CN119020257A - A perfusion culture medium for mammalian CHO cells and its use - Google Patents

Abstract

The invention provides a perfusion culture medium for mammalian CHO cells and application thereof, and belongs to the technical field of cell culture. The perfusion culture medium provided by the invention comprises metal compounds, saccharides, vitamins, amino acids, nucleoside/nucleoside compounds, fatty acids and other components. The perfusion culture medium provided by the invention can support the culture of cells of different subtypes, is beneficial to the production and subculture of CHO cells, ensures that the cells can maintain a good activity rate in a fermentation period, and is beneficial to the efficient expression of protein products; the produced substances are high in purity and less in charge variation, which is beneficial to the development of downstream plates of the biopharmaceuticals and saves the production cost of protein products; the culture medium is simple to prepare, and is favorable for commercial scale-up production.

Description

Perfusion culture medium for mammalian CHO cells and application thereof

Technical Field

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

Background

Mammalian cell culture is an upstream ring node of biopharmaceuticals, and is indispensable in the fields of antibody production, vaccine development and the like, animal cells can be classified into suspension cells and adherent cells according to their characteristics, and in terms of suspension cells, the current culture process is mainly classified into a conventional fed-batch culture process and a perfusion (or so-called continuous flow) process.

Compared with the traditional fed-batch culture, the perfusion culture has the main advantages of novel culture technology, high expression level, small equipment occupation area, high total yield and the like, and can promote efficiency in the era of vigorous competition and is favored in the pharmaceutical industry in recent years. However, because the perfusion culture period is long and the general culture period is more than 20 days, it is important to develop a perfusion culture medium which can maintain a certain cell number, activity rate and productivity and can ensure the product quality.

The perfusion medium development on the market at present has the following problems: the main method is that a basic culture medium and a feed supplement culture medium used in the traditional fed-batch process are mixed according to a certain proportion, the mixed culture medium is used for perfusion culture, wherein the basic culture medium and the feed supplement culture medium are required to be prepared respectively, the operation is complex, and the production scale amplification culture is not facilitated. And lack the innovativeness of development by mixing commercial media in proportion. For example, in the patent application publication No. CN112592948A, the basal medium and the feed medium need to be mixed according to different volume ratios (80-90) at different stages of cultivation: (10-20) perfusion culture. In addition, patrick Mayrhofer and DAVID REINHART design DOE (design-of-experiment) based on commercial medium and feed supplement, and medium mixing ratio was selected as perfusion medium based on DOE results (Mayrhofer P,Reinhart D,Castan A,Kunert R.Rapid development of clone-specific,high-performing perfusion media from established feed supplements.Biotechnol Prog.2020Mar;36(2):e2933.).

Meanwhile, the high cost of the commercialized culture medium and the low yield of the biological product determine the high cost of the cell culture, which ultimately results in high cost of the commercialized production and high price of the biological product. In addition, the longer production period of perfusion culture has higher challenges for the quality stability of the product in the production period. Therefore, a culture medium which is low in cost, simple to operate, high in yield and stable in quality in the production period is developed, the overall price can be reduced, the product quality is ensured, and the culture medium is beneficial to human beings.

Disclosure of Invention

Problems to be solved by the invention

Aiming at the problems existing in the prior art, for example, a common development method is to mix a basic culture medium and a feed supplement culture medium used in fed-batch culture according to a certain proportion to be used as a culture medium for perfusion, and the development process lacks exploratory property; in practical use, the preparation is complex and is not beneficial to the enlarged production. The invention provides a culture medium for mammalian CHO cell culture and application thereof.

Solution for solving the problem

In view of the above problems of the prior art, the inventors have conducted intensive studies to develop a culture medium suitable for perfusion culture, which is simple to prepare and has clear chemical composition, based on a basal medium used for fed-batch culture, by optimizing the composition.

The technical scheme of the invention is as follows:

[1] a medium for CHO cell culture, wherein the medium for CHO cell culture is a chemically defined medium;

the culture medium comprises metal compounds, saccharides, vitamins, amino acids, nucleoside/nucleoside compounds, fatty acids and other components;

wherein the metal compound comprises: aluminum trichloride hexahydrate, ammonium metavanadate, barium acetate, calcium chloride, chromium (III) chloride hexahydrate, cobalt (II) chloride hexahydrate, copper sulfate pentahydrate, cyanocobalamine, manganese sulfate monohydrate, nickel (II) sulfate hexahydrate, potassium bromide, potassium chloride, potassium iodide, rubidium chloride, sodium fluoride, sodium metasilicate nonahydrate, sodium phosphate monobasic, sodium pyruvate, tin (II) chloride dihydrate, zinc sulfate heptahydrate, zirconium oxychloride octahydrate, and ferric ammonium citrate;

The saccharide comprises glucose; the vitamins include: folic acid, nicotinamide, vitamin C, inositol, pyridoxine hydrochloride, riboflavin, and alpha-tocopheryl acetate;

The amino acids include: l-arginine, L-asparagine monohydrate, L-aspartic acid, L-cysteine Shan Yan acid salt monohydrate, L-cystine dihydrochloride, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine monohydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt, and L-valine;

the nucleoside/nucleoside compounds include thymidine, hypoxanthine monosodium salt, cytosine, guanine, thymine, adenine, deoxycytidine 5' -monophosphate, 5' -monophosphate adenosine monohydrate and 2' -deoxyguanosine monohydrate;

The fatty acid comprises: arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid;

the other components include: choline chloride, dextran sulfate sodium salt, ethanolamine hydrochloride and 1, 4-butanediamine dihydrochloride.

[2] The medium for CHO cell culture according to [1], wherein the metal compound contained in the medium comprises: 0.02-0.08mg/L of aluminum trichloride hexahydrate, 0.0005-0.0045mg/L of ammonium metavanadate, 0.05-0.15mg/L of barium acetate, 500-1500mg/L of calcium chloride, 0.002-0.006mg/L of chromium (III) chloride hexahydrate, 0.05-1.05mg/L of cobalt (II) chloride hexahydrate, 0.5-1.5mg/L of copper sulfate pentahydrate, 10.00-20.00mg/L of cyanocobalamine, 0.025-0.08mg/L of manganese sulfate monohydrate, 0.005-0.02mg/L of nickel (II) sulfate hexahydrate, 0.003-0.02mg/L of potassium bromide, 2000-4500mg/L of potassium chloride, 0.053-1.68mg/L of potassium iodide, 0.08-0.85mg/L of rubidium chloride, 3000-1.5 mg/L of sodium chloride, 0.025-0.08mg/L of sodium chloride, 0.1200-0.9 mg/L of sodium hydroxide, 0.05-0.25 mg/L of sodium hydroxide, 0.25 mg-0.25 mg/L of sodium hydroxide, 0.05-0.25 mg/L of sodium hydroxide sulfate, 0.05-0.02 mg/L of sodium hydroxide;

Preferably comprises: 0.03-0.07mg/L aluminum trichloride hexahydrate, 0.001-0.003mg/L ammonium metavanadate, 0.08-0.12mg/L barium acetate, 700-1300mg/L calcium chloride, 0.0035-0.0045mg/L chromium (III) chloride hexahydrate, 0.3-0.7mg/L cobalt (II) chloride hexahydrate, 0.35-1.25mg/L copper sulfate pentahydrate, 12.00-17.00mg/L cyanocobalamine, 0.03-0.075mg/L manganese sulfate monohydrate, 0.008-0.175mg/L nickel (II) sulfate hexahydrate, 0.008-0.012mg/L potassium bromide 2300-4000mg/L potassium chloride, 0.082-1.23mg/L potassium iodide, 0.30-0.75mg/L rubidium chloride, 4500-6400mg/L sodium chloride, 0.05-0.11mg/L sodium fluoride, 4.0-8.5mg/L sodium metasilicate nonahydrate, 200-500mg/L monobasic sodium phosphate, 450-1000mg/L sodium pyruvate, 0.0006-0.0016mg/L tin (II) chloride dihydrate, 35.0-60.0mg/L zinc sulfate heptahydrate, 0.10-0.25mg/L zirconium oxychloride octahydrate, and 20-60mg/L ferric ammonium citrate;

