the initial culture weight on

days

3, 5, 7, 9 and 11, detecting the protein expression amount from day 7, and harvesting on day 15.

And (3) test results: as shown in fig. 11 to 13, the various additives were significantly different in Fed-Batch, but the concentrations of the various additives were not significantly improved in terms of viable cell density, cell viability maintenance, and protein expression compared to the control (the control group was a group to which the above additives were not added), so that the above additives were not added to the final medium.

Example 5: optimization of content of trace elements in culture medium

The trace elements are of various types, and a suitable application range is determined by gradient screening of 3 combinations of trace elements (see table 10).

Experimental design and experimental method referring to example 4, the mother liquors of trace elements were mixed in the optimized basal medium and fed-batch medium, respectively, in proportions such that: TE 110X, TE 20X and TE 40X are added into the basic culture medium, TE 140X, TE 80X and TE 160X are added into the corresponding feeding culture medium (namely, the addition amount of the trace elements in the feeding culture medium is 4 times of the addition amount of the trace elements in the basic culture medium), TE2 and TE3 are combined, and the like, and the control group is a group without the trace elements.

Table 10: trace element component

And (3) test results: as shown in FIGS. 14 to 16, the difference of the effect of the combination of 3 trace elements on the cell growth is significant, the viable cell density and the cell viability rate are both reduced along with the increase of the TE1 concentration, and the protein expression level is also in negative correlation with the TE1 concentration; different concentrations of TE2 had no significant effect on cell growth and protein expression; TE3 significantly affected cell growth and protein expression, and the combined trace elements were toxic to cells and not suitable for subsequent use in Fed-Batch.

According to the comprehensive evaluation of indexes such as viable cell density, cell viability maintenance, protein expression and the like, TE 210 multiplied by trace elements are finally determined to be added into the basal medium, and TE 240 multiplied by trace elements are added into the fed-batch medium to be used as the optimized addition amount of the trace elements in the medium (note: legend is named by the addition amount of the trace elements in the basal medium, for example, "TE 110 multiplied by the basal medium, and TE 140 multiplied by the fed-batch medium").

Example 6: optimization of additional tyrosine addition in fed-batch medium

The research process of the inventor finds that the Fed-Batch culture can be obviously influenced by additionally adding tyrosine into a feeding culture medium, so that different concentration gradient screening is carried out on tyrosine, the test design and the test method refer to example 4, tyrosine mother liquor is respectively added in the 3 rd, 5 th, 7 th, 9 th and 11 th days of the feeding culture, and the total adding concentration is 0.2-1.6 g/L.

And (3) test results: as shown in FIGS. 17-19, the addition of low concentrations of tyrosine (i.e., 0.2g/L and 0.6g/L tyrosine) significantly affected viable cell density, cell viability and product concentration, and the addition of 0.8-1.6 g/L tyrosine concentrate to the fed-batch medium did not significantly differ in cell growth, viability maintenance and protein expression effects.

And (4) test conclusion: in the batch culture process of the fed-batch culture medium, 0.8-1.6 g/L of tyrosine concentrated solution needs to be additionally added, the tyrosine content can obviously improve the expression quantity of a target product, and the adding time of the tyrosine can be consistent with that of the fed-batch culture medium.

Example 7: suitability and stability of the Medium in different types of CHO cells

CHO cells are widely applied to cell culture in the biopharmaceutical industry, CHO cell subtypes with different genetic characteristics are generated along with research and development of the CHO cells, four existing CHO cell subtypes (CHO-S, CHO-K1, CHO-GS and CHO-DG44) on the market are researched, and the wide applicability and stability of the culture medium with optimized components provided by the invention are verified.

Verification of the primary passage stability performance of the medium provided by the present invention in the existing CHO platform project cells CHO-S (which contains therein the gene sequence encoding the anti-OX 40 monoclonal antibody), CHO-K1 (which contains therein the gene sequence encoding the anti-CD 20 monoclonal antibody), CHO-GS (which contains therein the gene sequence encoding the anti-TIGIT monoclonal antibody) and CHO-DG44 (which contains therein the gene sequence encoding the anti-TNF α monoclonal antibody): the cells were cultured in a medium of 0.4X 10⁶The cells/ml were diluted to a new flask and placed in 36.5 ℃ 6% CO₂One generation was transferred every 3 days in a carbon dioxide shaker at 130 rpm. As shown in FIGS. 20-22, the doubling time of each cell line is 18-35 hours, the viable cell density is kept stably increasing in different cell generations, the cell viability is maintained at more than 99%, and the cell lines of each platform project are stable.

