CN113088480B

CN113088480B - Culture medium for CHO cells and application thereof

Info

Publication number: CN113088480B
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: 2022-10-11
Anticipated expiration: 2039-12-23
Also published as: CN113088480A

Abstract

The invention provides a culture medium for CHO cells and application thereof. The culture medium is characterized by comprising a basic culture medium and a fed-batch culture medium with the final concentration of 120-150g/L, wherein the culture medium is definite in chemical components and does not contain serum, ornithine, hydroxyproline, p-aminobenzoic acid, thymine, thymidine, uracil, guanine and cytosine. The culture medium for CHO cells provided by the invention can support long-term subculture growth of CHO cells of different subtypes; 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 the engineering cells maintain higher viable cell density, higher cell viability and longer culture time, so 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 dimer, 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 the batch difference is inevitable. Therefore, the development of serum-free, chemically defined media is a hot spot 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 large-scale production of animal cell-expressed products, which is chemically defined and free of any animal origin, but the specific growth rate of each example of the invention in the logarithmic growth phase of cells in a batch culture process is only 0.41 to 0.55 days ^-1 The highest viable cell density is only 10.71 multiplied by 10 ⁶ The integral value of the cells/ml and the living cells to the time is only 56.6 multiplied by 10 at most ⁹ Cell 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 more and more 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 above problems in the prior art, the present inventors have conducted extensive research and repeated experiments, and have completed the present invention by carrying out basic medium screening, fed-batch medium screening, amino acid content optimization in the medium, tyrosine content optimization in the fed-batch medium, additive and trace element screening optimization, etc. on the basis of an excellent medium development platform, and respectively carrying out effect verification on CHO host cell platforms of different subtypes, such as CHO-S, CHO-K1, CHO-GS, and CHO-DG44, etc., and finally screening a medium with definite chemical components suitable for high-density growth of CHO cells, stable maintenance of CHO cell viability, and high-expression of desired products. Namely, the present invention is as follows:

the invention provides a culture medium for CHO cells, which is characterized in that the culture medium is a culture medium which is specific in chemical composition and does not contain serum, ornithine, hydroxyproline, p-aminobenzoic acid, thymine, thymidine, uracil, guanine and cytosine, and comprises a basal medium and a fed-batch culture medium with the 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, nicotinamide, 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 block 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, cadmium chloride 5/2 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 stannous chloride dihydrate, silver nitrate, potassium bromide, 5/2 cadmium chloride hydrate and potassium iodide.

Furthermore, the contents of sodium bicarbonate, glucose and dextran sulfate contained in the basic culture 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 1500-2500mg/L, 2000-7000mg/L and 20-35mg/L respectively; 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-containing monohydrate in the fed-batch culture medium is 500-8000mg/L; preferably, the content of the asparagine-monohydrate is 1000-5500mg/L; more preferably, the content of the asparagine-monohydrate is 4363mg/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 ranges in column 3 of 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 ranges in column 3 of table 1; most preferably, as indicated in table 1 by the content ranges in column 4. The contents of the amino acid, the vitamin, the inorganic salt and other components 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 sum of the concentrations of all the components in the fed-batch medium (i.e., the final concentration) was 140g/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. The 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 attribute 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 invention provides a culture medium which is applicable to CHO cells and has definite chemical components, mainly comprising a Basal medium (Basal Media) and a fed-batch medium (Feed Media), wherein 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; sodium bicarbonate 1000-3000mg/L as buffering agent; contains 0-8000mg/L glucose, and each trace element contained 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 the recombinant CHO cell and performing shake flask amplification culture;

(2) According to the initial inoculation density (1.0 +/-0.3) multiplied by 10 ⁶ Inoculating the recombinant CHO cells in the step (1) into the basic culture medium in the first aspect of the invention, wherein DO is controlled to be 20-80%, and pH is controlled to be 6.80-7.20;

(3) The feeding medium as described in the first aspect of the present invention is fed to the culture until

days

3, 5, 7, 9, 11, respectively, at 4% to 5% (w/w) of the initial culture weight.

Further, the specific operations of the resuscitation and the shake flask amplification culture in the step (1) are as follows: resuscitating the seed cells from liquid nitrogen or a freezer at-80 ℃ and inoculating the seed cells into a shake flask containing the basal medium of any one of the first aspect of the invention, wherein the culture temperature is controlled to 36.0-37.0 ℃, the CO content is controlled to 5-7% ₂ 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 10 ⁶ Carrying out shake flask gradual amplification and passage at the density of each/ml, wherein each stage of amplificationCulturing for 2-4 days until the cell density reaches (1.0-7.0) x 10 ⁶ Inoculating 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 of batch culture comprises the following steps: resuscitating and shake flask expanding the seed cells according to the initial inoculation density (1.0 + -0.3). Times.10 ⁶ Inoculating recombinant CHO cells containing encoding foreign proteins into a basal culture medium at a temperature of between 32.0 and 34.0 ℃ in 5 days of culture, wherein DO is controlled to be between 20 and 80 percent, pH is controlled to be between 6.80 and 7.20, and the temperature of the early culture is between 36.0 and 37.0 ℃. 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 5.0g/L. The total amount of Antifoam (including but not limited to Antifoam (Hyclone, SH 30897.01)) cannot exceed 100ppm, depending on the actual amount of foam added manually. Cultivation processSampling the cell fluid every day, detecting the pH, the viable cell density, the cell viability and other biochemical indexes (such as the glucose content, the lactic acid content and the ammonia content in the culture fluid), and culturing until 12-16 days.

