Preparation method and application of long-acting erythropoietin

Abstract

The present application relates to the field of biopharmaceutical technology, and in particular, to a method for preparing a long-acting erythropoietin and its application. Specifically, the present application provides a method for preparing a long-acting erythropoietin, comprising the steps of: cell activation, resuscitation of a CHO cell line capable of expressing recombinant human erythropoietin; cell culture, adding the revived CHO cells to a CHO fermentation medium for cultivation, controlling the temperature at 36-38°C, the pH at 6.8-7.2, and the dissolved oxygen content at 40-60%; protein purification, purifying the EPO in the culture fluid; wherein the CHO fermentation medium comprises a basal medium and an additive, the additive comprising composition A, the composition A consisting of fatty acids, lecithin, and adjusting the fatty acid concentration to 2-6mg / L and the lecithin concentration to 1-3mg / L. Using the above technical solution, by supplementing the additives of the CHO fermentation medium with an appropriate amount of fatty acids and lecithin components, the survival rate of CHO cells in reactor culture and the stability of protein expression can be improved.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceutical technology, and in particular to a preparation method and application of long-acting erythropoietin. Background Art

[0002] Erythropoietin (EPO) was discovered in 1906. It is a glycoprotein mainly produced by the kidneys with a molecular weight of approximately 35KD. It is the main hormone regulating erythroid hematopoiesis in the human body and plays an important regulatory role in the proliferation, differentiation and maturation of erythroid progenitor cells.

[0003] In existing technologies, DNA recombination technology can be used to construct the EPO gene into Chinese hamster ovary cells (CHO cells) to form CHO cells that can express the recombinant human erythropoietin (rhEPO) gene.

[0004] However, in recent production and testing by the applicant, it was found that the constructed CHO cell line capable of expressing rhEPO is relatively sensitive to the external environment, which may affect the final expression of EPO.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this application is to provide a preparation method of long-acting erythropoietin and its application to solve at least one technical problem described in the background technology.

[0007] Specifically, the first aspect of the present application provides a method for preparing a long-acting erythropoietin, comprising the steps of:

[0008] Cell activation, reviving CHO cell lines capable of expressing recombinant human erythropoietin;

[0009] Cell culture: add the revived CHO cells to CHO fermentation medium for cultivation, controlling the temperature at 36-38°C, pH at 6.8-7.2, and dissolved oxygen content at 40-60%;

[0010] Protein purification: purify EPO in the culture medium;

[0011] The CHO fermentation medium includes a basal medium and additives, wherein the additives include composition A, which is composed of fatty acids and lecithin, and the fatty acid concentration is adjusted to 2-6 mg / L and the lecithin concentration is adjusted to 1-3 mg / L.

[0012] By adopting the above technical solution, by adding appropriate amounts of fatty acids and lecithin components to the additives of the CHO fermentation medium, the survival rate of CHO cells during culture in the reactor and the stability of protein expression can be improved.

[0013] Preferably, the fatty acid concentration is 2.5-4 mg / L, and the lecithin concentration is 1.5-2 mg / L.

[0014] Preferably, the fatty acid consists of oleic acid, arachidonic acid and linolenic acid, wherein the ratio of the three is 1-2:0.2-1:1.

[0015] Preferably, the additive further comprises a composition B, wherein the composition B comprises the following components:

[0016] AEO-9 100-300mg / L;

[0017] Insulin 2-8 mg / L;

[0018] IGF-I 5-15 mg / L;

[0019] Transferrin 5-8 mg / L;

[0020] Ethanolamine 1-5mg / L.

[0021] More preferably, the composition B comprises the components:

[0022] AEO-9 150-200mg / L;

[0023] Insulin 4-6 mg / L;

[0024] IGF-I 8-12 mg / L;

[0025] Transferrin 6-7 mg / L;

[0026] Ethanolamine 2-4 mg / L.

[0027] Preferably, the basal culture medium comprises sugars, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), amino acids, vitamins, and inorganic salts.

[0028] Preferably, the sugars include:

[0029] D-glucose, galactose, fructose or a combination of them.

[0030] Preferably, the sugars include:

[0031] D-glucose 3000-5000 mg / L, galactose 200-300 mg / L, fructose 200-300 mg / L.

[0032] Preferably, the HEPES concentration is 1000-1500 mg / L.

[0033] Preferably, the amino acids include:

[0034] One or a combination of glycine, alanine, isoleucine, leucine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0035] Preferably, the amino acids include:

[0036] One or a combination of the following: glycine 20-40 mg / L, alanine 60-100 mg / L, isoleucine 20-50 mg / L, leucine 100-200 mg / L, arginine 200-400 mg / L, asparagine 10-30 mg / L, aspartic acid 20-50 mg / L, cysteine ​​25-100 mg / L, glutamic acid 5-20 mg / L, glutamine 100-300 mg / L, histidine 25-50 mg / L, lysine 100-350 mg / L, methionine 25-50 mg / L, phenylalanine 15-60 mg / L, proline 2-10 mg / L, serine 5-50 mg / L, threonine 50-100 mg / L, tryptophan 5-10 mg / L, tyrosine 10-30 mg / L, and valine 50-80 mg / L.