More preferably, the method comprises: 0.05mg/L of aluminum trichloride hexahydrate, 0.002mg/L of ammonium metavanadate, 0.1mg/L of barium acetate, 1000mg/L of calcium chloride, 0.005mg/L of chromium (III) chloride hexahydrate, 0.5mg/L of cobalt (II) chloride hexahydrate, 1mg/L of copper sulfate pentahydrate, 15.00mg/L of cyanocobalamine, 0.05mg/L of manganese sulfate monohydrate, 0.01mg/L of nickel (II) sulfate hexahydrate, 0.01mg/L of potassium bromide, 3000mg/L of potassium chloride, 1mg/L of potassium iodide, 0.5mg/L of rubidium chloride, 5900mg/L of sodium chloride, 0.1mg/L of sodium fluoride, 5.6mg/L of sodium metahydrate, 350mg/L of sodium phosphate monobasic, 800mg/L of sodium pyruvate, 0.001mg/L of tin (II) chloride dihydrate, 50.2mg/L of zinc sulfate dihydrate, and 0.50 mg/L of zinc sulfate octa.

[3] The medium for CHO cell culture according to [1] or [2], wherein the vitamins contained in the medium comprise: 20-70mg/L folic acid, 15-80mg/L nicotinamide, 0.05-1.20mg/L vitamin C, 100-170mg/L inositol, 3.0-25.0mg/L pyridoxine hydrochloride, 0.5-5.6mg/L riboflavin, and 0.025-0.095mg/L alpha-tocopheryl acetate;

Preferably comprises: 20-50mg/L folic acid, 30-75mg/L nicotinamide, 0.5-1.0mg/L vitamin C, 120-160mg/L inositol, 8.0-12.0mg/L pyridoxine hydrochloride, 1.25-3.50mg/L riboflavin, and 0.04-0.07mg/L alpha-tocopheryl acetate;

More preferably, the method comprises: 30mg/L folic acid, 50mg/L nicotinamide, 0.8mg/L vitamin C, 150mg/L inositol, 19mg/L pyridoxine hydrochloride, 2mg/L riboflavin, and 0.056mg/L alpha-tocopheryl acetate.

[4] The medium for CHO cell culture according to any one of [1] to [3], wherein the amino acids contained in the medium comprise: 500-1000 mg/L-arginine, 900-2500 mg/L-asparagine monohydrate, 500-1500 mg/L-aspartic acid, 50.0-500 mg/L-cysteine Shan Yan acid salt monohydrate, 100-600 mg/L-cystine dihydrochloride, 200-1200 mg/L-glutamic acid, 500-1500 mg/L-histidine, 500-1500 mg/L-isoleucine, 500-1800 mg/L-leucine, 800-2000 mg/L-lysine monohydrochloride, 100-700 mg/L-methionine, 200-500 mg/L-phenylalanine, 150-1000 mg/L-proline, 500-1500 mg/L-serine, 400-1200 mg/L-threonine, 200-1000 mg/L-tryptophan, 225-1350 mg/L-disodium salt and 100-300 mg/L-valine;

Preferably comprises 500-800 mg/L-arginine, 1000-2000 mg/L-asparagine monohydrate, 700-1000 mg/L-aspartic acid, 100-250 mg/L-cysteine Shan Yan acid salt monohydrate, 100-400 mg/L-cystine dihydrochloride, 400-1000 mg/L-glutamic acid, 700-1100 mg/L-histidine, 600-1300 mg/L-isoleucine, 900-1600 mg/L-leucine, 1000-1600 mg/L-lysine monohydrochloride, 300-600 mg/L-methionine, 250-400 mg/L-phenylalanine, 300-900 mg/L-proline, 700-1000 mg/L-serine, 650-900 mg/L-threonine, 400-950 mg/L-tryptophan, 500-900 mg/L-disodium salt and 150-275 mg/L-valine;

More preferably, the pharmaceutical composition comprises 600 mg/L-arginine, 1800 mg/L-asparagine monohydrate, 800 mg/L-aspartic acid, 200mg/L of L-cysteine Shan Yan acid hydrochloride monohydrate, 300mg/L of L-cystine dihydrochloride, 500 mg/L-glutamic acid, 800 mg/L-histidine, 700mg/L of L-isoleucine 1500 mg/L-leucine, 1500 mg/L-lysine monohydrochloride, 500 mg/L-methionine, 300 mg/L-phenylalanine, 600 mg/L-proline 800mg/L of L-serine, 800mg/L of L-threonine, 500mg/L of L-tryptophan, 700mg/L of L-tyrosine disodium salt and 200mg/L of L-valine.

[5] The medium for CHO cell culture according to any one of [1] to [4], wherein the nucleoside/nucleoside compound contained in the medium comprises: 0.05-0.75mg/L thymidine, 5.00-11.0mg/L hypoxanthine monosodium salt, 1.25-9.5mg/L cytosine, 2.5-9.0mg/L guanine, 1.3-8.0mg/L thymine, 0.5-8.5mg/L adenine, 1.0-12mg/L deoxycytidine 5' -monophosphate, 1.5-6.5 mg/L5 ' -monophosphate adenosine monohydrate, and 2.00-8.00 mg/L2 ' -deoxyguanosine monohydrate;

Preferably comprises 0.10-0.55mg/L of thymidine, 8.0-10.0mg/L of hypoxanthine monosodium salt, 3.5-5.5mg/L of cytosine, 3.0-4.5mg/L of guanine, 2.5-6.0mg/L of thymine, 1.0-6.0mg/L of adenine, 3.0-7.0mg/L of deoxycytidine 5' -monophosphate, 2.0-5.0mg/L of 5' -monophosphate adenosine monohydrate and 2.0-6.0mg/L of 2' -deoxyguanosine monohydrate;

More preferably, the composition comprises 0.2mg/L of thymidine, 10mg/L of hypoxanthine monosodium salt, 4mg/L of cytosine, 4mg/L of guanine, 4mg/L of thymine, 4mg/L of adenine, 4mg/L of deoxycytidine 5' -monophosphate, 4mg/L of 5' -monophosphate adenosine monohydrate and 4mg/L of 2' -deoxyguanosine monohydrate;

the fatty acids contained in the medium include: 0.0005-0.0030mg/L of arachidonic acid, 0.0020-0.10mg/L of linoleic acid, 0.0035-0.016mg/L of linolenic acid, 0.0028-0.035mg/L of myristic acid, 0.0045-0.018mg/L of oleic acid, 0.0045-0.020mg/L of palmitic acid and 0.005-0.012mg/L of stearic acid;

Preferably comprises 0.0010-0.0020mg/L of arachidonic acid, 0.0035-0.10mg/L of linoleic acid, 0.0055-0.009mg/L of linolenic acid, 0.004-0.01mg/L of myristic acid, 0.0065-0.010mg/L of oleic acid, 0.005-0.015mg/L of palmitic acid and 0.006-0.0012mg/L of stearic acid;

More preferably comprises 0.0016mg/L arachidonic acid, 0.008mg/L linoleic acid, 0.008mg/L linolenic acid, 0.008mg/L myristic acid, 0.008mg/L oleic acid, 0.008mg/L palmitic acid and 0.008mg/L stearic acid;

Other substances contained in the medium include: 100-300mg/L of choline chloride, 20-70mg/L of dextran sulfate sodium salt, 70-130mg/L of ethanolamine hydrochloride and 0.06-1.1mg/L of 1, 4-butanediamine dihydrochloride;

Preferably comprises 150-250mg/L choline chloride, 40-70mg/L dextran sulfate sodium salt, 80-110mg/L ethanolamine hydrochloride and 0.4-0.9 mg/L1, 4-butanediamine dihydrochloride;

More preferably comprises 200mg/L choline chloride, 60mg/L dextran sulfate sodium salt, 100mg/L ethanolamine hydrochloride and 0.5 mg/L1, 4-butanediamine dihydrochloride;

The saccharide contained in the medium comprises 5000-15000mg/L glucose, preferably 8000-11000mg/L glucose, more preferably 10000mg/L glucose.