And (3) test results: according to the primary passage stability results of the cells of each platform project, the culture medium provided by the invention has good adaptability in the cells of the four platform projects of CHO-S, CHO-K1, CHO-GS and CHO-DG44, the cells grow well, the growth rate is stable, and no obvious difference exists among generations.

Example 8: application of culture medium in different types of CHO cell culture

Test materials: recombinant CHO-S cells comprising a gene sequence encoding an anti-OX 40 monoclonal antibody, recombinant CHO-K1 cells comprising a gene sequence encoding an anti-CD 20 monoclonal antibody, recombinant CHO-GS cells comprising a gene sequence encoding an anti-TIGIT monoclonal antibody and CHO-DG44 cells comprising a gene sequence encoding an anti-TNF α monoclonal antibody.

The test method comprises the following steps: (1) resuscitating and shake flask expansion culturing the recombinant CHO cell according to the method described in example 1; and the Dynamis culture medium is replaced by the basal culture medium with optimized components, namely the basal culture medium with the optimal content of each component in the table 1.

(2) According to the initial inoculation density (1.0 +/-0.3) multiplied by 10⁶And (2) inoculating the recombinant CHO cells in the step (1) into a basal culture with the optimal content of each component in the table 1, controlling DO to be 20-80% and pH to be 6.80-7.20. Controlling the culture temperature at 36.0-37.0 ℃ on the 1-4 th day of culture, and reducing the culture temperature to 32.0-34.0 ℃ on the 5 th day of culture.

(3) Culturing for 3 rd, 5 th, 7 th, 9 th and 11 th days, respectively adding 4-5% (w/w) of fed-batch culture medium with optimal content of each component as described in Table 2, and adding tyrosine concentrate with final concentration of 0.8-1.6 g/L simultaneously with the fed-batch culture medium.

And (4) test conclusion: the culture medium provided by the invention has better performance in Fed-Batch Fed-Batch culture of recombinant cell strains of CHO platform cells CHO-S, CHO-K1, CHO-GS and CHO-DG44 (see particularly FIGS. 23-30), and can reach higher living cell density (particularly can reach (15-30) × 10⁶Viable cell density per ml), and the cell viability is maintained well, and finally the concentration of the target product of each platform also reaches a higher level (2000-8000 mg/l).

Claims

1. A culture medium for CHO cells, characterized in that the culture medium is a chemically defined and serum-free, ornithine, hydroxyproline, p-aminobenzoic acid, thymine, thymidine, uracil, guanine and cytosine-free medium, comprising a basal medium and a fed-batch medium with a final concentration of 120-150g/L,

2. The culture medium for CHO cells according to claim 1, wherein:

other components in the basic culture medium also comprise glucose; the trace elements in the basic culture medium further comprise: one or more of the group consisting of stannous chloride dihydrate, silver nitrate, potassium bromide, 5/2 cadmium chloride hydrate and potassium iodide.

3. The culture medium for CHO cells according to claim 2, wherein the contents of sodium bicarbonate, glucose and dextran sulfate contained in the basal medium are 1000-3000mg/L, 0-8000mg/L and 15-40mg/L, respectively; preferably, the contents of the sodium bicarbonate, the glucose and the dextran sulfate are respectively 1500-2500mg/L, 2000-7000mg/L and 20-35 mg/L; more preferably, the contents of the sodium bicarbonate, the glucose and the dextran sulfate are 2000mg/L, 6000mg/L and 25mg/L respectively;

the content of the asparagine-linked monohydrate contained in the fed-batch culture medium is 500-8000 mg/L; preferably, the content of the asparagine-monohydrate is 1000-5500 mg/L; more preferably, the content of asparagine-monohydrate is 4363 mg/L.

