A culture medium and fermentation method for producing anti-CD47 antibodies

Abstract

The present invention belongs to the field of biopharmaceuticals, and specifically relates to a culture medium for producing anti-CD47 antibodies and a fermentation method thereof. The basic culture medium of the present invention contains amino acids, glucose, vitamins, trace elements, inorganic salts and other substances; during the fermentation process, feed culture is carried out in a timely manner, and the feed culture medium includes amino acid components. The present invention replaces the commercial culture medium with an independently optimized culture medium, which better promotes cell growth and effectively increases the expression of anti-CD47 antibodies. The feed process is adjusted during the fermentation process, which effectively reduces the protein acid-base peak content and improves the purity of the target protein.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a culture medium and a fermentation method for producing anti-CD47 antibodies. Background Art

[0002] Monoclonal antibodies have the characteristics of high specificity, low side effects and excellent effects, and are increasingly widely used in treatment and diagnosis. Cluster of differentiation protein 47 (CD47), also known as integrin-associated protein (IAP), ovarian cancer antigen OA3 and Rh-related antigen, is a member of the pentaspan transmembrane superfamily that is widely expressed on the cell membrane surface. CD47 interacts with SIRP-α (signal regulatory protein-α) on macrophages to inhibit phagocytosis. Cancer cells in this pathway escape phagocytosis and thereby promote cancer cell survival (Jaiswal, S. et al., Trends in Immunol 31: 212-219, 2010), which is the latest discovered tumor immune escape mechanism, so targeting CD47 in therapy has been widely used in a variety of cancers.

[0003] CD47 is widely expressed on the cell surface and can interact with SIRPα, thrombospondin-1 (TSP1) and integrins to mediate a series of reactions such as apoptosis, proliferation and immunity. TSP1 is related to cell proliferation, growth and differentiation. The binding of CD47 to TSP1 plays an important role in regulating cell migration, cell proliferation and apoptosis, as well as promoting angiogenesis and inflammatory response. In addition, CD47 is an important self-recognition marker on the cell surface. CD47 can bind to SIRPα on the surface of macrophages, phosphorylate its immunoreceptor tyrosine inhibitory motif (ITIM), and then recruit SHP-1 protein, resulting in a series of cascade reactions that inhibit the phagocytosis of macrophages.

[0004] Different studies have shown that almost all tumor cells and tissues highly express CD47. CD47 highly expressed on the surface of tumor cells binds to SIRPα on the surface of macrophages, releasing the "don't eat me" signal, causing macrophages in the tumor tissue infiltration area to not only live in harmony with tumor cells, but also promote the proliferation of blood vessels in the tumor, inhibit the role of effector T cells, and promote the proliferation and growth of tumor cells.

[0005] Currently, several anti-CD47 antibodies have been reported. U.S. Patent US2015 / 0183874A reported a human IgG1 chimeric monoclonal antibody derived from B6H12 and a humanized B6H12 antibody produced by CDR transplantation, which has lower immunogenicity than known antibodies. U.S. Patent US9045541 reported an anti-CD47 antibody that does not significantly cause hemagglutination, and compared with known antibodies, the antibody is significantly effective in tumor models, such as improving the ability of macrophages to phagocytose tumor cells.

[0006] There are few reports on the production methods of anti-CD47 antibodies in the prior art. CHO cells (Chinese Hamster Ovary cells) are the most widely used host cells for the production of monoclonal antibody protein drugs. Nearly 70% of the therapeutic proteins that have been marketed are produced by CHO cells. Compared with other cell expression systems, CHO cells have advantages such as post-translational modifications similar to humans, clear historical background, robust cells, and rapid growth.

[0007] However, due to the complex macromolecular structure of monoclonal antibodies, a large number of modifications will occur during the production and storage process. For example, glycosylation, oxidation, deamidation, etc., cause antibodies to produce a large amount of heterogeneity, also known as charge isomers. Analysis using CEX-HPLC or CIEF methods will result in a series of peaks, which are mainly divided into three categories: acidic peaks, main peaks, and alkaline peaks. Charge isomers have an important influence on the stability of monoclonal antibodies and the performance of biological functions. Therefore, they are called very important quality attributes that need to be controlled in the production process of monoclonal antibodies, and are also important reaction indicators for the stability of the production process. Charge isomers produce acid-base peaks, and the alkaline peaks mainly come from incomplete removal of C-terminal lysine, N-terminal pyroglutamine cyclization amine, etc. The acidic peak generally comes from sialylation modification of N-sugar end, deamidation of amino acid residues, etc. However, the charge isomers corresponding to this acidic peak and alkaline peak have similar chemical properties. It is difficult to control charge heterogeneity through later purification and separation, and the method of controlling antibody charge heterogeneity by controlling the cell culture process is also challenging, which has become a problem that has been difficult to solve in this field.