[6] The medium for CHO cell culture according to any one of [1] to [5], wherein the medium further comprises a buffer substance and a shear resistant substance;

Preferably, the buffer substance comprises sodium bicarbonate and the shear resistant substance comprises PluronicF68;

Optionally, the sodium bicarbonate is contained in the medium in an amount of 1000-3700mg/L, preferably 1500-3500mg/L, more preferably 2000mg/L;

Optionally, the culture medium comprises PluronicF68 in an amount of 1250-3600mg/L, preferably 1600-2500mg/L, more preferably 2000mg/L.

[7] A method of culturing CHO cells, the method comprising the steps of:

(1) Cell resuscitating the CHO cells;

(2) Performing seed amplification culture on the cells recovered in the step (1);

(3) Culturing in a first stage: inoculating CHO cells obtained by seed amplification culture in the step (2) into a seed culture medium for culture according to the initial inoculation density (1.1+/-0.3) multiplied by 10 ⁶/mL, wherein the temperature is 36.5+/-0.5 ℃, the stirring speed is 180-220 r/min, starting perfusion culture with the seed culture medium for the next day of culture, culturing until the viable cell density reaches more than 20.0×10 ⁶/mL, and performing second-stage culture;

(4) Culturing in the second stage: perfusion culturing with the culture medium of any one of [ 1-6 ], cooling to 33-35 ℃ when the density of living cells is more than 60.0X10 ⁶/mL, adjusting the rotating speed to 230-280 r/min, and maintaining the density of living cells to 100.0X10 ⁶/mL;

Preferably, the seed medium is a CD CHO medium;

optionally, dissolved oxygen is controlled to be 20% -80%, preferably 50% in the culture process; the pH value is controlled to be 7.00+/-0.35;

alternatively, the first stage incubation time is 4 to 6 days in total.

[8] The method for culturing CHO cells according to [7], wherein,

The specific operation of cell resuscitation in the step (1) is as follows: resuscitating the seed cells from the refrigerator at-80 ℃, inoculating the seed cells into a CD CHO culture medium, and culturing for 2-4 days at 36.5+/-0.5 ℃ and 6.0+/-1.0% CO ₂ and 130+/-10 r/min;

The specific operation of the seed amplification culture in the step (2) is as follows: inoculating the resuscitated cells obtained in the step (1) into a shake flask containing a CD CHO culture medium according to the density of 0.3-0.7X10- ⁶/mL, culturing for 2-4 days at 36.5+/-0.5 ℃ and 6.0+/-1.0% CO ₂ and 130+/-10 r/min, and expanding and culturing seed cells step by step in the shake flask of 250-2000 mL according to the step;

In the step (3), the next day of culture starts to irrigate culture with a seed culture medium at a rate of 0.5VVD, and then the irrigated rate is increased by 0.5VVD to not more than 2.0VVD every day;

In the step (4), the medium of any one of [1] to [6] is used for perfusion culture at a rate of 0.5 VVD; when the glucose concentration in the reaction system is lower than 2.00g/L, glucose is fed in a flowing way so as to maintain the glucose concentration in the reaction system not lower than 2.00g/L, and when the glucose concentration is higher than 3.00g/L, the feeding amount of the glucose is gradually reduced until the feeding is stopped; alternatively, when the glucose concentration is lower than 2.00g/L, the fed-batch amount of glucose is sequentially increased in units of 1.00 g/L/day to a concentration no longer lower than 2.00g/L; alternatively, when the glucose concentration is more than 3.00g/L, the fed-batch amount of glucose is sequentially decreased in units of 1.00 g/L/day.

[9] The method for culturing a CHO cell according to [7] or [8], wherein the CHO cell is a CHO cell containing a foreign protein encoding; preferably, the CHO cell is CHO-K1, CHO-S, CHO-K1SV, 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] The use of the medium for CHO cell culture of any one of [1] to [6] for CHO cell expression of a foreign protein; preferably, the CHO cell is CHO-K1, CHO-S, CHO-K1SV, 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 has definite chemical components and simple preparation, is beneficial to production and subculture of CHO cells, is beneficial to efficient expression and commercial large-scale production of protein products, and saves the production cost of the protein products.

(2) The culture medium provided by the invention is suitable for culturing CHO cell line cells, such as CHO-K1, CHO-S, CHO-K1SV and the like, and has wide application range and high universality.

(3) The culture medium provided by the invention can maintain a good cell activity rate and has a high expression capacity in a fermentation period in an actual perfusion culture process, is simple to operate, meets the period requirement of perfusion culture, and also meets the high productivity requirement of the current biological industry, wherein the maintenance of the high cell activity rate and the simple preparation of the culture medium are more beneficial to commercial scale-up production.

(4) The culture medium provided by the invention ensures the quality of cultured biological products, the metabolism of cells and the quality of products are stable in the perfusion fermentation period, the produced substances are high in purity and less in charge variation, the development of downstream plates of biopharmaceuticals is facilitated, and meanwhile, the cost is saved to a certain extent.

Drawings

FIG. 1 shows a graph of the density of living cells in the experimental and control groups over time in the examples.

FIG. 2 shows a graph of cell viability over time in the experimental and control groups of the examples.

Fig. 3 shows a graph of glucose concentration over time in the experimental and control groups in the examples.

Fig. 4 shows a graph of NH ₄ ⁺ concentration versus time in the experimental and control groups in the examples.

Fig. 5 shows a graph of cumulative yield over time in experimental and control groups in the examples.

Fig. 6 shows a graph of the specific productivity over time in the experimental group and the control group in the examples.

Fig. 7 shows the main component ratios measured by the size exclusion chromatography in the experimental group and the control group in the examples.

FIG. 8 shows the principal component ratios measured by CE-SDS in the experimental group and the control group in the examples.

Fig. 9 shows the ratio of acid charge variant in the experimental group to the control group in the examples.

Detailed Description

Various exemplary embodiments, features and aspects of the invention are described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, well known methods, procedures, means, equipment and steps have not been described in detail so as not to obscure the present invention.

Definition of the definition

The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or specification may refer to "one" but may also refer to "one or more", "at least one" and "one or more".

As used in the claims and specification, the words "comprise," "have," "include" or "contain" mean including or open-ended, and do not exclude additional, unrecited elements or method steps.

As used herein, the term "about" means: one value includes the standard deviation of the error of the device or method used to determine the value. Illustratively, the foregoing standard deviation is generally within a range of 20-30% of the original value.

Although the disclosure supports the definition of the term "or" as being inclusive of alternatives and "and/or", the term "or" in the claims means "and/or" unless expressly indicated otherwise as being exclusive of each other, as defined by the alternatives or alternatives. In this specification, the term "and/or" when used to connect two or more selectable items is understood to mean any one of the selectable items or any two or more of the selectable items.