The specific operation of "resuscitating and shake flask expanding seed cells" described above is: recovering seed cells from liquid nitrogen or-80 deg.C in refrigerator, inoculating into 125ml shake flask containing basic culture medium, supplementing basic culture medium to 20-30 ml, controlling culture temperature at 36.0-37.0 deg.C, and adjusting CO content at 5-7% ₂ Under the condition of (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 10 ⁶ Carrying 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 10 ⁶ When 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, does not contain any animal source, does not contain serum components, and is qualified in detection of bacteria, heat sources, pH and titer. 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 generation of CHO cells of different subtypes (including CHO-S, CHO-K1, CHO-GS and CHO-DG 44)Long and in vitro suspension culture. 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) ⁶ cells/ml-30X 10 ⁶ Cell/ml), maintenance of the later cell viability (the cell viability is more than or equal to 90 percent at days 12 to 16) and high expression of the target product (namely the target protein).

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 as a function of 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 is a graph showing the respective test groups in the additive screening test and how the viable cell density in the groups was changed with time.

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 respective test groups in the additive screening test and how the concentrations of the products in the groups were changed with time.

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 additions of different concentrations of tyrosine to the fed medium.

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

Figure 19 shows a graph of the product concentration over time with additional additions 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 survival rate with cell passage when CHO cells of different subtypes are cultured in the optimized medium of the present invention.

FIG. 23 shows a graph of the change of viable cell density and cell viability rate with time during fed-batch culture of CHO-K1 recombinant cell lines.

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

FIG. 25 shows a graph of the viable cell density and cell viability rate of CHO-S recombinant cell lines over time during fed 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 is a graph showing the change of viable cell density and cell viability over time during the fed culture of the CHO-DG44 recombinant cell line.

FIG. 30 shows a graph of the change in product concentration over time during fed-culture of a 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 alterations and modifications are intended to be included within the scope of the present invention, without departing from the spirit and scope of the 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 U.S.A
		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 (components of a screening basic culture medium are shown in a table 5), and experimental design is carried out by utilizing DOE software (specifically shown in a table 6); dynamis (Gbico, lot A26175) was used as the medium for the Control group, and duplicate experiments (i.e., control-1 and Control-2 shown in Table 6) were set for the Control group.

Test materials: CHO-S recombinant cells integrated with the coding sequence of the anti-CTLA-4 monoclonal antibody 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 in water bath at 37.0 deg.C + -0.5 deg.C for 2min, thawing, transferring the cell suspension into a centrifuge tube containing 8.5ml Dynamis culture medium, centrifuging at 1000r/min for 5min, removing supernatant, resuspending the cells with Dynamis culture medium and transferring to 125ml shake flask, and supplementing to 30ml. Placing the shake flask at 36.0-37.0 deg.C, 5-7% of CO ₂ And culturing for 2-4 days under the condition of 120-140 r/min. The shaking flask amplification step is as follows: the cells are cultured in Dynamis medium at a ratio of 0.3-0.7X 10 ⁶ Diluting the individual/ml density into a new shake flask, the CO content is 5-7% at 36.0-37.0 deg.C ₂ And 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 10 ⁶ The 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; 3 days later, each group of cells were cultured with classical Batch at 0.8X 10 ⁶ Cell density inoculation of cells/ml, culture volume 30ml, at 36.5 ℃,6% ₂ 、200r/minCell culture under 80% humidity condition, 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 are shown for each group of basal media, 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 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 basic Medium for screening

Table 6: basic culture medium mixing design

Example 2: screening of fed-batch Medium

And (3) experimental design: the 10 th group obtained by screening in example 1 was used as a basic medium, and 4 feeding media Feed1, feed2, feed3 and Feed4 with different components were screened by mixing design, wherein the components of the 4 feeding media for screening are specifically shown in table 7, and the mixing design is shown in table 8. The Control medium was a combination of the basal medium Dynamis and the Feed medium Feed C +, and the Control was set up for repeat experiments (i.e., control-1 and Control-2 as shown in Table 8).