[0037] Preferably, the vitamins include:

[0038] One or a combination of vitamin C, vitamin H, vitamin PP, vitamin B6, vitamin B2, vitamin B1, vitamin B12, calcium pantothenate, folic acid, and inositol.

[0039] Preferably, the vitamins include:

[0040] One or a combination of the following: vitamin C 0.05-0.1 mg / L, vitamin H 0.02-0.05 mg / L, vitamin PP 0.1-1 mg / L, vitamin B6 0.5-2.5 mg / L, vitamin B2 0.01-0.08 mg / L, vitamin B1 0.5-2 mg / L, vitamin B12 0.05-0.2 mg / L, calcium pantothenate 0.2-2 mg / L, folic acid 2-10 mg / L, inositol 0.5-5 mg / L.

[0041] Preferably, the inorganic salt comprises:

[0042] One or a combination of sodium chloride, sodium bicarbonate, disodium hydrogen phosphate, magnesium chloride, calcium chloride, ferric nitrate, zinc sulfate, sodium selenate, sodium metasilicate, copper sulfate, chromium sulfate, aluminum chloride, ammonium vanadate, barium acetate, nickel chloride, stannous chloride, and manganese chloride.

[0043] Preferably, the additive further comprises an inorganic salt, and the inorganic salt comprises:

[0044] Sodium chloride 2000-3000 mg / L, sodium bicarbonate 800-1200 mg / L, disodium hydrogen phosphate 100-300 mg / L, magnesium chloride 25-100 mg / L, calcium chloride 5-10 mg / L, ferric nitrate 0.25-1.5 0.001-0.005 mg / L, ammonium vanadate 0.001-0.005 mg / L, barium acetate 0.001-0.003 mg / L, nickel chloride 0.00005-0.0001 mg / L, stannous chloride 0.00005-0.0001 mg / L, and manganese chloride 0.00001-0.00005 mg / L, or a combination of the following:

[0045] Preferably, the additive does not include cholesterol, or the cholesterol content does not exceed 0.005 mg / L.

[0046] Preferably, in the cell culturing step,

[0047] The revived CHO cells were seeded at a density of 2-4×10 5 Cells / ml were added into the culture medium for cultivation.

[0048] More preferably, in the cell culturing step,

[0049] The revived CHO cells were seeded at a density of 2.5-3×10 5 Cells / ml were added into the culture medium for cultivation.

[0050] Preferably, in the cell culture step, the temperature variation does not exceed 0.5°C / 6h.

[0051] Preferably, the CHO cell is CHO-K1.

[0052] Preferably, the expression vector may be one of pQkX2, pSV2, pEGFP-N1, and pCMVp-NEO-BAN. More preferably, the expression vector is pQKX2.

[0053] Preferably, in the cell culture step, when the cell density in the culture medium reaches 4-6×10 6 After 100 μg / ml, 2-3% of the culture medium was fed every 24 h.

[0054] Preferably, the protein purification comprises the steps of:

[0055] a supernatant collection step, wherein cells are separated by centrifugation or filtration, and the supernatant is collected;

[0056] In the affinity chromatography step, the supernatant is loaded and eluted using an affinity chromatography column;

[0057] a hydrophobic chromatography step, wherein the affinity chromatography eluate is passed through a hydrophobic chromatography column for elution;

[0058] In the anion exchange chromatography step, the eluate obtained in the previous step is passed through an anion exchange chromatography column for elution.

[0059] Preferably, the affinity chromatography column is Blue Sepharose 6 FF (GE) 1.6×2.5 cm 5 ml.

[0060] Preferably, the hydrophobic chromatography column is HiTrap Butyl S FF (GE) 1ml.

[0061] Preferably, the hydrophobic chromatography step comprises the steps of:

[0062] Sample pretreatment was performed by adjusting the affinity eluate with 4 M ammonium sulfate to obtain an affinity eluate containing 1.5 M ammonium sulfate, and then performing hydrophobic chromatography.

[0063] Preferably, the anion exchange chromatography column is DEAE FF (GE) 0.7×2.5 cm 1 ml.

[0064] The second aspect of the present application provides a use of the long-acting erythropoietin as described in the first aspect of the present application in treating anemia symptoms.

[0065] In summary, this application has the following beneficial effects:

[0066] 1. The long-acting erythropoietin preparation method provided in this application can improve the survival rate of CHO cells and the stability of protein expression during reactor culture by adding appropriate amounts of fatty acids and lecithin components to the culture medium additives.

[0067] 2. The long-acting erythropoietin preparation method provided in this application achieves stable growth of CHO cells during culture by controlling the inoculation amount of CHO cells, thereby ensuring the expression of erythropoietin over a longer period of time.