When used in the claims or specification, the term "numerical range" is intended to include both the numerical endpoints of the range and all natural numbers covered in the middle of the numerical endpoints relative to the numerical endpoints.

In the present invention, the term "CHO cell" refers to Chinese Hamster Ovary (CHO) cells, which are the most widely used mammalian cell expression system for recombinant drug protein production, and are also the hot spot for biotechnology and drug development research. The CHO cell culture process is very robust and enables large scale high level production of a variety of recombinant glycoproteins.

In the present invention, the term "seeding density" refers to the initial cell density of the seeding flask or bioreactor.

In the present invention, the term "cell culture" refers to a method of simulating in vitro an in vivo environment (sterility, proper temperature, pH, certain nutritional conditions, etc.) to survive, grow, reproduce and maintain the main structure and function.

In the present invention, the term "perfusion cell culture" or "perfusion culture" means that after cells and a culture medium are added together to a reactor, a part of a conditioned medium is continuously taken out during cell growth and product formation while new medium is continuously perfused.

In the present invention, the term "fed-batch culture" means that a specific fed-batch medium is continuously or intermittently fed into a reactor to supplement new nutrients according to the metabolic demand of cells during the culture.

In the present invention, the term "bioreactor" refers to a container for culturing cells, which is a device system for performing biochemical reactions in vitro using biological functions possessed by enzymes or organisms (e.g., microorganisms), and is a biological function simulator.

In the present invention, the term "dissolved oxygen" or "DO" is the percentage of dissolved oxygen present in a given liquid (such as a cell culture medium) on a saturated air basis.

In the present invention, the term "cell discard (cell bleeding)" means that a portion of the culture fluid is removed from the tank during the perfusion culture process, and the cell growth rate is determined to maintain high cell activity, preventing the blocking of the cell trapping module of the perfusion system.

In the present invention, the term "viable cell density" refers to the number of viable cells per unit area or volume, which is used to reflect the growth of cells.

In the present invention, the term "cell viability" refers to the percentage of viable cells in the total cell population.

In the present invention, the term "cumulative yield" refers to the total yield in one culture period.

In the present invention, the term "perfusion flow rate" is a key parameter of the process performance, which is generally defined as perfusion rate (daily tank volume, VVD, L/(l·day)) or cell-specific perfusion rate (CELL SPECIFIC perfusion rate, CSPR, nL/cell/day).

In the present invention, the term "VCD (viable CELL DENSITY)" or "viable cell density" refers to the number of viable cells contained in a volume of medium, and is expressed in units of "cells/mL" or "individual/mL". In the present invention, the term "VIB (Viability)" or "cell viability" means the ratio of the total number of living cells contained in a certain volume of medium to the total number of cells, expressed in units of "%".

In the present invention, the term "antibody expression level" refers to the total amount of antibody produced in a volume of cell culture, expressed in units of "g/L".

In the present invention, the term "specific productivity" means the calculation of growth rate and productivity in units of pick protein produced per cell per day, the calculation of which is shown below:

Specific productivity = antibody yield/cumulative viable cell density;

The "IVCD (integrated viable CELL DENSITY)" or "cumulative viable cell density" is calculated as follows:

Where t is the culture time (unit: day), VCD is the viable cell density (unit: cell/mL), VCD _t is the viable cell density at the t-th day of culture, V is the culture volume (unit: mL), V _t is the culture volume at the t-th day of culture, and V ₀ is the initial culture volume.

In the present invention, the term "size exclusion chromatography" is a method of analyzing a solute by utilizing the uniqueness of a porous gel stationary phase, mainly based on the relative relationship between the pore size of gel pores and the coil size of polymer sample molecules.

In the present invention, the term "CE-SDS" refers to a sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method for quantitatively determining the purity of a recombinant monoclonal antibody product by capillary electrophoresis under reducing and non-reducing conditions, depending on the molecular weight.

In the present invention, the higher the purity of the "main component", i.e., the target product, the more the target product is obtained.

The "charge variant" according to the present invention is called a charge variant because the protein product undergoes post-translational modification and degradation events in the cell, resulting in heterogeneity with biophysical properties, and a difference in charge.

As used herein, an "acidic charge variant" is a charge variant that is earlier or later than the main peak in the different assays. The formation of acidic charge variants is more likely due to the presence of modifications that affect the biological activity of the product, and therefore the level of acidic charge variants is generally used as a diagnostic indicator of the quality of the antibody product.

First aspect

In a first aspect the invention provides a medium for CHO cell culture comprising metal compounds, sugars, vitamins, amino acids, nucleoside/nucleoside compounds, fatty acids and other ingredients.

Specifically, the culture medium provided by the invention comprises a carbon source, a nitrogen source, amino acids, vitamins, metal compounds, nucleoside/nucleoside compounds, fatty acids, buffers, shearing-resistant substances, trace elements and other nutrients to promote the growth, maintenance and expression of products of CHO cells.

In the invention, the carbon source is taken as the most main energy source substance, provides needed energy for biological synthesis and a framework for synthesizing products, and different carbon source forms have different functions, and commonly used glucose, galactose, mannose, fucose, sodium pyruvate, maltose, isomaltose, fructose, sucrose, arabinose, fructo-oligosaccharide, xylo-oligosaccharide and the like. Nitrogen sources are the major tissue parts contributing to protein synthesis in organisms, and common nitrogen sources include various ammonium salts including sodium nitrate, potassium nitrate, magnesium nitrate, aluminum nitrate, zinc nitrate, iron nitrate, copper nitrate or manganese nitrate, nitrates, peptones, fish meal, and the like, and ammonium salts including ammonium chloride, ammonium sulfate or ammonium nitrate. In some specific embodiments, the carbon source comprises glucose, preferably D-glucose.

In the invention, amino acid is an important basis for cell growth and metabolism, is a basic unit for constituting protein, is an energy source and a signal molecule, and the amino acid with different component contents directly influences the growth and maintenance of cells and the expression of protein products, and even influences the quality attributes of the products; commonly used amino acids are classified into essential amino acids and non-essential amino acids, the essential amino acids being not synthesized by the cells themselves and must be supplied by means of a culture broth, the essential amino acids including L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-valine, wherein L-glutamine is an amino acid essential for the synthesis of nucleic acids and proteins by the cells; the amino acid requirements for different CHO cell types are also different. In some specific embodiments, the amino acid comprises L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-alanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. Optionally, the amino acid is added to the culture medium in the form of a monohydrate, a hydrochloride, and/or a sodium salt.

In the invention, vitamins provide a large amount of coenzyme factors for organisms and play an important role in the metabolism process; most vitamins are sensitive to intense light and heat and are easily oxidized. In some specific embodiments, the vitamin comprises at least one of folic acid, nicotinamide, vitamin C, inositol, pyridoxine hydrochloride, riboflavin, and alpha-tocopheryl acetate.

In the invention, metal ions are generated after the metal compounds in the culture medium are dissolved in water, and the metal ions can participate in cell division, thus having an inherent important effect on maintaining the cell structure and on lipoprotein membranes; the metal ions also directly affect the structure of the ribosome and thus the normal synthesis process of the protein. In some specific embodiments, the metal ion comprises an aluminum ion, an iron ion, a vanadium ion, a divalent cobalt ion, a trivalent cobalt ion, a barium ion, a calcium ion, a trivalent chromium ion, a copper ion, a potassium ion, a rubidium ion, a sodium ion, a stannous ion, a zinc ion, a zirconium ion, a ferric ion. In some specific embodiments, the metal compound comprises aluminum trichloride hexahydrate, ammonium metavanadate, barium acetate, calcium chloride, chromium (III) chloride hexahydrate, cobalt (II) chloride hexahydrate, copper sulfate pentahydrate, cyanocobalamine, manganese sulfate monohydrate, nickel (II) sulfate hexahydrate, potassium bromide, potassium chloride, potassium iodide, rubidium chloride, sodium fluoride, sodium metasilicate nonahydrate, sodium phosphate monobasic (i.e., naH ₂PO₄), sodium pyruvate, tin (II) chloride dihydrate, zinc sulfate heptahydrate, zirconium oxychloride octahydrate, and ferric ammonium citrate.