4. A culture medium for CHO cells according to claim 2 or 3, characterized in that the contents of trace elements contained in the basal medium are: 0.0005 to 0.003mg/L of manganese sulfate monohydrate, 0.005 to 0.04mg/L of sodium silicate nonahydrate, 0.05 to 0.3mg/L of ammonium molybdate tetrahydrate, 0.001 to 0.002mg/L of ammonium metavanadate, 0.0001 to 0.008mg/L of nickel sulfate hexahydrate, 0 to 0.001mg/L of stannous chloride dihydrate, 0.0007 to 0.002mg/L of aluminum chloride hexahydrate, 0 to 0.0008mg/L of silver nitrate, 0.00008 to 0.001mg/L of barium acetate, 0-0.0007mg/L of potassium bromide, 0-0.01mg/L of 5/2 hydrated cadmium chloride, 0.002-0.02mg/L of cobalt chloride hexahydrate, 0.0005-0.004mg/L of chromium chloride, 0.005-0.05mg/L of sodium fluoride, 0.0005-0.0025mg/L of germanium oxide, 0-0.0005mg/L of potassium iodide, 0.0006-0.01mg/L of rubidium chloride and 0.002-0.05mg/L of zirconium oxychloride octahydrate; preferably, the contents of the trace elements contained in the basic culture medium are respectively as follows: 0.001-0.002mg/L of manganese sulfate monohydrate, 0.008-0.03mg/L of sodium silicate nonahydrate, 0.08-0.2mg/L of ammonium molybdate tetrahydrate, 0.001-0.0018mg/L of ammonium metavanadate, 0.001-0.005mg/L of nickel sulfate hexahydrate, 0.0001-0.0009mg/L of stannous chloride dihydrate, 0.001-0.002mg/L of aluminum chloride hexahydrate, 0.000001-0.00008mg/L of silver nitrate, 0.0001-0.0008mg/L of barium acetate, 0.00001-0.0001mg/L potassium bromide, 0.0001-0.0085 mg/L5/2 hydrated cadmium chloride, 0.003-0.01mg/L cobalt chloride hexahydrate, 0.0008-0.002mg/L chromium chloride, 0.008-0.03mg/L sodium fluoride, 0.0008-0.002mg/L germanium oxide, 0.00001-0.0003mg/L potassium iodide, 0.0008-0.008mg/L rubidium chloride and 0.003-0.01mg/L zirconium oxychloride octahydrate; more preferably, the contents of the trace elements contained in the basic culture medium are respectively as follows: 0.0015mg/L manganese sulfate monohydrate, 0.015mg/L sodium silicate nonahydrate, 0.1mg/L ammonium molybdate tetrahydrate, 0.0015mg/L ammonium metavanadate, 0.003mg/L nickel sulfate hexahydrate, 0.00025mg/L stannous chloride dihydrate, 0.0015mg/L aluminum chloride hexahydrate, 0.000055mg/L silver nitrate, 0.00035mg/L barium acetate, 0.00006mg/L potassium bromide, 0.0065 mg/L5/2 cadmium chloride hydrate, 0.0055mg/L cobalt chloride hexahydrate, 0.00155mg/L chromium chloride, 0.012mg/L sodium fluoride, 0.0012mg/L germanium oxide, 0.00015mg/L potassium iodide, 0.0035mg/L rubidium chloride and 0.007mg/L zirconium oxychloride octahydrate;