[0008] CN105779394A discloses a method for adding glutamine to a culture medium to reduce the content of an acidic peak. However, glutamine has a very adverse effect on the stability of the later process. After adding glutamine during the culture process, glutamine is easily degraded, and a large amount of ammonium ions are generated, which has an adverse effect on cell growth and expression. In addition, for a CHO expression system, adding glutamine can only reduce the content of the acidic peak of the secreted antibody, and cannot increase the content of the antibody acidic peak as required.

[0009] CN10328259A discloses a method for culturing animal cells, wherein after culturing at a normal culture temperature for a certain period of time, the culture temperature is lowered to 25-35°C and the culture is continued to inhibit the generation of the acidic peak. The inventor tried this method, but it is not applicable to anti-CD47 antibodies.

[0010] CN104560882A discloses a CHO cell culture process for reducing the acidic variant content of recombinant human tumor necrosis factor receptor-Fc recombinant protein (rhTNFR-Fc) antibody, which reduces the acidic peak level of the target protein by using different commercially available basal culture media and concentrated culture media during the CHO cell culture process. The commercially available culture media have fixed components and are not targeted at specific target proteins. For anti-CD47 antibodies, the reduction of the acidic peak level is limited, and the yield of the target protein cannot be increased.

[0011] CN108823267A discloses a method for regulating the acidic peak content of antibodies secreted by a CHO-K1 expression system, wherein CHO-K1 cells are inoculated into a culture medium for cell culture, and tyrosine and / or cysteine ​​are added during the cell culture process to increase or decrease the acidic peak content of the antibody.

[0012] Mammalian cell culture has very strict requirements on the culture environment and the quality requirements on the expressed antibodies, so it is particularly important to optimize the process conditions to achieve the product quality requirements. How to increase the expression of antibodies while reducing the content of acidic and basic variants is one of the problems that has been difficult to solve in this field. Summary of the invention

[0013] Based on the deficiencies of the prior art, the present invention aims to provide a CHO cell culture process capable of reducing the content of acidic and basic variants, and in particular to a cell culture process for anti-CD47 antibodies based on a CHO expression system.

[0014] In order to achieve the above-mentioned object, the present invention provides a culture medium for producing anti-CD47 antibodies, and a method for producing anti-CD47 antibodies using the culture medium. The present invention optimizes the culture medium to effectively increase the expression level of anti-CD47 antibodies and reduce the content of acidic and basic variants.

[0015] The technical solution of the present invention is as follows:

[0016] A culture medium for producing an anti-CD47 antibody, the culture medium comprising 8-12 g / L of amino acids, 6-10 g / L of glucose, 0.8-1.2 g / L of vitamins, 0.1-0.2 g / L of trace elements, 6-12 g / L of inorganic salts, and 2-4 g / L of other substances.

[0017] Preferably, the amino acids in the culture medium include L-aspartic acid, L-glutamic acid, L-asparagine, L-serine, L-glutamine, L-glycine, L-histidine, L-arginine, L-threonine, L-alanine, L-proline, phosphorylated-L-tyrosine, L-valine, L-methionine, S-sulfo-L-cysteine, L-isoleucine, L-leucine, L-phenylalanine, L-tryptophan, and L-lysine.

[0018] Preferably, the vitamins in the culture medium are biotin, D-calcium pantothenate, folic acid, cyclohexanehexol, nicotinamide, pyridoxine hydrochloride, riboflavin, vitamin B12, thiamine hydrochloride, lipoic acid, and lutein.

[0019] Preferably, the trace elements in the culture medium are ferric chloride hexahydrate, choline chloride, sodium selenite, manganese chloride, ammonium molybdate tetrahydrate, anhydrous zinc chloride, cupric chloride dihydrate, cobalt chloride, copper sulfate, potassium iodide, and aluminum chloride.

[0020] Preferably, the inorganic salts in the culture medium are anhydrous sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, magnesium sulfate, potassium chloride, calcium chloride, trisodium citrate dihydrate, potassium sulfate, and magnesium chloride.