In the present invention, lipid substances are the main components of cell membranes, which provide both energy and signal pathways in cells, and fatty acids, including arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid, phospholipids (e.g., ethanolamine), cholesterol, and the like are commonly used.

In the invention, the nucleoside/nucleoside compound is used as a raw material for cell division and DNA replication, so that the DNA replication of the cells is smoothly carried out, and the normal division of the cells is ensured. Illustratively, the nucleoside/nucleoside compounds include thymidine, hypoxanthine monosodium salt, cytosine, guanine, thymine, adenine, deoxycytidine 5' -monophosphate, 5' -monophosphate adenylmonohydrate, and 2' -deoxyguanylmonohydrate.

Further, the culture medium also contains other components, including energy substances, hormones and serum substitutes, which play an important role in maintaining normal cell growth and expression. In some specific embodiments, the other ingredients include choline chloride, dextran sulfate sodium salt, ethanolamine hydrochloride, and 1, 4-butanediamine dihydrochloride.

In the invention, the buffer substances in the culture medium can regulate the permeability of cell membranes, maintain the normal osmotic pressure and acid-base balance inside and outside the cells, and promote the growth of the cells. In some embodiments of the invention, the buffer substance comprises sodium bicarbonate.

In the present invention, the shearing resistant material in the culture medium, including nonionic surfactant and the like, can protect cells from damage by shearing force applied during the culture. In some specific embodiments, the shear resistant material is PluronicF68.

Second aspect

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

Cell resuscitation is carried out on CHO cells, and the specific operation is as follows: the seed cells are taken out from a refrigerator at the temperature of minus 80 ℃ for resuscitation, inoculated into a CD CHO culture medium and cultured for 2 to 4 days under the conditions of 36.5+/-0.5 ℃ and 6.0+/-1.0 percent CO ₂ and 130+/-10 r/min.

Further, the cells recovered in the steps are subjected to seed expansion culture, and the specific operation is as follows: the resuscitated cells obtained in the above steps are inoculated into shake flasks containing CD CHO medium according to a density of 0.3-0.7X10- ⁶/mL, cultured for 2-4 days at 36.5+ -0.5 ℃ and 6.0+ -1.0% CO ₂ and 130+ -10 r/min, and subjected to gradual expansion culture until the number of cells meets the perfusion inoculation requirement of a 2L reactor, and the cell concentration can reach 0.8-1.4X10 ⁶/mL in a reactor with a working volume of 1.0-1.3L, preferably 1.1X10 ⁶/mL in a reactor with a working volume of 1.1L. In some specific embodiments, the step-wise expansion culture is an expansion culture of seed cells in shake flasks of 250mL, 500mL, 1000mL, and 2000mL in sequence.

Further, after the seed expansion culture, the first stage culture is performed, specifically comprising the following steps: inoculating CHO cells obtained by seed amplification culture in the step (2) into a seed culture medium for culture according to the initial inoculation density (1.1+/-0.3) multiplied by 10 ⁶/mL, wherein the temperature is 36.5+/-0.5 ℃, the stirring speed is 180-220 r/min, starting perfusion culture with the seed culture medium for the next day of culture, culturing until the viable cell density reaches more than 20.0×10 ⁶/mL, and performing second-stage culture; in some embodiments, the next day of culture begins perfusion culture with seed medium at a rate of 0.5VVD, followed by daily increases in the rate of perfusion by 0.5VVD to no more than 2.0VVD (i.e., perfusion culture at a rate of 0.5VVD, 1.0VVD, 1.5VVD, 2.0VVD, respectively); in some alternative embodiments, the first stage incubation time is 4 to 6 days total, preferably 6 days.

Further, after the first-stage cultivation, the second-stage cultivation is performed, and the specific steps are as follows: perfusion culturing with the culture medium, cooling to 33-35 deg.c when the density of living cell is greater than 60.0X10- ⁶/mL, regulating rotation speed to 230-280 r/min and maintaining the density of living cell at 100.0X10- ⁶/mL; in some embodiments, the medium described above is used to perfuse culture at a rate of 0.5 VVD; when the glucose concentration in the reaction system is lower than 2.00g/L, glucose is fed in a flowing way so as to maintain the glucose concentration in the reaction system not lower than 2.00g/L, and when the glucose concentration is higher than 3.00g/L, the feeding amount of the glucose is gradually reduced until the feeding is stopped; in some alternative embodiments, when the in-tank glucose concentration is below 2.00g/L, starting glucose feeding with an initial glucose feeding of 1.00 g/L/day followed by increasing the glucose feeding in a gradient of 1.00 g/L/day until the in-tank glucose concentration is no longer below 2.00g/L; when the glucose concentration in the tank is more than 3.00g/L, the fed-batch amount of glucose is decreased in a gradient of 1.00 g/L/day.

In some preferred embodiments, the seed medium is CD CHO medium.

In some alternative embodiments, dissolved oxygen is controlled to be 20% to 80%, preferably 50% during the culturing process; the pH value is controlled to 7.00+/-0.35.

In some embodiments, the CHO cell is a CHO cell comprising a polypeptide encoding a heterologous protein; preferably, the CHO cell is CHO-K1, CHO-S, CHO-K1SV, 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.

Third aspect of the invention

The third aspect of the invention provides the application of the culture medium for CHO cell culture in expressing exogenous proteins in CHO cells.

In some preferred embodiments, the CHO cell is CHO-K1, CHO-S, CHO-K1SV, CHO-GS or CHO-DG44 and the foreign protein is an antibody.

In some more preferred embodiments, the exogenous protein is a monoclonal antibody or a bispecific antibody.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The key instrumentation, materials and consumables used in the examples of the present invention are shown in tables 1 and 2 below.

TABLE 1 Key instrumentation for experiments

Device name	Place of origin and brand
		Cell counter	Beckman Coulter in U.S
Full-automatic biochemical analyzer	Switzerland Roche
		High performance liquid chromatograph	Agilent in America
Biological safety cabinet	Suzhou Sujing Antai
		Carbon dioxide shaking table	Swiss Kuhner
Bioreactor	Netherlands Applikon
		My-control	Netherlands Applikon
Centrifugal machine	Hunan Xiang instrument
		Centrifugal machine	U.S. Thermo FISHER SCIENTIFIC
Pipe connecting machine	Terumo U.S.A
		Tube sealing machine	Germany Startorius

TABLE 2 materials for experiments and Critical consumables

Example 1: perfusion culture in 2L reactor

Experimental materials: the corresponding antibodies were expressed using a suspension cell line CHO-K1SV GS-KO cell line, which was acclimatized to CHO-K1 (ECACC) serum-free suspension, containing an electrotransfer plasmid and a gene sequence encoding an anti-VEGF monoclonal antibody (Bevacizumab).

Perfusion medium: according to the invention, through integral optimization, the optimal content and the better content of each component of the perfusion culture medium are obtained. The detailed formulation is shown in Table 3 below.

TABLE 3 Components and contents of perfusion Medium

The cell culture process mainly comprises 3 steps of cell resuscitating, seed amplifying and 2L reactor perfusion culture. CD CHO seed medium was used in both cell resuscitation and seed expansion stages, and perfusion medium with the optimal component content of the present invention or control commercial perfusion medium was used in 2L reactor perfusion culture stage.