[0068] 3. The long-acting erythropoietin preparation method provided in this application can fully exert the effect of the culture medium and maintain the stable growth of CHO cells by controlling the addition of no additional cholesterol components to the culture medium additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is a temperature fluctuation cycle based on 37°C and 34°C in Experimental Example 4;

[0070] Figure 2 This is a temperature fluctuation cycle based on 37°C and 34°C in Experimental Example 5;

[0071] Figure 3 The cell density after 7 days of culture of technical solutions AJ in Experimental Example 7;

[0072] Figure 4 The EPO expression level after 7 days of culture in technical solution AJ in Experimental Example 7;

[0073] Figure 5 is the cell density of Examples 1-6 and Comparative Examples 1-2 (unit: 106 cells / ml);

[0074] Figure 6 It is the EPO expression level of Example 1-6 and Comparative Example 1-2. DETAILED DESCRIPTION

[0075] Exemplary embodiments will be described in detail herein, with examples illustrated in the accompanying drawings. In the following description, when referring to the drawings, identical numerals in different figures represent identical or similar elements, unless otherwise indicated. The embodiments described in the following exemplary embodiments are not intended to represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.

[0076] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. As used in this application and the appended claims, the singular forms "a," "an," "the," and "the" are intended to include the plural forms, unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0077] The present application will be described in detail below through examples.

[0078] Erythropoietin (EPO) is a glycoprotein mainly produced by the kidneys with a molecular weight of approximately 35KD. It is the main hormone regulating erythroid hematopoiesis in the human body and plays an important regulatory role in the proliferation, differentiation and maturation of erythroid progenitor cells.

[0079] In existing technologies, DNA recombination technology can be used to construct the EPO gene into Chinese hamster ovary cells (CHO cells) to form CHO cells that can express the recombinant human erythropoietin (rhEPO) gene.

[0080] However, during recent production and testing, the applicant unexpectedly discovered that the constructed rhEPO-expressing CHO cell line is sensitive to the external environment, which may affect the final EPO expression. Specifically, the applicant discovered that when the culture temperature fluctuates drastically, the EPO expression of CHO cells grown in conventional culture medium will decrease significantly.

[0081] In view of this, the inventive concept of the present application is to provide a method for preparing long-acting erythropoietin, comprising the following steps: cell activation, resuscitating a CHO cell line capable of expressing recombinant human erythropoietin; cell culture, adding the revived CHO cells to a CHO fermentation medium for culture, controlling the temperature at 36-38°C, the pH at 6.8-7.2, and the dissolved oxygen content at 40-60%; and protein purification, purifying EPO in the culture medium; wherein the CHO fermentation medium comprises a basal medium and additives, the additives comprising composition A, the composition A comprising fatty acids and lecithin, and adjusting the fatty acid concentration to 2-5 mg / L and the lecithin concentration to 1-3 mg / L. By adopting the above technical solution, by supplementing the additives of the CHO fermentation medium with appropriate amounts of fatty acids and lecithin components, the survival rate of CHO cells during reactor culture and the stability of protein expression can be improved.

[0082] To better understand the above technical solution, the following will be described in detail with reference to specific embodiments. Those skilled in the art will also understand that the reaction times and drug dosing times involved in this application cannot be achieved with absolute precision in actual production processes, but are within the allowable error range. For example, if a sample is expected to be heated for 30 minutes, the actual operation may be 1 second over or under 30 minutes; if a 30g sample is expected to be weighed, the actual weighing may be 30.001g or 29.998g.

[0083] Experimental Example 1 CHO cell line capable of expressing recombinant human erythropoietin

[0084] Unless otherwise specified, the CHO cells used in the experimental examples and embodiments of this application are the cell lines described in these experimental examples. The CHO cells are selected from CHO-K1 cells, which contain the expression vector pQKX2, which carries a gene sequence capable of expressing an EPO unit, the gene sequence being SEQ ID NO. 1 in CN111019907B, the prior art disclosed by the applicant.

[0085] Specifically, the CHO cells are also prepared using the prior art method of CN111019907B:

[0086] A cloning vector containing the sequence SEQ ID NO. 1 is prepared and corresponding restriction sites are added; an expression vector pQKX2 is prepared; the cloning vector and the expression vector are simultaneously digested with restriction endonucleases SapI and EcoRI; the cloning vector is digested to produce a target sequence, the target sequence comprising the sequence SEQ ID NO. 1; the target sequence is ligated into the digested expression vector to obtain a recombinant expression vector;

[0087] The recombinant expression vector was digested with PvuI to obtain a linearized recombinant plasmid, which was then transfected into CHO cells using a Bio-Rad electroporator to obtain CHO cells capable of expressing recombinant EPO.

[0088] Experimental Example 2 Blank Control CHO Cell Line

[0089] CHO-K1 cells were purchased from ATCC (58995535).

[0090] Experimental Example 3 EPO protein purification method

[0091] The EPO protein purification steps include:

[0092] a supernatant collection step, wherein cells are separated by centrifugation or filtration, and the supernatant is collected;

[0093] In the affinity chromatography step, the supernatant is loaded and eluted using an affinity chromatography column; wherein the affinity chromatography column is Blue Sepharose 6 FF (GE) 1.6×2.5 cm 5 ml;

[0094] In the hydrophobic chromatography step, the affinity eluate is adjusted with 4 M ammonium sulfate to obtain an affinity eluate containing 1.5 M ammonium sulfate, and the affinity eluate is passed through a hydrophobic chromatography column for elution; wherein the hydrophobic chromatography column is a HiTrap Butyl S FF (GE) 1 ml;

[0095] In the anion exchange chromatography step, the eluate obtained in the previous step is passed through an anion exchange chromatography column for elution; wherein the anion exchange chromatography column is DEAE FF (GE) 0.7×2.5cm 1ml.