[0021] Preferably, other substances in the culture medium are P-F68 (polyoxypropylene polyoxyethylene block polyether), dibutylene glycol, ethanolamine, and sodium pyruvate.

[0022] Further preferably, the culture medium comprises the components shown in Table 1 below:

[0023] Table 1 Culture medium formula

[0024]

[0025]

[0026] In a preferred experimental scheme, the culture medium includes the components shown in Table 2:

[0027] Table 2

[0028]

[0029]

[0030] A feed medium for producing an anti-CD47 antibody comprises the following components: L-aspartic acid, L-glutamic acid, L-asparagine, L-serine, L-glutamine, L-glycine, L-histidine, L-arginine, L-threonine, L-alanine, L-proline, phosphorylated-L-tyrosine, L-valine, L-methionine, S-sulfo-L-cysteine, L-isoleucine, L-leucine, L-phenylalanine, L-tryptophan and L-lysine.

[0031] Preferably, the concentration of each component in the feed medium is 5 to 12 times the concentration of the corresponding component in Table 1.

[0032] The second object of the present invention is to provide a method for producing an anti-CD47 antibody, which overcomes the negative effects of cell growth by using the above-mentioned culture medium, effectively increases the expression level of the antibody, and reduces the content of the acid-base peak, thereby effectively improving the purity of the target antibody.

[0033] A method for producing an anti-CD47 antibody comprises the following steps: (a) providing CHO cells containing a target gene encoding an anti-CD47 monoclonal antibody; (b) culturing the cells in a stepwise manner in the culture medium of the present invention; and (c) transferring the cells to a fermenter for high-density culture, and adding feed culture medium to the fermentation system when the cells grow to a certain stage until the culture ends.

[0034] Preferably, the high-density culturing step in step (c) comprises the following three stages:

[0035] (1) Cell growth stage: Cells are suspended and cultured using the culture medium of the present invention, with an initial density of about 2 to 6 × 10 5 cells / mL, the initial culture temperature was 37±0.5℃, and the pH was 7.2±0.1.

[0036] (2) Cell production of antibodies: When the cell density grows to 5-7×10 6 cells / mL, the cell culture enters the stage of cell protein production, and the feed medium is added to promote the cell expression of antibody protein. The daily feed volume is 2-3% of the culture volume. When the cell density reaches 13-18×10 6 cells / mL and lower the culture temperature to 32±0.5℃ and pH to 6.90±0.05.

[0037] (3) When the cells were cultured for 14 days or the cell viability dropped below 90%, the feed medium was stopped and the culture was terminated.

[0038] Preferably, in (2) the cell antibody production stage, the feed medium components are the amino acids in the basal medium of the present invention, and the concentration is 5 to 12 times the concentration of the amino acids in the basal medium.

[0039] Preferably, the feed medium components and concentrations are as described in Table 3.

[0040] Table 3 Feed medium formula

[0041] Components Concentration (mM) Components Concentration (mM) L-Aspartic Acid 25～60 L-Proline 15～36 L-Glutamic Acid 20～48 Phospho-L-tyrosine 10～24 L-Asparagine 25～60 L-Valine 15～36 L-Serine 25～60 L-Methionine 7～16.8 L-Glutamine 30～72 S-Sulfo-L-cysteine 5～12 L-Glycine 1.5～3.6 L-Isoleucine 15～36 L-Histidine 5～12 L-Leucine 20～48 L-Arginine 10～24 L-Phenylalanine 5～12 L-Threonine 15～36 L-Tryptophan 10～24 L-Alanine 10～24 L-Lysine 20～48

[0042] Further preferably, the feed medium is based on the medium described in Table 2 of the present invention, and the amino acid concentration is controlled to be 5 to 12 times the amino acid concentration in the basal medium, and more preferably the amino acid concentration in the feed medium is 8 to 10 times.

[0043] Preferably, in (2) the cell antibody production stage, the feed medium is added in a semi-continuous manner; further, the amount of feed medium added on the first day is 2% of the culture volume, and 3% of the culture volume is added every day thereafter.

[0044] Preferably, in (2) the cell antibody production stage, the stirring speed is 180-250 rpm, and the dissolved oxygen content is not less than 35%, and more preferably, the dissolved oxygen content is 35-60%.