The following set values of the cell culture process parameters are optimal parameter conditions, and each parameter condition has an acceptable range.

(1) Cell resuscitation: a frozen cell is taken out from the refrigerator at-80 ℃ and is quickly put in water bath for not more than 2min at 37.0+/-0.5 ℃. The cells after thawing in water bath were gently mixed, transferred to a centrifuge tube containing 8.5ml of CD CHO seed medium, centrifuged at 1000rpm for 5min, the supernatant was discarded, the pellet was gently suspended using about 5ml of CD CHO medium and transferred to a shake flask containing 25ml of CD CHO medium, and incubated in a shaker at 36.5℃with an acceptable range of 36.0-37.0 ℃), 6.0% CO ₂ (acceptable range of 5.0% -7.0% CO ₂), 130r/min (acceptable range of 120-140 r/min) for 3 days (acceptable range of 2-4 days).

(2) And (3) seed amplification: inoculating cells recovered in the step (1) into a new shake flask with a CD CHO seed medium at a density of 0.5X10 ⁶/ml (acceptable range is 0.3-0.7X10 ⁶/ml), and placing in a carbon dioxide shake flask under the following culture conditions: 36.5 ℃ (acceptable range is 36.0-37.0 ℃), 6.0% CO ₂ (acceptable range is 5.0% -7.0% CO ₂), 130r/min (acceptable range is 120-140 r/min) and 3 days (acceptable range is 2-4 days). And (3) carrying out seed amplification by using shake flasks with the specifications of 250ml, 500ml, 1000ml and 2000ml step by step, and gradually amplifying the amplification volume until the number of cells in the shake flasks meets the inoculation requirement of 2L reactor perfusion culture, wherein the inoculation requirement of 2L reactor perfusion culture is shown in the step (3).

(3) 2L reactor perfusion culture: the perfusion medium of the present invention and that purchased from Merck were used separatelyThe INV-PM perfusion culture medium is used for carrying out parallel experiments, and other materials and technological parameters are consistent except the perfusion culture medium. The perfusion culture process is specifically as follows:

Cells obtained by stepwise expansion in step (2) above were inoculated with CD CHO medium at a density of 1.1X10 ⁶ cells/ml (acceptable range 0.8-1.4X10 ⁶ cells/ml) into a 2L Applikon perfusion bioreactor controlled by my-control with a working volume of 1.1L. The culture medium used in the first stage is a CD CHO seed culture medium, the culture temperature is 36.5 ℃ (the acceptable range is 36.0-37.0 ℃), the initial setting value of the stirring speed is 200r/min, the pH value is set to 7.00+/-0.30, and the range is 6.65-7.35; dissolved oxygen was set to 50% > (acceptable range was 20% -80%). The 2 nd day after inoculation starts to culture with CD CHO seed medium perfused with 0.5VVD, then 0.5VVD to 2.0VVD is added daily for 6 days (acceptable range is 4-6 days). When the viable cell density reached 20.0X10 ⁶/ml, the second stage was entered and perfusion culture with 2.0VVD was started using perfusion medium. Setting the pH value to 7.00 plus or minus 0.30 and the range to 6.65-7.35; DO is 50% and the range is 20% -80%; the culture temperature is 36.5 ℃, the range is 36.0-37.0 ℃, and when the VCD exceeds 60 multiplied by 10 ⁶/ml, the temperature is reduced to 35 ℃. The rotation speed is regulated to 250r/min (230-280 r/min) according to the cell density. When the viable cell density is greater than 100.0X10 ⁶/ml, a cell discarding (cell bleeding) procedure is performed to maintain the viable cell density at 100.0X10 ⁶/ml.

And in the perfusion culture stage using the perfusion culture medium, glucose is supplemented according to the daily detection result of the glucose concentration, and when the glucose concentration is lower than 2.00g/L, the automatic feeding of glucose is started, and the feeding rate is 1.00 g/L/day. In the process of starting glucose feeding, when the glucose concentration is lower than 2.00g/L, the concentration is sequentially increased to be no lower than 2.00g/L by taking 1.00g/L as a unit; when the glucose concentration was higher than 3.00g/L, the glucose feeding was successively decreased to stop in units of 1.00 g/L/day. The defoamer is added according to the actual foam amount, and the total amount cannot exceed 200ppm.

One hour after inoculation was completed, samples were taken, and the time interval between sampling per day was controlled to 24 hours (acceptable range 20-28 hours). Cell density, activity, glucose concentration, lactic acid concentration, NH ₄ ⁺ concentration, osmotic pressure and other growth metabolism indexes are detected, protein yield is measured, specific productivity is calculated, and the proportion of main components in the protein is measured by using size exclusion chromatography and CE-SDS.

Experimental results: comprehensively evaluating the invention by monitoring the growth metabolism, the protein expression capacity and the quality index of the product of the cells, and particularly referring to fig. 1 to 9, wherein the solid line represents the performance of the perfusion medium of the invention; the dotted line is MerckThe performance of the INV-PM perfusion medium was defined as the control group.

Compared with the control medium:

As shown in FIG. 1, cells grow faster in the perfusion medium of the invention in the early stage, and the highest viable cell density is slightly higher, reaching 149.1X10 ⁶ cells/ml; the cell densities at the late stage of the culture tended to be uniform, and the cell density at the end of the culture (day 26) was 110.1X10 ⁶/ml, and the control was 104.5X10 ⁶/ml.

As shown in FIG. 2, in the perfusion medium of the present invention, the cell viability was maintained at a high level, and the cell viability at day 26 was 94.2% and the control was 93.0%.

In terms of glucose metabolism, the cells cultured in the medium of the present invention have high glucose utilization (fig. 3); the NH ₄ ⁺ level was lower than that of the control group throughout the culture period of the medium of the invention, and the cell culture microenvironment was relatively good (FIG. 4).

In terms of antibody expression ability (i.e., protein yield), the cell cultured in the medium of the present invention has high antibody expression ability (FIG. 5), the cumulative yield of the present invention is up to 101.35g/L, the cumulative yield of the control is 79.14g/L, and the total yield is increased by 28% compared with the Merck medium; and, the specific cell productivity of the cell antibody cultured in the medium of the present invention was higher than that of the control group (FIG. 6).

As shown in fig. 7 to 9, the quality was stable during the culture period, and the overall purity at different detection points was higher than Merck medium and the acidic charge variant was lower than Merck medium.

The comprehensive results show that the culture medium of the invention not only reduces the production cost, but also can maintain better cell growth capacity and activity rate and higher antibody expression capacity, and the quality of protein products is stable in the production period, and the products have high purity and fewer charge variants influencing the biological activity of the products.

It should be noted that, although the technical solution of the present invention is described in specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the technical improvements in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Claims (10)

1. A culture medium for CHO cell culture, wherein the culture medium for CHO cell culture is of defined chemical composition;

the culture medium comprises metal compounds, saccharides, vitamins, amino acids, nucleoside/nucleoside compounds, fatty acids and other components;

wherein the metal compound comprises: aluminum trichloride hexahydrate, ammonium metavanadate, barium acetate, calcium chloride, chromium (III) chloride hexahydrate, cobalt (II) chloride hexahydrate, copper sulfate pentahydrate, cyanocobalamine, manganese sulfate monohydrate, nickel (II) sulfate hexahydrate, potassium bromide, potassium chloride, potassium iodide, rubidium chloride, sodium fluoride, sodium metasilicate nonahydrate, sodium phosphate monobasic, sodium pyruvate, tin (II) chloride dihydrate, zinc sulfate heptahydrate, zirconium oxychloride octahydrate, and ferric ammonium citrate;

The saccharide comprises glucose; the vitamins include: folic acid, nicotinamide, vitamin C, inositol, pyridoxine hydrochloride, riboflavin, and alpha-tocopheryl acetate;

The amino acids include: l-arginine, L-asparagine monohydrate, L-aspartic acid, L-cysteine Shan Yan acid salt monohydrate, L-cystine dihydrochloride, L-glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine monohydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt, and L-valine;

The nucleoside/nucleoside compound includes: thymidine, hypoxanthine monosodium salt, cytosine, guanine, thymine, adenine, deoxycytidine 5' -monophosphate, 5' -monophosphate adenosine monohydrate and 2' -deoxyguanosine monohydrate;

The fatty acid comprises: arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid;

the other components include: choline chloride, dextran sulfate sodium salt, ethanolamine hydrochloride and 1, 4-butanediamine dihydrochloride.