[0096] Experimental Example 4 Temperature Fluctuation Simulation Test 1

[0097] The revived CHO cells were seeded at a density of 2.5×10 5The cells / ml were added to CHO fermentation medium for cultivation. The pH was controlled at 7.0±0.1, the dissolved oxygen content was 50±5%, the stirring speed was set at 60-70 r / min, and the culture was carried out for 7 days.

[0098] The initial culture temperature was 37°C, and when the cell density in the culture medium reached 6×10 6 After that, 2% culture medium was added every 24 h; when the cell density in the culture medium reached 8×10 6 After the reaction mixture was concentrated to 100 μg / ml, the reactor temperature was lowered to 34°C.

[0099] according to Figure 1 Example: a temperature fluctuation cycle between 37°C and 34°C.

[0100] Experimental Example 5 Temperature Fluctuation Simulation Test 2

[0101] The revived CHO cells were seeded at a density of 2.5×10 5 The cells / ml were added to CHO fermentation medium for cultivation. The pH was controlled at 7.0±0.1, the dissolved oxygen content was 50±5%, the stirring speed was set at 60-70 r / min, and the culture was carried out for 7 days.

[0102] The initial culture temperature was 37°C, and when the cell density in the culture medium reached 6×10 6 After that, 2% culture medium was added every 24 h; when the cell density in the culture medium reached 8×10 6 After the reaction mixture was concentrated to 100 μg / ml, the reactor temperature was lowered to 34°C.

[0103] according to Figure 2 Example: a temperature fluctuation cycle between 37°C and 34°C.

[0104] Experimental Example 6 Conventional culture test

[0105] The revived CHO cells were seeded at a density of 2.5×10 5 The cells were added to CHO fermentation medium for 7 days at a controlled temperature of 37 ± 0.1°C, a pH of 7.0 ± 0.1, a dissolved oxygen content of 50 ± 5%, and a stirring speed of 60-70 r / min.

[0106] When the cell density in the culture medium reached 5×10 6 After that, 2% culture medium was added every 24 h.

[0107] Experimental Example 7 CHO cell culture test in different culture media

[0108] Technical Solution A

[0109] CHO fermentation medium OPM-CHO CD07 was purchased from Shanghai Aopuma Biotechnology Co., Ltd.

[0110] Technical Solution B

[0111] CHO fermentation medium DMEM / F12 was purchased from GIBCO.

[0112] Technical Solution C

[0113] CHO fermentation medium, including:

[0114] Basal culture medium, including: sugars, HEPES, amino acids, vitamins, inorganic salts;

[0115] Wherein, the sugars include: D-glucose 4000 mg / L, galactose 250 mg / L, and fructose 250 mg / L.

[0116] The HEPES concentration is 1000 mg / L.

[0117] The amino acids include: glycine 30 mg / L, alanine 80 mg / L, isoleucine 30 mg / L, leucine 150 mg / L, arginine 300 mg / L, asparagine 20 mg / L, aspartic acid 30 mg / L, cysteine ​​50 mg / L, glutamic acid 10 mg / L, glutamine 200 mg / L, histidine 30 mg / L, lysine 200 mg / L, methionine 30 mg / L, phenylalanine 30 mg / L, proline 5 mg / L, serine 10 mg / L, threonine 60 mg / L, tryptophan 8 mg / L, tyrosine 20 mg / L, and valine 60 mg / L.

[0118] The vitamins include: vitamin C 0.06 mg / L, vitamin H 0.04 mg / L, vitamin PP 0.2 mg / L, vitamin B6 1.0 mg / L, vitamin B2 0.04 mg / L, vitamin B1 1.0 mg / L, vitamin B12 0.1 mg / L, calcium pantothenate 0.5 mg / L, folic acid 5 mg / L, and inositol 1.0 mg / L.

[0119] The inorganic salts include: 2500 mg / L sodium chloride, 1000 mg / L sodium bicarbonate, 200 mg / L disodium hydrogen phosphate, 50 mg / L magnesium chloride, 6 mg / L calcium chloride, 0.5 mg / L ferric nitrate, 1.0 mg / L zinc sulfate, 0.05 mg / L sodium selenate, 0.005 mg / L sodium metasilicate, 0.002 mg / L copper sulfate, 0.002 mg / L chromium sulfate, 0.002 mg / L aluminum chloride, 0.004 mg / L ammonium vanadate, 0.002 mg / L barium acetate, 0.00006 mg / L nickel chloride, 0.00008 mg / L stannous chloride, and 0.00004 mg / L manganese chloride.

[0120] Technical Solution D

[0121] CHO fermentation medium, including:

[0122] Basic culture medium, the same as that of technical solution C;

[0123] additives, including composition B;

[0124] The composition B comprises: 200 mg / L AEO-9; 4 mg / L insulin; 10 mg / L IGF-I; 6 mg / L transferrin; and 2 mg / L ethanolamine.