[0045] A preferred experimental scheme, a method for producing an anti-CD47 antibody, comprises the following steps: providing CHO cells containing a target gene encoding an anti-CD47 monoclonal antibody, subculturing and amplifying the cells in a culture medium, amplifying the cells once every three days, and amplifying the cells through three-stage roller bottles of 30 mL, 150 mL, and 500 mL until the cell density reaches 2 to 3×10 6 cells / mL, and transferred to a fermenter; in the fermenter, a high-density culture stage was carried out with a stirring speed of 180-250 rpm, a dissolved oxygen content greater than 35%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3-4 days of culture, when the cell density reached 5-7×10 6 When the cell density reached 13-18×10 cells / mL, feed medium was added semi-continuously. The amount of feed medium added on the first day was 2% of the culture volume, and 3% of the culture volume was added every day thereafter. After 6-7 days of culture, when the cell density reached 13-18×10 6 When the cell / mL was 1.547 μg / mL, the culture temperature was lowered to 32±0.5℃ and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. When the culture was 14 days or the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. Samples were taken and the expression amount of the anti-CD47 monoclonal antibody, protein SEC purity, and acid-base peak were determined after affinity chromatography purification.

[0046] The present invention replaces the commercial culture medium with the self-optimized culture medium, which better promotes cell growth and effectively increases the expression of anti-CD47 monoclonal antibodies. The feed process is adjusted during the fermentation process, which effectively reduces the protein acid-base peak content and improves the purity of the target protein. DETAILED DESCRIPTION

[0047] The present invention is explained below in conjunction with specific implementation cases. It should be understood that the following embodiments are only for illustrative purposes and do not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0048] Unless otherwise specified, the reagents and raw materials used in the following examples can be obtained through commercial channels.

[0049] Example 1

[0050] Provide CHO cells containing a gene encoding an anti-CD47 monoclonal antibody; culture at 0.5×10 6 The cells were subcultured and expanded every three days. After three-stage amplification in 30 mL, 150 mL, and 500 mL flasks, the cell density reached 2×10 cells / mL. 6 cells / mL, and transferred to a 5L fermenter; in the 5L fermenter, the high-density culture stage was carried out with an initial culture volume of 3L, a stirring speed of 180-250rpm, a dissolved oxygen content of 35%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3-4 days of culture, when the cell density reached 6×10 6 When the cell density reached 18×10 cells / mL, the feed medium described in Table 4 was added. The amount of feed on the first day was 2% of the culture volume, and 3% of the culture volume was added every day thereafter. After 6 to 7 days of culture, when the cell density reached 18×10 6 When the cell / mL was 1.547 μg / mL, the culture temperature was lowered to 32±0.5°C and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. After 14 days of culture or when the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. The results of cell detection during the culture process are shown in Table 5. After the culture was completed, samples were taken and purified by affinity chromatography to determine the expression level of the anti-CD47 monoclonal antibody, the protein SEC purity, and the acid-base peak.

[0051] Table 4 Feed medium formula

[0052] Components Concentration (mM) Components Concentration (mM) L-Aspartic Acid 40 L-Proline 24 L-Glutamic Acid 32 Phospho-L-tyrosine 16 L-Asparagine 40 L-Valine 24 L-Serine 40 L-Methionine 11.2 L-Glutamine 48 S-Sulfo-L-cysteine 8 L-Glycine 2.4 L-Isoleucine 24 L-Histidine 8 L-Leucine 32 L-Arginine 16 L-Phenylalanine 8 L-Threonine 24 L-Tryptophan 16 L-Alanine 16 L-Lysine 32

[0053] Table 5 Cell detection results of Example 1

[0054]

[0055] Example 2

[0056] Provide CHO cells containing a gene encoding an anti-CD47 monoclonal antibody; culture at 0.5×10 6 The cells were subcultured and expanded every three days. After three-stage amplification in 30 mL, 150 mL, and 500 mL flasks, the cell density reached 3×10 cells / mL. 6 cells / mL, and transferred to a 5L fermenter; in the 5L fermenter, the high-density culture stage was carried out with an initial culture volume of 3L, a stirring speed of 180-250rpm, a dissolved oxygen content of 60%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3-4 days of culture, when the cell density reached 7×10 6 cells / mL, feed medium as described in Table 7 was added. The amount added on the first day was 2% of the culture volume, and 3% of the culture volume was added every day thereafter. After 6 to 7 days of culture, when the cell density reached 13×10 6 When the cell / mL was 1.547 μg / mL, the culture temperature was lowered to 32±0.5°C and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. After 14 days of culture or when the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. The results of cell detection during the culture process are shown in Table 8. After the culture was completed, samples were taken and purified by affinity chromatography to determine the expression level of the anti-CD47 monoclonal antibody, the protein SEC purity, and the acid-base peak.