2. The medium for CHO cell culture according to claim 1, wherein the metal compounds comprised in the medium comprise: 0.02-0.08mg/L of aluminum trichloride hexahydrate, 0.0005-0.0045mg/L of ammonium metavanadate, 0.05-0.15mg/L of barium acetate, 500-1500mg/L of calcium chloride, 0.002-0.006mg/L of chromium (III) chloride hexahydrate, 0.05-1.05mg/L of cobalt (II) chloride hexahydrate, 0.5-1.5mg/L of copper sulfate pentahydrate, 10.00-20.00mg/L of cyanocobalamine, 0.025-0.08mg/L of manganese sulfate monohydrate, 0.005-0.02mg/L of nickel (II) sulfate hexahydrate, 0.003-0.02mg/L of potassium bromide, 2000-4500mg/L of potassium chloride, 0.053-1.68mg/L of potassium iodide, 0.08-0.85mg/L of rubidium chloride, 3000-1.5 mg/L of sodium chloride, 0.025-0.08mg/L of sodium chloride, 0.1200-0.9 mg/L of sodium hydroxide, 0.05-0.25 mg/L of sodium hydroxide, 0.25 mg-0.25 mg/L of sodium hydroxide, 0.05-0.25 mg/L of sodium hydroxide sulfate, 0.05-0.02 mg/L of sodium hydroxide;

Preferably comprises: 0.03-0.07mg/L aluminum trichloride hexahydrate, 0.001-0.003mg/L ammonium metavanadate, 0.08-0.12mg/L barium acetate, 700-1300mg/L calcium chloride, 0.0035-0.0045mg/L chromium (III) chloride hexahydrate, 0.3-0.7mg/L cobalt (II) chloride hexahydrate, 0.35-1.25mg/L copper sulfate pentahydrate, 12.00-17.00mg/L cyanocobalamine, 0.03-0.075mg/L manganese sulfate monohydrate, 0.008-0.175mg/L nickel (II) sulfate hexahydrate, 0.008-0.012mg/L potassium bromide 2300-4000mg/L potassium chloride, 0.082-1.23mg/L potassium iodide, 0.30-0.75mg/L rubidium chloride, 4500-6400mg/L sodium chloride, 0.05-0.11mg/L sodium fluoride, 4.0-8.5mg/L sodium metasilicate nonahydrate, 200-500mg/L monobasic sodium phosphate, 450-1000mg/L sodium pyruvate, 0.0006-0.0016mg/L tin (II) chloride dihydrate, 35.0-60.0mg/L zinc sulfate heptahydrate, 0.10-0.25mg/L zirconium oxychloride octahydrate, and 20-60mg/L ferric ammonium citrate;

More preferably, the method comprises: 0.05mg/L of aluminum trichloride hexahydrate, 0.002mg/L of ammonium metavanadate, 0.1mg/L of barium acetate, 1000mg/L of calcium chloride, 0.005mg/L of chromium (III) chloride hexahydrate, 0.5mg/L of cobalt (II) chloride hexahydrate, 1mg/L of copper sulfate pentahydrate, 15.00mg/L of cyanocobalamine, 0.05mg/L of manganese sulfate monohydrate, 0.01mg/L of nickel (II) sulfate hexahydrate, 0.01mg/L of potassium bromide, 3000mg/L of potassium chloride, 1mg/L of potassium iodide, 0.5mg/L of rubidium chloride, 5900mg/L of sodium chloride, 0.1mg/L of sodium fluoride, 5.6mg/L of sodium metasilicate nonahydrate, 350mg/L of sodium phosphate monobasic, 800mg/L of sodium pyruvate, 0.001mg/L of tin (II) chloride dihydrate, 50mg/L of zinc sulfate dihydrate, 50mg/L of zinc oxide, and 50mg/L of zinc sulfate octa hydrate.

3. The medium for CHO cell culture according to claim 1 or 2, wherein the vitamins contained in the medium comprise: 20-70mg/L folic acid, 15-80mg/L nicotinamide, 0.05-1.20mg/L vitamin C, 100-170mg/L inositol, 3.0-25.0mg/L pyridoxine hydrochloride, 0.5-5.6mg/L riboflavin, and 0.025-0.095mg/L alpha-tocopheryl acetate;

Preferably comprises: 20-50mg/L folic acid, 30-75mg/L nicotinamide, 0.5-1.0mg/L vitamin C, 120-160mg/L inositol, 8.0-12.0mg/L pyridoxine hydrochloride, 1.25-3.50mg/L riboflavin, and 0.04-0.07mg/L alpha-tocopheryl acetate;

More preferably, the method comprises: 30mg/L folic acid, 50mg/L nicotinamide, 0.8mg/L vitamin C, 150mg/L inositol, 19mg/L pyridoxine hydrochloride, 2mg/L riboflavin, and 0.056mg/L alpha-tocopheryl acetate.

4. A medium for CHO cell culture according to any one of claims 1 to 3, comprising amino acids comprising: 500-1000 mg/L-arginine, 900-2500 mg/L-asparagine monohydrate, 500-1500 mg/L-aspartic acid, 50.0-500 mg/L-cysteine Shan Yan acid salt monohydrate, 100-600 mg/L-cystine dihydrochloride, 200-1200 mg/L-glutamic acid, 500-1500 mg/L-histidine, 500-1500 mg/L-isoleucine, 500-1800 mg/L-leucine, 800-2000 mg/L-lysine monohydrochloride, 100-700 mg/L-methionine, 200-500 mg/L-phenylalanine, 150-1000 mg/L-proline, 500-1500 mg/L-serine, 400-1200 mg/L-threonine, 200-1000 mg/L-tryptophan, 225-1350 mg/L-disodium salt and 100-300 mg/L-valine;

Preferably comprises 500-800 mg/L-arginine, 1000-2000 mg/L-asparagine monohydrate, 700-1000 mg/L-aspartic acid, 100-250 mg/L-cysteine Shan Yan acid salt monohydrate, 100-400 mg/L-cystine dihydrochloride, 400-1000 mg/L-glutamic acid, 700-1100 mg/L-histidine, 600-1300 mg/L-isoleucine, 900-1600 mg/L-leucine, 1000-1600 mg/L-lysine monohydrochloride, 300-600 mg/L-methionine, 250-400 mg/L-phenylalanine, 300-900 mg/L-proline, 700-1000 mg/L-serine, 650-900 mg/L-threonine, 400-950 mg/L-tryptophan, 500-900 mg/L-disodium salt and 150-275 mg/L-valine;

More preferably, the pharmaceutical composition comprises 600 mg/L-arginine, 1800 mg/L-asparagine monohydrate, 800 mg/L-aspartic acid, 200mg/L of L-cysteine Shan Yan acid hydrochloride monohydrate, 300mg/L of L-cystine dihydrochloride, 500 mg/L-glutamic acid, 800 mg/L-histidine, 700mg/L of L-isoleucine 1500 mg/L-leucine, 1500 mg/L-lysine monohydrochloride, 500 mg/L-methionine, 300 mg/L-phenylalanine, 600 mg/L-proline 800mg/L of L-serine, 800mg/L of L-threonine, 500mg/L of L-tryptophan, 700mg/L of L-tyrosine disodium salt and 200mg/L of L-valine.