the contents of the trace elements contained in the fed-batch culture medium are respectively as follows: 0.0005-0.05mg/L of manganese sulfate monohydrate, 0.0005-5.0mg/L of sodium silicate nonahydrate, 0.005-0.05mg/L of ammonium molybdate tetrahydrate, 0.0001-0.04mg/L of ammonium metavanadate, 0.0005-0.05mg/L of nickel sulfate hexahydrate, 0.00001-0.005mg/L of stannous chloride dihydrate, 0.00001-0.005mg/L of aluminum chloride hexahydrate, 0.00001-0.005mg/L of silver nitrate, 0.0001-0.08mg/L of barium acetate, 0.00005-0.005mg/L potassium bromide, 0.005-0.08 mg/L5/2 hydrated cadmium chloride, 0.005-0.08mg/L cobalt chloride hexahydrate, 0.0005-0.01mg/L chromium chloride, 0.005-0.1mg/L sodium fluoride, 0.0005-0.05mg/L germanium oxide, 0.0005-0.008mg/L potassium iodide, 0.005-0.1mg/L rubidium chloride and 0.005-0.01mg/L zirconium oxychloride octahydrate; preferably, the contents of the trace elements contained in the fed-batch culture medium are respectively as follows: 0.0009 to 0.02mg/L of manganese sulfate monohydrate, 0.001 to 2.5mg/L of sodium silicate nonahydrate, 0.008 to 0.02mg/L of ammonium molybdate tetrahydrate, 0.0005 to 0.02mg/L of ammonium metavanadate, 0.0006 to 0.03mg/L of nickel sulfate hexahydrate, 0.0001 to 0.004mg/L of stannous chloride dihydrate, 0.001 to 0.01mg/L of aluminum chloride hexahydrate, 0.00005 to 0.004mg/L of silver nitrate, 0.0008 to 0.05mg/L of barium acetate, 0.00006-0.001mg/L potassium bromide, 0.01-0.06 mg/L5/2 hydrated cadmium chloride, 0.008-0.05mg/L cobalt chloride hexahydrate, 0.001-0.009mg/L chromium chloride, 0.007-0.08mg/L sodium fluoride, 0.001-0.015mg/L germanium oxide, 0.0005-0.005mg/L potassium iodide, 0.0065-0.08mg/L rubidium chloride and 0.0065-0.08mg/L zirconium oxychloride octahydrate; more preferably, the contents of the trace elements contained in the fed-batch culture medium are respectively as follows: 0.006mg/L manganese sulfate monohydrate, 0.06mg/L sodium silicate nonahydrate, 0.4mg/L ammonium molybdate tetrahydrate, 0.006mg/L ammonium metavanadate, 0.012mg/L nickel sulfate hexahydrate, 0.001mg/L stannous chloride dihydrate, 0.006mg/L aluminum chloride hexahydrate, 0.00022mg/L silver nitrate, 0.0014mg/L barium acetate, 0.00024mg/L potassium bromide, 0.026 mg/L5/2 cadmium chloride hydrate, 0.022mg/L cobalt chloride hexahydrate, 0.0062mg/L chromium chloride, 0.048mg/L sodium fluoride, 0.0048mg/L germanium oxide, 0.0006mg/L potassium iodide, 0.014mg/L rubidium chloride and 0.028mg/L zirconium oxychloride octahydrate.

5. A culture medium for CHO cells according to any one of claims 2 to 4, wherein the content of each of amino acids, vitamins, inorganic salts and other components contained in the basic medium is: alanine 40-100mg/L, arginine 500-2000mg/L, asparagine 1000-2000mg/L, aspartic acid 200-1000mg/L, cysteine hydrochloride 20-85mg/L, cystine hydrochloride 50-300mg/L, glutamic acid 50-350mg/L, glycine 20-50mg/L, histidine 100-450mg/L, isoleucine 500-1500mg/L, leucine 1000-2000mg/L, lysine hydrochloride 500-1500mg/L, methionine 80-350mg/L, phenylalanine 200-800mg/L, proline 600-1450mg/L, serine 600-2200mg/L, threonine 200-800mg/L, 300mg/L tryptophan, 50-400mg/L tyrosine, 300mg/L valine, 0.05-0.5mg/L biotin, 2-12mg/L calcium pantothenate, 50-400mg/L choline chloride, 1-8mg/L folic acid, 50-350mg/L inositol, 0.5-7.5mg/L nicotinamide, 1.5-4.5mg/L pyridoxine hydrochloride, 0.08-0.5mg/L riboflavin, 1-8mg/L thiamine hydrochloride, 120.8-5.8 mg/L vitamin B, 500-2000mg/L monosodium phosphate monohydrate, 400mg/L magnesium sulfate, 50-250mg/L calcium chloride, 1000mg/L potassium chloride, 1-10mg/L zinc heptasulfate hydrate, 1-10mg/L valine, vitamin A, vitamin B, vitamin A, and vitamin B, 0.2-0.6mg/L of copper sulfate pentahydrate, 5-50mg/L of ferric ammonium citrate, 0.01-0.05mg/L of sodium selenite, 50-250mg/L of putrescine dihydrochloride, 50-150mg/L of ethanolamine and 68600-2000 mg/L of block polyether F; preferably, the content of each substance in the amino acid, the vitamin, the inorganic salt and other components contained in the basic culture medium is respectively alanine 50-80mg/L, arginine 1200-1600mg/L, asparagine 1150-hydrate 1750mg/L, aspartic acid 350-950mg/L, cysteine hydrochloride 25-65mg/L, cystine dihydrochloride 70-200mg/L, glutamic acid 100-250mg/L, glycine 30-45mg/L, histidine 200-300mg/L, isoleucine 650-900mg/L, leucine 1300-1800mg/L, lysine 700-1200mg/L, methionine 100-300mg/L, phenylalanine 350-650mg/L, lysine, 1000-1400mg/L proline, 850-1800mg/L serine, 350-650mg/L threonine, 100-200mg/L tryptophan, 100-300mg/L tyrosine, 350-650mg/L valine, 0.1-0.3mg/L biotin, 3-10mg/L calcium pantothenate, 200-400mg/L choline chloride, 2-7mg/L folic acid, 80-300mg/L inositol, 1.5-6.5mg/L nicotinamide, 2-4mg/L pyridoxine hydrochloride, 0.1-0.4mg/L riboflavin, 2-6mg/L thiamine hydrochloride, 122.0-4.0 mg/L vitamin B, 1000-1800mg/L sodium dihydrogen phosphate monohydrate, 280mg/L magnesium sulfate, 280mg/L, 100mg/L of calcium chloride, 800mg/L of potassium chloride, 2.5-6.5mg/L of zinc sulfate heptahydrate, 0.3-0.5mg/L of copper sulfate pentahydrate, 10-40mg/L of ferric ammonium citrate, 0.01-0.03mg/L of sodium selenite, 80-150mg/L of putrescine dihydrochloride, 80-120mg/L of ethanolamine and 68800-1500 mg/L of segmented polyether F; more preferably, the content of each of the amino acids, vitamins, inorganic salts and other components contained in the basic culture medium is: alanine 60mg/L, arginine 1300mg/L, asparagine.monohydrate 1450mg/L, aspartic acid 800mg/L, cysteine.HCl.35 mg/L, cystine.dihydrochloride 120mg/L, glutamic acid 180mg/L, glycine 35mg/L, histidine 255mg/L, isoleucine 850mg/L, leucine 1600mg/L, lysine.HCl 850mg/L, methionine 180mg/L, phenylalanine 455mg/L, proline 1250mg/L, serine 1000mg/L, threonine 550mg/L, tryptophan 150mg/L, tyrosine 250mg/L, valine 450mg/L, biotin 0.15mg/L, calcium pantothenate 8mg/L, choline chloride 250mg/L, 4.5mg/L folic acid, 210mg/L inositol, 3.5mg/L nicotinamide, pyridoxine hydrochloride 2.5mg/L, riboflavin 0.2mg/L thiamine hydrochloride 3mg/L vitamin B123.5mg/L, monobasic sodium phosphate monohydrate 1500mg/L, magnesium sulfate 255mg/L, calcium chloride 125mg/L potassium chloride 550mg/L zinc sulfate heptahydrate 4mg/L, copper sulfate pentahydrate 0.4mg/L, ferric ammonium citrate 35mg/L, sodium selenite 0.015mg/L, putrescine dihydrochloride 110mg/L ethanolamine 100mg/L, segmented polyether F681000 mg/L;