[0057] Table 6 Basic culture medium formula

[0058]

[0059]

[0060] Table 7 Feed medium formulation

[0061] Components Concentration (mM) Components Concentration (mM) L-Aspartic Acid 36 L-Proline 24 L-Glutamic Acid 30 Phospho-L-tyrosine 18 L-Asparagine 42 L-Valine 18 L-Serine 42 L-Methionine 9.6 L-Glutamine 48 S-Sulfo-L-cysteine 7.2 L-Glycine 2.4 L-Isoleucine 24 L-Histidine 7.2 L-Leucine 36 L-Arginine 14.4 L-Phenylalanine 9.6 L-Threonine 31.2 L-Tryptophan 18 L-Alanine 18 L-Lysine 18

[0062] Table 8 Example 2 Cell detection results

[0063]

[0064]

[0065] Example 3

[0066] Provide CHO cells containing a gene encoding an anti-CD47 monoclonal antibody; culture at 0.5×10 6 The cells were subcultured and expanded every three days. After three-stage amplification in 30 mL, 150 mL, and 500 mL flasks, the cell density reached 2×10 cells / mL.6 cells / mL, and transferred to a 5L fermenter; in the 5L fermenter, the high-density culture stage was carried out with an initial culture volume of 3L, a stirring speed of 180-250rpm, a dissolved oxygen content of 40%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3-4 days of culture, when the cell density reached 5×10 6 cells / mL, feed medium as described in Table 10 was added. The amount added on the first day was 2% of the culture volume, and 3% of the culture volume was added every day thereafter. After 6 to 7 days of culture, when the cell density reached 18×10 6 When the cell / mL was 1.547 cells / mL, the culture temperature was lowered to 32±0.5°C and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. After 14 days of culture or when the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. The results of cell detection during the culture process are shown in Table 11. After the culture was completed, samples were taken and purified by affinity chromatography to determine the expression level of the anti-CD47 monoclonal antibody, the protein SEC purity, and the acid-base peak.

[0067] Table 9 Basic culture medium formula

[0068]

[0069]

[0070] Table 10 Feed medium formulation

[0071] Components Concentration (mM) Components Concentration (mM) L-Aspartic Acid 30 L-Proline 30 L-Glutamic Acid 37.5 Phospho-L-tyrosine 22.5 L-Asparagine 52.5 L-Valine 22.5 L-Serine 52.5 L-Methionine 12 L-Glutamine 60 S-Sulfo-L-cysteine 9 L-Glycine 3 L-Isoleucine 30 L-Histidine 9 L-Leucine 45 L-Arginine 18 L-Phenylalanine 12 L-Threonine 39 L-Tryptophan 22.5 L-Alanine 22.5 L-Lysine 22.5

[0072] Table 11 Cell detection results of Example 3

[0073]

[0074] Example 4

[0075] Provide CHO cells containing a gene encoding an anti-CD47 monoclonal antibody; culture at 0.5×10 6 The cells were subcultured and expanded every three days. After three-stage amplification in 30 mL, 150 mL, and 500 mL flasks, the cell density reached 3×10 cells / mL. 6 cells / mL, and transferred to a 5L fermenter; in the 5L fermenter, the high-density culture stage was carried out with an initial culture volume of 3L, a stirring speed of 180-250rpm, a dissolved oxygen content of 35%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3-4 days of culture, when the cell density reached 6×10 6When the cell density reached 18×10 cells / mL, the feed medium described in Table 12 was added. The amount added on the first day was 2% of the culture volume, and 3% of the culture volume was added every day thereafter. After 6 to 7 days of culture, when the cell density reached 18×10 6 When the cell / mL was 1.547 cells / mL, the culture temperature was lowered to 32±0.5°C and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. After 14 days of culture or when the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. The cell detection results during the culture process are shown in Table 13. After the culture was completed, samples were taken and purified by affinity chromatography to determine the expression level of the anti-CD47 monoclonal antibody, the protein SEC purity, and the acid-base peak.