[0125] Technical Solution E

[0126] CHO fermentation medium, including:

[0127] Basic culture medium, the same as that of technical solution C;

[0128] Additives, including composition A and composition B;

[0129] The composition A comprises: 1 mg / L of oleic acid, 0.5 mg / L of arachidonic acid, and 1 mg / L of linolenic acid;

[0130] The composition B comprises: 200 mg / L AEO-9; 4 mg / L insulin; 10 mg / L IGF-I; 6 mg / L transferrin; and 2 mg / L ethanolamine.

[0131] Technical Solution F

[0132] CHO fermentation medium, including:

[0133] Basic culture medium, the same as that of technical solution C;

[0134] Additives, including composition A and composition B;

[0135] The composition A comprises: 1 mg / L oleic acid, 1 mg / L linolenic acid, and 2 mg / L lecithin;

[0136] The composition B comprises: 200 mg / L AEO-9; 4 mg / L insulin; 10 mg / L IGF-I; 6 mg / L transferrin; and 2 mg / L ethanolamine.

[0137] Technical Solution G

[0138] CHO fermentation medium, including:

[0139] Basic culture medium, the same as that of technical solution C;

[0140] Additives, including composition A and composition B;

[0141] The composition A comprises: 1 mg / L oleic acid, 0.5 mg / L arachidonic acid, 1 mg / L linolenic acid, and 2 mg / L lecithin;

[0142] The composition B comprises: 200 mg / L AEO-9; 4 mg / L insulin; 10 mg / L IGF-I; 6 mg / L transferrin; and 2 mg / L ethanolamine.

[0143] Technical Solution H

[0144] CHO fermentation medium, including:

[0145] Basic culture medium, the same as that of technical solution C;

[0146] Additives, including composition A and composition B;

[0147] The composition A comprises: oleic acid 1 mg / L, arachidonic acid 0.5 mg / L, linolenic acid 1 mg / L, lecithin 2 mg / L, and cholesterol 1 mg / L;

[0148] The composition B comprises: 200 mg / L AEO-9; 4 mg / L insulin; 10 mg / L IGF-I; 6 mg / L transferrin; and 2 mg / L ethanolamine.

[0149] Technical Solution I

[0150] CHO fermentation medium, including:

[0151] Basic culture medium, the same as that of technical solution C;

[0152] Additives, including composition A and composition B;

[0153] The composition A comprises: oleic acid 1 mg / L, arachidonic acid 0.5 mg / L, linolenic acid 1 mg / L, lecithin 2 mg / L, and cholesterol 0.005 mg / L;

[0154] The composition B comprises: 200 mg / L AEO-9; 4 mg / L insulin; 10 mg / L IGF-I; 6 mg / L transferrin; 2 mg / L ethanolamine; and 0.1 mg / L linoleic acid.

[0155] Technical Solution J

[0156] It is basically the same as Technical Solution G, except that:

[0157] The revived CHO cells were seeded at a density of 6 × 10 5 cells / ml were added into CHO fermentation medium for cultivation.

[0158] The CHO fermentation medium provided by Technical Solutions A to Technical Solutions J was applied to the culture conditions in Experimental Examples 4, 5, and 6, and the method of Experimental Example 3 was used to purify the EPO protein, and the amount of the purified protein was calculated.

[0159] The cell density of technical solutions A-technical solutions J after 7 days of culture is shown in Figure 3 shown.

[0160] The EPO expression levels of technical solutions A to J after 7 days of culture are shown in Figure 4 shown.

[0161] The research basis of this application is that the applicant discovered that the temperature fluctuated greatly due to unexpected circumstances during the fermentation of the strain, and after the temperature fluctuation, the EPO production showed a significant downward trend. Based on this, the applicant simulated a fluctuating temperature environment and tested the tolerance of CHO cells under different culture media under this condition.

[0162] according to Figure 3-Figure 4 Those skilled in the art will understand that:

[0163] First, under temperature fluctuations, the cell growth status and EPO expression level of the experimental CHO cells under the culture conditions of commercially available culture media (Technical Solutions A and B) were significantly lower than those of the culture media provided in this application (Technical Solutions CJ), indicating that the culture media and preparation methods provided in this application are more tolerant to external environmental fluctuations, especially temperature fluctuations.

[0164] Second, for Technical Solution CG, when composition A includes appropriate amounts of oleic acid, arachidonic acid, linolenic acid, and lecithin components, the growth state and EPO expression level of CHO cells are significantly higher (Technical Solution G);

[0165] Third, the applicant found (Technical Solution HI) that when excessive cholesterol is added to Composition A, it will have an adverse effect on the growth and protein expression of CHO cells. Unless otherwise specified, it is recommended not to add cholesterol to the culture medium. Even if it is added, referring to Technical Solution I, the applicant recommends that the added amount should not exceed 0.005 mg / L.

[0166] Fourth, for Technical Solutions G and J, in an environment with temperature fluctuations, a high inoculation density of CHO cells will not increase EPO production, but will have a certain adverse effect. It is speculated that a high inoculation density under temperature fluctuations will easily lead to insufficient nutrient intake in the early stage of cell culture. Therefore, the applicant recommends that the inoculation density should not exceed 6×10 5 / ml, or more preferably not more than 6×10 5 pcs / ml.