the contents of amino acid, vitamins, inorganic salt and other components in the fed-batch culture medium are respectively as follows: 8000mg/L of arginine 1500-, 30-450mg/L calcium pantothenate, 100-650mg/L choline chloride, 10-100mg/L folic acid, 50-850mg/L inositol, 10-80mg/L nicotinamide, 5-55mg/L pyridoxine hydrochloride, 0.5-8.5mg/L riboflavin, 10-85mg/L thiamine hydrochloride, 124.5-35 mg/L vitamin B, 500mg/L sodium pyruvate, 4500mg/L monosodium phosphate monohydrate 2000mg/L, 5-50mg/L zinc sulfate heptahydrate, 0.5-5mg/L copper sulfate pentahydrate, 20-300mg/L ferric ammonium citrate, 0.001-0.05mg/L sodium selenite, 20-620mg/L putrescine dihydrochloride, 550mg/L ethanolamine 100-, Block polyether F681000-4000 mg/L, glucose 20000-100000mg/L, hydroxyethyl piperazine ethanethiosulfonic acid (HEPES)1000-4500 mg/L; preferably, the contents of amino acids, vitamins, inorganic salts and other components in the fed-batch culture medium are respectively as follows: arginine 2000-7500mg/L, aspartic acid 3500-6500mg/L, cysteine hydrochloride 100-200mg/L, cystine dihydrochloride 80-350mg/L, glutamic acid 4500-8500mg/L, histidine 800-3000mg/L, isoleucine 3000-5500mg/L, leucine 4500-8500mg/L, lysine hydrochloride 4000-8500mg/L, methionine 500-2000mg/L, phenylalanine 1500-4500mg/L, proline 2000-4000mg/L, serine 6000-12000mg/L, threonine 2000-5500mg/L, tryptophan 500-2500mg/L, tyrosine 100-320mg/L, valine-7500 mg/L2500 mg/L, 100-400mg/L calcium pantothenate, 150-550mg/L choline chloride, 25-75mg/L folic acid, 150-650mg/L inositol, 25-65mg/L nicotinamide, 10-40mg/L pyridoxine hydrochloride, 1.0-6.5mg/L riboflavin, 15-65mg/L thiamine hydrochloride, 128-25 mg/L vitamin B, 800-3500mg/L sodium pyruvate, 2500-2500 monohydrate, 6500mg/L zinc sulfate heptahydrate, 10-45mg/L blue vitriol, 1.0-3.5mg/L bluestone pentahydrate, 100-300mg/L ammonium iron citrate, 0.003-0.02mg/L sodium selenite, 50-350mg/L putrescine dihydrochloride, 150-350mg/L ethanolamine, Block polyether F681100-2700 mg/L, glucose 30000-90000mg/L, hydroxyethyl piperazine ethanesulfonic acid (HEPES)1500-3500 mg/L; more preferably, the contents of amino acids, vitamins, inorganic salts and other components in the fed-batch culture medium are respectively as follows: 5500mg/L arginine, 5550mg/L aspartic acid, cysteine, hydrochloride, 110mg/L monohydrate, cystine, dihydrochloride 270mg/L, glutamic acid 5050mg/L, histidine 2250mg/L, isoleucine 5250mg/L isoleucine, leucine 8150mg/L, lysine, hydrochloride, 7500mg/L methionine, 650mg/L methionine, 3500mg/L phenylalanine, 3500mg/L proline, 10500mg/L serine, 2500mg/L threonine, 2100mg/L tryptophan, 280mg/L tyrosine, 5500mg/L valine, 350mg/L calcium pantothenate, 400mg/L choline chloride, 60mg/L folic acid, 510mg/L inositol, 45mg/L nicotinamide, pyridoxine, hydrochloride 15mg/L, riboflavin 4.5mg/L, Thiamine hydrochloride 25mg/L, vitamin B1216 mg/L, sodium pyruvate 2050mg/L, monobasic sodium phosphate monohydrate 4500mg/L, zinc sulfate heptahydrate 30mg/L, copper sulfate pentahydrate 2.5mg/L, ferric ammonium citrate 200mg/L, sodium selenite 0.009mg/L, putrescine dihydrochloride 150mg/L, ethanolamine 200mg/L, segmented polyether F682000 mg/L, glucose 55000mg/L, and hydroxyethyl piperazine ethanesulfonic acid (HEPES)3000 mg/L.