[0076] Table 12 Feed medium formulation

[0077]

[0078]

[0079] Table 13 Cell detection results of Example 4

[0080]

[0081] Example 5

[0082] Provide CHO cells containing a gene encoding an anti-CD47 monoclonal antibody; culture at 0.5×10 6 The cells were subcultured and expanded every three days. After three-stage amplification in 30 mL, 150 mL, and 500 mL flasks, the cell density reached 2×10 cells / mL. 6 cells / mL, and transferred to a 5L fermenter; in the 5L fermenter, the high-density culture stage was carried out with an initial culture volume of 3L, a stirring speed of 180-250rpm, a dissolved oxygen content of 35%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3-4 days of culture, when the cell density reached 6×10 6 cells / mL, feed medium as described in Table 14 was added at a rate of 3% of the culture volume per day. After 6 to 7 days of culture, when the cell density reached 18×10 6 When the cell / mL was 1.547 cells / mL, the culture temperature was lowered to 32±0.5°C and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. After 14 days of culture or when the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. The results of cell detection during the culture process are shown in Table 15. After the culture was completed, samples were taken and purified by affinity chromatography to determine the expression level of the anti-CD47 monoclonal antibody, the protein SEC purity, and the acid-base peak.

[0083] Table 14 Feed medium formula

[0084] Components Concentration (mM) Components Concentration (mM) L-Aspartic Acid 15 L-Proline 9 L-Glutamic Acid 12 Phospho-L-tyrosine 6 L-Asparagine 15 L-Valine 9 L-Serine 15 L-Methionine 4.2 L-Glutamine 18 S-Sulfo-L-cysteine 3 L-Glycine 0.9 L-Isoleucine 9 L-Histidine 3 L-Leucine 12 L-Arginine 6 L-Phenylalanine 3 L-Threonine 9 L-Tryptophan 6 L-Alanine 6 L-Lysine 12

[0085] Table 15 Cell detection results of Example 5

[0086]

[0087]

[0088] Comparative Example 1

[0089] Provide CHO cells containing the target gene encoding anti-CD47 monoclonal antibody; in CD01 medium at 0.5×10 6 The cells were subcultured and expanded every three days. After three-stage amplification in 30 mL, 150 mL, and 500 mL flasks, the cell density reached 2×10 cells / mL. 6 cells / mL, and transferred to a 5L fermenter; in the 5L fermenter, the high-density culture stage was carried out with an initial culture volume of 3L, a stirring speed of 250rpm, a dissolved oxygen content of 35%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3 to 4 days of culture, when the cell density reached 6×10 6 When the cell density reached 18×10 cells / mL, feed medium PFF06 was added semi-continuously. The amount of feed medium was 2% of the culture volume on the first day and 3% of the culture volume every day thereafter. After 6 to 7 days of culture, when the cell density reached 18×10 6 When the cell / mL was 1.547 μg / mL, the culture temperature was lowered to 32±0.5°C and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. After 14 days of culture or when the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. The results of cell detection during the culture process are shown in Table 16. After the culture was completed, samples were taken and purified by affinity chromatography to determine the expression level of the anti-CD47 monoclonal antibody, the protein SEC purity, and the acid-base peak.

[0090] Table 16 Comparative Example 1 Cell Detection Results

[0091]

[0092] Comparative Example 2

[0093] Provide CHO cells containing the target gene encoding anti-CD47 monoclonal antibody; in Actipro medium at 0.5×10 6 The cells were subcultured and expanded every three days. After three-stage amplification in 30 mL, 150 mL, and 500 mL flasks, the cell density reached 2×10 cells / mL. 6cells / mL, and transferred to a 5L fermenter; in the 5L fermenter, the high-density culture stage was carried out with an initial culture volume of 3L, a stirring speed of 250rpm, a dissolved oxygen content of 35%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3 to 4 days of culture, when the cell density reached 6×10 6 cells / mL, feed medium CB7a+CB7b was added semi-continuously, with 2% of the culture volume added on the first day and 3% of the culture volume added every day thereafter. After 6 to 7 days of culture, when the cell density reached 18×10 6 When the cell / mL was 1.547 cells / mL, the culture temperature was lowered to 32±0.5°C and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. After 14 days of culture or when the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. The results of cell detection during the culture process are shown in Table 17. After the culture was completed, samples were taken and purified by affinity chromatography to determine the expression level of the anti-CD47 monoclonal antibody, the protein SEC purity, and the acid-base peak.