[0167] Example 1

[0168] Cell activation: Take the CHO cells in Experimental Example 1 and resuscitate them;

[0169] Cell culture: CHO cells were revived and seeded at a density of 2 × 10 5 Cells were added into CHO fermentation medium for cultivation at a controlled temperature of 36 ± 0.1 °C, a pH of 6.8 ± 0.1, a dissolved oxygen content of 45 ± 5%, and a stirring speed of 60 r / min for 13 days. When the cell density in the culture medium reached 4 × 10 6 After the culture medium has reached 1000 μg / ml, 2-3% of the culture medium is fed every 24 h.

[0170] Protein purification: EPO in the culture medium was purified according to the method of Experimental Example 3;

[0171] Wherein, the CHO fermentation medium includes a basal medium and additives;

[0172] The additive includes composition A, which consists of fatty acids and lecithin, and the fatty acid concentration is adjusted to 2 mg / L and the lecithin concentration is 1 mg / L; the fatty acid consists of oleic acid, arachidonic acid, and linolenic acid, wherein the ratio of the three is 2:0.2:1.

[0173] The additive includes composition B, which includes 100 mg / L AEO-9, 2 mg / L insulin, 5 mg / L IGF-I, 5 mg / L transferrin, and 1 mg / L ethanolamine.

[0174] The basic culture medium includes sugars, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), amino acids, vitamins, and inorganic salts.

[0175] Wherein, the sugars include: D-glucose 3000 mg / L, galactose 200 mg / L, and fructose 200 mg / L.

[0176] The HEPES concentration is 1000 mg / L.

[0177] The amino acids include: glycine 20 mg / L, alanine 60 mg / L, isoleucine 20 mg / L, leucine 100 mg / L, arginine 200 mg / L, asparagine 10 mg / L, aspartic acid 20 mg / L, cysteine ​​25 mg / L, glutamic acid 5 mg / L, glutamine 100 mg / L, histidine 25 mg / L, lysine 100 mg / L, methionine 25 mg / L, phenylalanine 15 mg / L, proline 2 mg / L, serine 5 mg / L, threonine 50 mg / L, tryptophan 5 mg / L, tyrosine 10 mg / L, and valine 50 mg / L.

[0178] The vitamins include: vitamin C 0.05 mg / L, vitamin H 0.02 mg / L, vitamin PP 0.1 mg / L, vitamin B6 0.5 mg / L, vitamin B2 0.01 mg / L, vitamin B1 0.5 mg / L, vitamin B12 0.05 mg / L, calcium pantothenate 0.2 mg / L, folic acid 2 mg / L, and inositol 0.5 mg / L.

[0179] The inorganic salts include: sodium chloride 2000 mg / L, sodium bicarbonate 800 mg / L, disodium hydrogen phosphate 100 mg / L, magnesium chloride 25 mg / L, calcium chloride 5 mg / L, ferric nitrate 0.25 mg / L, zinc sulfate 0.5 mg / L, sodium selenate 0.01 mg / L, sodium metasilicate 0.003 mg / L, copper sulfate 0.001 mg / L, chromium sulfate 0.001 mg / L, aluminum chloride 0.001 mg / L, ammonium vanadate 0.001 mg / L, barium acetate 0.001 mg / L, nickel chloride 0.00005 mg / L, stannous chloride 0.00005 mg / L, and manganese chloride 0.00001 mg / L.

[0180] Example 2

[0181] Cell activation: Take the CHO cells in Experimental Example 1 and resuscitate them;

[0182] Cell culture: CHO cells were revived and inoculated at a density of 2.5 × 10 5Cells were added into CHO fermentation medium for cultivation at a controlled temperature of 37 ± 0.1 °C, a pH of 7.0 ± 0.1, a dissolved oxygen content of 50 ± 5%, and a stirring speed of 65 r / min for 13 days. When the cell density in the culture medium reached 5 × 10 6 After the culture medium has reached 1000 μg / ml, 2-3% of the culture medium is fed every 24 h.

[0183] Protein purification: EPO in the culture medium was purified according to the method of Experimental Example 3;

[0184] Wherein, the CHO fermentation medium includes a basal medium and additives;

[0185] The additive includes composition A, which consists of fatty acids and lecithin, and the fatty acid concentration is adjusted to 4 mg / L and the lecithin concentration is 2 mg / L; the fatty acid consists of oleic acid, arachidonic acid, and linolenic acid, wherein the ratio of the three is 1:0.5:1.

[0186] The additive includes composition B, which includes 150 mg / L of AEO-9, 4 mg / L of insulin, 8 mg / L of IGF-I, 6 mg / L of transferrin, and 2 mg / L of ethanolamine.

[0187] The basic culture medium includes sugars, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), amino acids, vitamins, and inorganic salts.

[0188] Wherein, the sugars include: D-glucose 4000 mg / L, galactose 250 mg / L, and fructose 250 mg / L.

[0189] The HEPES concentration is 1000 mg / L.