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

The test method comprises the following steps: the CHO-K1 recombinant cells were subjected to recovery and shake flask expansion culture as described in example 1, followed by inoculation of the cells into the preferred basal medium selected in example 1 for 3 days of subculture adaptation, followed by 0.8X 10 ⁶ The 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 carried out on Asn, namely a low level (2252 mg/L, called as Asn (-1) group), a high level (6475 mg/L, called as Asn (1) group) and a central point (4363 mg/L, called as Asn (0) group), as shown in figure 7, the increase of the concentration of Asn is beneficial to the growth of cells at the early stage of batch culture, but the cell density, the activity rate and the protein expression quantity are obviously reduced at the later stage of batch culture along with the increase of the concentration of Asn, and the Asn has obvious influence on the batch culture, so that the concentration confirmation experiment is carried out on Asn, the low level, the high level and the central point of the DOE screening experiment are respectively selected to carry out batch culture single factor confirmation again, and the result shows that the low level, the high level and the central point are consistent with the DOE result. 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 4363mg/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 and cytosine are necessary for the synthesis of DNA, which is a nucleic acid precursor. 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 (uradine), 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 10 ⁶ Cells/ml were seeded in 250ml baffled shake flasks with 80ml volume. The parameters of shaking table culture are as follows: 6% of CO ₂ Sampling at 36.5 deg.C, 130r/min, 3, 5, 7, 9, 11, 14 days to test viable cell density and cellsThe activity rate is monitored by a biochemical analyzer, and the glucose content is monitored when the glucose concentration is high<Increasing the final concentration of glucose to 6g/L when the concentration is 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 variety of the trace elements is various, and a proper application range is determined by performing gradient screening on 3 combined 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 1X 10, TE 20X and TE 40X are added into a basic culture medium, TE 1X 40X 80X and TE 160X are added into a corresponding fed-batch culture medium (namely, the addition amount of trace elements in the fed-batch culture medium is 4 times of that of the trace elements in the basic culture medium), TE2 and TE3 are combined by analogy, and a control group is a group without the addition of the trace elements.

Table 10: trace element component

And (3) test results: as shown in fig. 14 to 16, the difference of the effect of the combination of 3 trace elements on cell growth is significant, as the TE1 concentration increases, the viable cell density and the cell viability decrease, and the protein expression level is also negatively correlated with the TE1 concentration; the different concentrations of TE2 have no significant influence on cell growth and protein expression; TE3 significantly affects cell growth and protein expression, and the combined trace elements are toxic to cells and are not suitable for being used in subsequent Fed-Batch.

According to the comprehensive evaluation of indexes such as living cell density, cell viability maintenance and protein expression, TE2 Xtrace elements are finally added into the basal medium, TE2 Xtrace elements are finally determined to be added into the fed-batch medium 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 1X is TE 1X added into the basal medium, TE 1X is added into 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 expression quantity of a target product can be obviously improved by the tyrosine with the content, and the adding time of the tyrosine can be consistent with that of the fed-batch culture medium in batch culture.

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-DG 44) on the market are researched, and the wide applicability and stability of the culture medium subjected to component optimization provided by the invention are verified.

The culture medium provided by the invention is applied to the existing CHO platformVerification of Primary passage stability Performance in the cells of interest CHO-S (comprising therein the gene sequence encoding an anti-OX 40 monoclonal antibody), CHO-K1 (comprising therein the gene sequence encoding an anti-CD 20 monoclonal antibody), CHO-GS (comprising therein the gene sequence encoding an anti-TIGIT monoclonal antibody) and CHO-DG44 (comprising therein the gene sequence encoding an anti-TNF α monoclonal antibody): the cells were cultured in a medium of 0.4X 10 ⁶ Diluting the cells/ml density to a new shake flask, placing at 36.5 degrees, 6% CO ₂ One generation was grown every 3 days in a carbon dioxide shaker at 130 rpm. As shown in FIGS. 20-22, the doubling time of each cell line was 18-35 hours, the viable cell density was stably increased in different cell generations, the cell viability was maintained at 99% or more, and the cell lines of each platform item were 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 between 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 with the optimal content of each component in the table 1.

(2) According to the initial inoculation density (1.0 +/-0.3) multiplied by 10 ⁶ Inoculating the recombinant CHO cells in the step (1) into a basic culture with the optimal content of each component in the table 1, controlling DO to be 20-80%, and controlling pH to be 6.80E7.20. Controlling the culture temperature at 36.0-37.0 ℃ on the 1 st-4 th days of culture, and reducing the culture temperature to 32.0-34.0 ℃ from the 5 th day of culture.