5. The medium for CHO cell culture according to any one of claims 1 to 4, wherein the nucleoside/nucleoside compound comprised in the medium comprises: 0.05-0.75mg/L thymidine, 5.00-11.0mg/L hypoxanthine monosodium salt, 1.25-9.5mg/L cytosine, 2.5-9.0mg/L guanine, 1.3-8.0mg/L thymine, 0.5-8.5mg/L adenine, 1.0-12mg/L deoxycytidine 5' -monophosphate, 1.5-6.5 mg/L5 ' -monophosphate adenosine monohydrate, and 2.00-8.00 mg/L2 ' -deoxyguanosine monohydrate;

Preferably comprises 0.10-0.55mg/L of thymidine, 8.0-10.0mg/L of hypoxanthine monosodium salt, 3.5-5.5mg/L of cytosine, 3.0-4.5mg/L of guanine, 2.5-6.0mg/L of thymine, 1.0-6.0mg/L of adenine, 3.0-7.0mg/L of deoxycytidine 5' -monophosphate, 2.0-5.0mg/L of 5' -monophosphate adenosine monohydrate and 2.0-6.0mg/L of 2' -deoxyguanosine monohydrate;

More preferably, the composition comprises 0.2mg/L of thymidine, 10mg/L of hypoxanthine monosodium salt, 4mg/L of cytosine, 4mg/L of guanine, 4mg/L of thymine, 4mg/L of adenine, 4mg/L of deoxycytidine 5' -monophosphate, 4mg/L of 5' -monophosphate adenosine monohydrate and 4mg/L of 2' -deoxyguanosine monohydrate;

the fatty acids contained in the medium include: 0.0005-0.0030mg/L of arachidonic acid, 0.0020-0.10mg/L of linoleic acid, 0.0035-0.016mg/L of linolenic acid, 0.0028-0.035mg/L of myristic acid, 0.0045-0.018mg/L of oleic acid, 0.0045-0.020mg/L of palmitic acid and 0.005-0.012mg/L of stearic acid;

Preferably comprises 0.0010-0.0020mg/L of arachidonic acid, 0.0035-0.10mg/L of linoleic acid, 0.0055-0.009mg/L of linolenic acid, 0.004-0.01mg/L of myristic acid, 0.0065-0.010mg/L of oleic acid, 0.005-0.015mg/L of palmitic acid and 0.006-0.0012mg/L of stearic acid;

More preferably comprises 0.0016mg/L arachidonic acid, 0.008mg/L linoleic acid, 0.008mg/L linolenic acid, 0.008mg/L myristic acid, 0.008mg/L oleic acid, 0.008mg/L palmitic acid and 0.008mg/L stearic acid;

Other substances contained in the medium include: 100-300mg/L of choline chloride, 20-70mg/L of dextran sulfate sodium salt, 70-130mg/L of ethanolamine hydrochloride and 0.06-1.1mg/L of 1, 4-butanediamine dihydrochloride;

Preferably comprises 150-250mg/L choline chloride, 40-70mg/L dextran sulfate sodium salt, 80-110mg/L ethanolamine hydrochloride and 0.4-0.9 mg/L1, 4-butanediamine dihydrochloride;

More preferably comprises 200mg/L choline chloride, 60mg/L dextran sulfate sodium salt, 100mg/L ethanolamine hydrochloride and 0.5 mg/L1, 4-butanediamine dihydrochloride;

The saccharide contained in the medium comprises 5000-15000mg/L glucose, preferably 8000-11000mg/L glucose, more preferably 10000mg/L glucose.

6. The medium for CHO cell culture according to any one of claims 1 to 5, further comprising buffer and shear resistant substances;

Preferably, the buffer substance comprises sodium bicarbonate and the shear resistant substance comprises PluronicF68;

Optionally, the sodium bicarbonate is contained in the medium in an amount of 1000-3700mg/L, preferably 1500-3500mg/L, more preferably 2000mg/L;

Optionally, the culture medium comprises PluronicF68 in an amount of 1250-3600mg/L, preferably 1600-2500mg/L, more preferably 2000mg/L.

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

(1) Cell resuscitating the CHO cells;

(2) Performing seed amplification culture on the cells recovered in the step (1);

(3) Culturing in a first stage: inoculating CHO cells obtained by seed amplification culture in the step (2) into a seed culture medium for culture according to the initial inoculation density (1.1+/-0.3) multiplied by 10 ⁶/mL, wherein the temperature is 36.5+/-0.5 ℃, the stirring speed is 180-220 r/min, starting perfusion culture with the seed culture medium for the next day of culture, culturing until the viable cell density reaches more than 20.0×10 ⁶/mL, and performing second-stage culture;

(4) Culturing in the second stage: perfusion culturing with the medium according to any one of claims 1 to 6, when the density of living cells is greater than 60.0X10 ⁶/mL, cooling to 33-35 ℃, adjusting the rotation speed to 230-280 r/min, and maintaining the density of living cells at 100.0X10 ⁶/mL;

Preferably, the seed medium is a CD CHO medium;

optionally, dissolved oxygen is controlled to be 20% -80%, preferably 50% in the culture process; the pH value is controlled to be 7.00+/-0.35;

alternatively, the first stage incubation time is 4 to 6 days in total.

8. The method for culturing CHO cells according to claim 7,

The specific operation of cell resuscitation in the step (1) is as follows: resuscitating the seed cells from the refrigerator at-80 ℃, inoculating the seed cells into a CD CHO culture medium, and culturing for 2-4 days at 36.5+/-0.5 ℃ and 6.0+/-1.0% CO ₂ and 130+/-10 r/min;

The specific operation of the seed amplification culture in the step (2) is as follows: inoculating the resuscitated cells obtained in the step (1) into a shake flask containing a CD CHO culture medium according to the density of 0.3-0.7X10- ⁶/mL, culturing for 2-4 days at 36.5+/-0.5 ℃ and 6.0+/-1.0% CO ₂ and 130+/-10 r/min, and expanding and culturing seed cells step by step in the shake flask of 250-2000 mL according to the step;

In the step (3), the next day of culture starts to irrigate culture with a seed culture medium at a rate of 0.5VVD, and then the irrigated rate is increased by 0.5VVD to not more than 2.0VVD every day;

In the step (4), the medium according to any one of claims 1 to 6 is used for perfusion culture at a rate of 0.5 VVD; when the glucose concentration in the reaction system is lower than 2.00g/L, glucose is fed in a flowing way so as to maintain the glucose concentration in the reaction system not lower than 2.00g/L, and when the glucose concentration is higher than 3.00g/L, the feeding amount of the glucose is gradually reduced until the feeding is stopped; alternatively, when the glucose concentration is lower than 2.00g/L, the fed-batch amount of glucose is sequentially increased in units of 1.00 g/L/day to a concentration no longer lower than 2.00g/L; alternatively, when the glucose concentration is more than 3.00g/L, the fed-batch amount of glucose is sequentially decreased in units of 1.00 g/L/day.

9. The method of culturing CHO cells of claim 7 or 8, wherein the CHO cells are CHO cells comprising a protein encoding a foreign protein; preferably, the CHO cell is CHO-K1, CHO-S, CHO-K1SV, 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 medium for CHO cell culture according to any one of claims 1 to 6 for CHO cell expression of foreign proteins; preferably, the CHO cell is CHO-K1, CHO-S, CHO-K1SV, 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.