[0190] The amino acids include: glycine 30 mg / L, alanine 80 mg / L, isoleucine 30 mg / L, leucine 150 mg / L, arginine 300 mg / L, asparagine 20 mg / L, aspartic acid 30 mg / L, cysteine ​​50 mg / L, glutamic acid 10 mg / L, glutamine 200 mg / L, histidine 30 mg / L, lysine 200 mg / L, methionine 30 mg / L, phenylalanine 30 mg / L, proline 5 mg / L, serine 10 mg / L, threonine 60 mg / L, tryptophan 8 mg / L, tyrosine 20 mg / L, and valine 60 mg / L.

[0191] The vitamins include: vitamin C 0.06 mg / L, vitamin H 0.04 mg / L, vitamin PP 0.2 mg / L, vitamin B6 1.0 mg / L, vitamin B2 0.04 mg / L, vitamin B1 1.0 mg / L, vitamin B12 0.1 mg / L, calcium pantothenate 0.5 mg / L, folic acid 5 mg / L, and inositol 1.0 mg / L.

[0192] The inorganic salts include: 2500 mg / L sodium chloride, 1000 mg / L sodium bicarbonate, 200 mg / L disodium hydrogen phosphate, 50 mg / L magnesium chloride, 6 mg / L calcium chloride, 0.5 mg / L ferric nitrate, 1.0 mg / L zinc sulfate, 0.05 mg / L sodium selenate, 0.005 mg / L sodium metasilicate, 0.002 mg / L copper sulfate, 0.002 mg / L chromium sulfate, 0.002 mg / L aluminum chloride, 0.004 mg / L ammonium vanadate, 0.002 mg / L barium acetate, 0.00006 mg / L nickel chloride, 0.00008 mg / L stannous chloride, and 0.00004 mg / L manganese chloride.

[0193] Example 3

[0194] Cell activation: Take the CHO cells in Experimental Example 1 and resuscitate them;

[0195] Cell culture: CHO cells were revived and seeded at a density of 4 × 10 5 Cells were added into CHO fermentation medium for cultivation at a controlled temperature of 38 ± 0.1 °C, a pH of 7.2 ± 0.1, a dissolved oxygen content of 55 ± 5%, and a stirring speed of 70 r / min for 13 days. When the cell density in the culture medium reached 6 × 10 6 After the culture medium has reached 1000 μg / ml, 2-3% of the culture medium is fed every 24 h.

[0196] Protein purification: EPO in the culture medium was purified according to the method of Experimental Example 3;

[0197] Wherein, the CHO fermentation medium includes a basal medium and additives;

[0198] The additive includes composition A, which consists of fatty acids and lecithin, and the fatty acid concentration is adjusted to 6 mg / L and the lecithin concentration is 3 mg / L; the fatty acid consists of oleic acid, arachidonic acid, and linolenic acid, wherein the ratio of the three is 1:1:1.

[0199] The additive includes composition B, which includes 300 mg / L AEO-9, 8 mg / L insulin, 15 mg / L IGF-I, 8 mg / L transferrin, and 5 mg / L ethanolamine.

[0200] The basic culture medium includes sugars, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), amino acids, vitamins, and inorganic salts.

[0201] Wherein, the sugars include: D-glucose 5000 mg / L, galactose 300 mg / L, and fructose 300 mg / L.

[0202] The HEPES concentration is 1000 mg / L.

[0203] The amino acids include: glycine 40 mg / L, alanine 100 mg / L, isoleucine 50 mg / L, leucine 200 mg / L, arginine 400 mg / L, asparagine 30 mg / L, aspartic acid 50 mg / L, cysteine ​​100 mg / L, glutamic acid 20 mg / L, glutamine 300 mg / L, histidine 50 mg / L, lysine 350 mg / L, methionine 50 mg / L, phenylalanine 60 mg / L, proline 10 mg / L, serine 50 mg / L, threonine 100 mg / L, tryptophan 10 mg / L, tyrosine 30 mg / L, and valine 80 mg / L.

[0204] The vitamins include: vitamin C 0.1 mg / L, vitamin H 0.05 mg / L, vitamin PP 1 mg / L, vitamin B6 2.5 mg / L, vitamin B2 0.08 mg / L, vitamin B1 2 mg / L, vitamin B12 0.2 mg / L, calcium pantothenate 2 mg / L, folic acid 10 mg / L, and inositol 5 mg / L.

[0205] The inorganic salts include: 3000 mg / L sodium chloride, 1200 mg / L sodium bicarbonate, 300 mg / L disodium hydrogen phosphate, 100 mg / L magnesium chloride, 10 mg / L calcium chloride, 1.5 mg / L ferric nitrate, 2 mg / L zinc sulfate, 0.1 mg / L sodium selenate, 0.006 mg / L sodium metasilicate, 0.004 mg / L copper sulfate, 0.006 mg / L chromium sulfate, 0.005 mg / L aluminum chloride, 0.005 mg / L ammonium vanadate, 0.003 mg / L barium acetate, 0.0001 mg / L nickel chloride, 0.0001 mg / L stannous chloride, and 0.00005 mg / L manganese chloride.

[0206] Example 4

[0207] It is basically the same as Example 2, except that:

[0208] The sugar does not contain galactose or fructose components;

[0209] The amino acids do not contain proline or tryptophan components;

[0210] The vitamins do not contain an inositol component;

[0211] The inorganic salt does not contain sodium metasilicate and nickel chloride components.