(3) After culturing for 3 rd, 5 th, 7 th, 9 th and 11 th days, feeding 4-5% (w/w) of feeding medium with optimal content of each component as stated in Table 2 based on the initial culture weight, and adding tyrosine concentrated solution with final concentration of 0.8-1.6 g/L while feeding the feeding 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 figures 23-30 in particular), and can reach higher living cell density (can reach (15-30) multiplied by 10 in particular) ⁶ Viable cell density per ml), and the cell viability rate is maintained better, 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,

<xnotran> 60mg/L L- , 1300mg/L L- , 1450mg/L L- · , 800mg/L L- , 35mg/L L- · · , 125mg/L L- · , 180mg/L L- , 35mg/L , 255mg/L L- , 850mg/L L- , 1600mg/L L- , 850mg/L L- · , 180mg/L L- , 455mg/L L- , 1250mg/L L- ,1000 mg/L L- , 550mg/L L- , 150mg/L L- , 250mg/L L- , 450mg/L L- , 0.15mg/L D- , 8mg/L D- , 250mg/L , 4.5mg/L , 210mg/L , 3.5mg/L , 2.5mg/L · , 0.2mg/L , 3mg/L · , 3.5mg/L B12, 110mg/L · ,100 mg/L ,1000 mg/L F68, 6000mg/L D- , 25mg/L , 1500mg/L , 255mg/L , </xnotran> 125mg/L calcium chloride, 550mg/L potassium chloride, 4mg/L zinc sulfate heptahydrate, 0.4mg/L copper sulfate pentahydrate, 35mg/L ferric ammonium citrate, 0.015mg/L sodium selenite, 2000mg/L sodium bicarbonate, 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/L cadmium chloride hydrate 5/2, 0.0055mg/L cobalt chloride hexahydrate, 0.00155 mg/L chromium chloride, 0.012mg/L sodium chloride, 0.0030.007 mg/L sodium chloride octahydrate, 0.0035mg/L zirconium iodide, 0.00115 mg/L rubidium chloride octahydrate, 0.007mg/L zirconium iodide octahydrate, 0.0015mg/L zirconium oxide, 0.0015/L zirconium oxide hexahydrate, 0.0015mg/L rubidium chloride octahydrate, 0.007;

<xnotran> 5500mg/L L- , 6475mg/L L- · , 5550mg/L L- , 110mg/L L- · · , 270mg/L L- · , 5050mg/L L- , 2250mg/L L- , 5250mg/L L- , 8150mg/L L- , 7500mg/L L- · , 650mg/L L- , 3500mg/L L- , 3500mg/L L- , 10500mg/L L- , 2500mg/L L- , 2100mg/L L- , 280mg/L L- , 5500mg/L L- , 350mg/L D- , 400mg/L , 60mg/L , 510mg/L , 45mg/L , 15mg/L · , 4.5mg/L , 25mg/L · , 16mg/L B12, 150mg/L · , 200mg/L , 2000mg/L F68, 55000mg/L D- , 3000mg/L , 2050mg/L , 4500mg/L , 30mg/L , 2.5mg/L , 200mg/L , </xnotran> 0.009mg/L sodium selenite, 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, 0.028mg/L zirconium chloride octahydrate.

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

(2) According to the initial inoculation density (1.0 +/-0.3) multiplied by 10 ⁶ Inoculating the recombinant CHO cells in the step (1) into the basic culture medium according to the claim 1, wherein DO is controlled to be 20-80%, and pH is controlled to be 6.80-7.20;

(3) Culturing for 3, 5, 7, 9 and 11 days, and respectively feeding 4-5% (w/w) of the initial culture weight of the fed-batch culture medium as claimed in claim 1.

3. The method of culturing recombinant CHO cells according to claim 2, 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 deg.C, inoculating into a shake flask containing the basic culture medium of claim 1, controlling the culture temperature to 36.0-37.0 deg.C, and the CO content to 5-7% ₂ 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 10 ⁶ Carrying 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 10 ⁶ Inoculating to the next stage when the seed/ml is required;

4. The method of culturing recombinant CHO cells according to claim 3, wherein the cell viability is not less than 85% during the seed recovery phase and not less than 90% during the shake flask expansion phase.

5. The method of culturing recombinant CHO cells according to any one of claims 2 to 4, characterized in that said recombinant CHO cells are CHO cells comprising a gene encoding a foreign protein.

6. The method of culturing recombinant CHO cells according to claim 5, wherein said CHO cells are CHO-S, CHO-K1, CHO-GS or CHO-DG44 and said foreign protein is an antibody.

7. The method of culturing recombinant CHO cells according to claim 6, wherein said foreign protein is a monoclonal antibody or a bispecific antibody.

8. Use of the culture medium for CHO cells according to claim 1 for expressing a foreign protein in CHO cells.

9. The use of claim 8, wherein the CHO cell is CHO-S, CHO-K1, CHO-GS, or CHO-DG44 and the exogenous protein is an antibody.

10. The use of claim 9, wherein the foreign protein is a monoclonal antibody or a bispecific antibody.