6. A culture medium for CHO cells according to any one of claims 1 to 5, characterized in that a final concentration of the fed-batch medium is 140 g/L.

7. A method of culturing recombinant CHO cells, the method comprising the steps of:

8. The method of culturing recombinant CHO cells according to claim 7, characterized in that,

the specific operations of the resuscitation and the shake flask amplification culture in the step (1) are as follows: recovering the seed cells from liquid nitrogen or a refrigerator at-80 ℃, inoculating the seed cells into a shake flask containing the basic culture medium of any one of claims 1-6, controlling the culture temperature to be 36.0-37.0 ℃ and 5-7% CO₂The shaking table speed is set to 120-140 r/min, the cultivation is carried out for 2-4 days according to (0.3-0.7) x 10⁶Carrying out shake flask gradual amplification passage at density of each/ml,each stage of amplification culture is carried out for 2-4 days, and when the cell density reaches (1.0-7.0) x 10⁶Inoculating to the next stage when the seed/ml is required; preferably, the cell viability rate is more than or equal to 85% in the seed recovery stage and more than or equal to 90% in the shake flask amplification stage;

9. The method of culturing recombinant CHO cells of claims 7 or 8, wherein the recombinant CHO cells are CHO cells comprising a heterologous protein encoding gene; preferably, the CHO cell is CHO-S, CHO-K1, CHO-GS or CHO-DG44, and the foreign protein is an antibody; more preferably, the foreign protein is a monoclonal antibody or a bispecific antibody.

10. Use of the culture medium for CHO cells according to any one of claims 1 to 6 for expression of foreign proteins in CHO cells; preferably, the CHO cell is CHO-S, CHO-K1, CHO-GS or CHO-DG44, and the foreign protein is an antibody; more preferably, the foreign protein is a monoclonal antibody or a bispecific antibody.