[0212] Example 5

[0213] It is basically the same as Example 2, except that:

[0214] The sugars do not contain fructose components;

[0215] The amino acids do not contain a serine component;

[0216] The vitamins do not contain a vitamin B12 component;

[0217] The inorganic salt does not contain stannous chloride components.

[0218] Example 6

[0219] It is basically the same as Example 2, except that:

[0220] The sugars do not contain fructose components;

[0221] The amino acids do not contain a tyrosine component;

[0222] The vitamins do not contain vitamin B2 components;

[0223] The inorganic salt does not contain copper sulfate components.

[0224] Comparative Example 1

[0225] The CHO fermentation medium was OPM-CHO CD07 medium, purchased from Shanghai Aopuma Biotechnology Co., Ltd.

[0226] Comparative Example 2

[0227] It is basically the same as Example 2, except that:

[0228] The CHO fermentation medium does not contain the composition A proposed in this application.

[0229] Example 7

[0230] The changes in cell density during culture of Examples 1-6 and Comparative Examples 1-2 were recorded respectively, and after the experiment, the amount of EPO protein obtained by fermentation of Examples 1-6 and Comparative Examples 1-5 was calculated. The results are shown in FIG. Figure 5、 Figure 6 shown.

[0231] according to Figure 5-Figure 6 Those skilled in the art will understand that:

[0232] First, compared with Comparative Example 1, the technical solution of the embodiment of the present application shows relatively normal trends in cell density and mechanical changes under normal culture conditions, and has no significant difference from commercial culture medium;

[0233] Second, compared to Comparative Example 1, the technical solution of the embodiment of the present application, under normal culture conditions, the expression level of EPO is not significantly different from that of the commercial culture medium;

[0234] Third, referring to Examples 1-3 and 4-6, the culture medium components of Examples 1-3 were slightly adjusted, and the EPO expression level decreased slightly, but was still within a controllable range;

[0235] Fourth, referring to Example 2 and Comparative Example 2, the expression level of EPO was reduced in the culture medium without Composition A, indicating that Composition A in the additive of the present application also has a certain positive effect on the expression of EPO.

[0236] In summary, the preparation method of long-acting erythropoietin provided in this application is based on the technical problem that CHO cell lines are more sensitive to the external environment, especially temperature changes. By improving the CHO cell culture medium, CHO is made more tolerant to changes in culture conditions during culture, and has good commercial prospects.

[0237] For ordinary technicians in this field, the technical features in the above embodiments can be freely combined, and the formed technical solutions also belong to the embodiments disclosed in this application.

[0238] Furthermore, without departing from the principles of the present application, several improvements and modifications may be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A method for preparing long-acting erythropoietin, characterized in that: Including steps: Cell activation, reviving CHO cell lines capable of expressing recombinant human erythropoietin; Cell culture: add the revived CHO cells to CHO fermentation medium for culture, control the temperature at 36-38°C, pH at 6.8-7.2, and dissolved oxygen content at 40-60%; wherein, the revived CHO cells are inoculated at a density of no more than 6×10 5 pcs / ml were added into the culture medium for cultivation; Protein purification: purify EPO in the culture medium; The CHO fermentation medium includes a basal medium and additives, wherein the additives include composition A and composition B, wherein composition A is composed of fatty acids and lecithin, and the fatty acid concentration is adjusted to 2-6 mg / L and the lecithin concentration is 1-3 mg / L; the composition B is composed of the following components: AEO-9 100-300 mg / L; insulin 2-8 mg / L; IGF-I 5-15 mg / L; transferrin 5-8 mg / L; and ethanolamine 1-5 mg / L. The fatty acids are composed of oleic acid, arachidonic acid and linolenic acid, wherein the ratio of the three is 1-2:0.2-1:

1.

2. The preparation method according to claim 1, wherein: The fatty acid concentration is 2.5-4 mg / L, and the lecithin concentration is 1.5-2 mg / L.

3. The preparation method according to claim 1, wherein: The basic culture medium includes sugars, HEPES, amino acids, vitamins and inorganic salts.

4. The preparation method according to claim 1, wherein: The additive does not include cholesterol, or the cholesterol content does not exceed 0.005 mg / L.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The revived CHO cells were seeded at a density of 2-4×10 5 Cells / ml were added into the culture medium for cultivation.

6. The preparation method according to claim 1, wherein: The protein purification comprises the steps of: a supernatant collection step, wherein cells are separated by centrifugation or filtration, and the supernatant is collected; In the affinity chromatography step, the supernatant is loaded and eluted using an affinity chromatography column; a hydrophobic chromatography step, wherein the affinity chromatography eluate is passed through a hydrophobic chromatography column for elution; In the anion exchange chromatography step, the eluate obtained in the previous step is passed through an anion exchange chromatography column for elution.

7. The preparation method according to claim 6, characterized in that: The hydrophobic chromatography step comprises the steps of: Sample pretreatment was performed by adjusting the affinity eluate with 4 M ammonium sulfate to obtain an affinity eluate containing 1.5 M ammonium sulfate, and then performing hydrophobic chromatography.

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