[0094] Table 17 Comparative Example 2 Cell Detection Results

[0095]

[0096] Comparative Example 3

[0097] Provide CHO cells containing the gene of interest encoding anti-CD47 monoclonal antibody; culture at 0.5×10 6 The cells were subcultured and expanded every three days. After three-stage amplification in 30 mL, 150 mL, and 500 mL flasks, the cell density reached 2×10 cells / mL. 6 cells / mL, and transferred to a 5L fermenter; in the 5L fermenter, the high-density culture stage was carried out with an initial culture volume of 3L, a stirring speed of 250rpm, a dissolved oxygen content of 35%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3 to 4 days of culture, when the cell density reached 6×10 6 When the cell density reached 18×10 cells / mL, feed medium Advanced Feed 1 was added semi-continuously. The feed volume on the first day was 2% of the culture volume, and then 3% of the culture volume was added every day. After 6 to 7 days of culture, when the cell density reached 18×10 6When the cell / mL was 1.547 μg / mL, the culture temperature was lowered to 32±0.5°C and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. After 14 days of culture or when the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. The cell detection results during the culture process are shown in Table 18. After the culture was completed, samples were taken and purified by affinity chromatography to determine the expression level of the anti-CD47 monoclonal antibody, the protein SEC purity, and the acid-base peak.

[0098] Table 18 Comparative Example 3 Cell Detection Results

[0099]

[0100] Comparative Example 4

[0101] Provide CHO cells containing the gene of interest encoding anti-CD47 monoclonal antibody; culture at 0.5×10 6 The cells were subcultured and expanded every three days. After three-stage amplification in 30 mL, 150 mL, and 500 mL flasks, the cell density reached 2×10 cells / mL. 6 cells / mL, and transferred to a 5L fermenter; in the 5L fermenter, the high-density culture stage was carried out with an initial culture volume of 3L, a stirring speed of 250rpm, a dissolved oxygen content of 35%, a temperature of 37±0.5℃, and a pH of 7.2±0.1. After 3 to 4 days of culture, when the cell density reached 6×10 6 When the cell density reached 18×10 cells / mL, feed medium was added semi-continuously, with 2% of the culture volume added on the first day and 3% of the culture volume added every day thereafter. After 6 to 7 days of culture, when the cell density reached 18×10 6 When the cell / mL was 1.547 cells / mL, the culture temperature was lowered to 32±0.5°C and the pH was 6.9±0.05. During the feeding culture, samples were taken every day to detect the cell density and viability. After 14 days of culture or when the cell viability was lower than 90%, the feeding medium was stopped and the culture was terminated. The results of cell detection during the culture process are shown in Table 19. After the culture was completed, samples were taken and purified by affinity chromatography to determine the expression level of the anti-CD47 monoclonal antibody, the protein SEC purity, and the acid-base peak.

[0102] Table 19 Comparative Example 4 Cell Detection Results

[0103]

[0104] The antibody determination results of Examples 1-5 and Comparative Examples 1-4 are shown in Table 20.

[0105] Table 20 Antibody fermentation test results of examples and comparative examples

[0106]

Claims

1. A culture medium for producing anti-CD47 antibodies, characterized in that The culture medium comprises a basal culture medium and a feed culture medium; wherein the basal culture medium is composed of the following components: The feed medium consisted of the following components: 。 2. A method for producing an anti-CD47 antibody, characterized in that: The method comprises the following steps: (a) providing CHO cells containing a target gene encoding an anti-CD47 antibody; (b) culturing the cells in a stepwise manner in the basal culture medium of claim 1; (c) transferring the cells to a fermenter for high-density culture; The high-density culture in step (c) includes the following three stages: (1) Cell growth stage: Cells are suspended and cultured using the basal medium described in claim 1, with an initial density of about 2 to 6 × 10 5 cells / ml, the initial culture temperature was 37±0.5℃, and the pH was 7.2±0.1; (2) Cell production antibody stage: When the cell density grows to 5~7×10 6 cells / ml, the cell culture enters the stage of cell protein production, and the feed medium of claim 1 is added to promote cell expression of antibody protein, and the daily addition amount is 2-3% of the culture volume; when the cell density reaches 13-18×10 6 cells / ml, lower the culture temperature to 32±0.5℃, pH to 6.90±0.05; (3) When the cells have been cultured for 14 days or the cell viability drops below 90%, stop adding feed medium and terminate the culture.

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