Preparation method and application of long-acting erythropoietin

Abstract

The invention relates to the technical field of biological medicines, in particular to a preparation method and application of long-acting erythropoietin. Specifically, the invention provides the preparation method of the long-acting erythropoietin, which comprises the following steps: activating cells, and recovering a CHO cell strain capable of expressing recombinant human erythropoietin; cell culture: the recovered CHO cells are added into a CHO fermentation culture medium to be cultured, the temperature is controlled to be 36-38 DEG C, the pH is 6.8-7.2, and the dissolved oxygen content is 40-60%; protein purification: purifying EPO in the culture solution; wherein the CHO fermentation culture medium comprises a basic culture medium and an additive, the additive comprises a composition A, the composition A is composed of fatty acid and lecithin, the concentration of the fatty acid is adjusted to be 2-6 mg / L, and the concentration of the lecithin is adjusted to be 1-3 mg / L. By adopting the technical scheme, a proper amount of fatty acid and lecithin components are supplemented into the additive of the CHO fermentation culture medium, so that the survival rate of CHO cells during culture in a reactor 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 of long-acting erythropoietin and its application. Background Art

[0002] Erythropoietin (EPO) was discovered in 1906. It is a glycoprotein mainly produced by the kidneys with a molecular weight of about 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 the existing technology, DNA recombination technology has been 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] 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 the present application is to provide a preparation method of long-acting erythropoietin and its application, so as 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: Cell activation, reviving CHO cell lines that can express recombinant human erythropoietin; Cell culture: adding the revived CHO cells to CHO fermentation medium for culture, controlling the temperature at 36-38°C, pH at 6.8-7.2, and dissolved oxygen content at 40-60%; Protein purification: purify EPO in the culture medium; 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.

[0008] By adopting the above technical scheme, the survival rate of CHO cells in reactor culture and the stability of protein expression can be improved by adding appropriate amounts of fatty acids and lecithin components to the additives of the CHO fermentation medium.

[0009] Preferably, the fatty acid concentration is 2.5-4 mg / L, and the lecithin concentration is 1.5-2 mg / L.

[0010] Preferably, the fatty acid is composed of oleic acid, arachidonic acid and linolenic acid, wherein the ratio of the three is 1-2:0.2-1:1.

[0011] Preferably, the additive further comprises a composition B, wherein the composition B comprises the following components: AEO-9 100-300mg / L; Insulin 2-8 mg / L; IGF-I 5-15 mg / L; Transferrin 5-8 mg / L; Ethanolamine 1-5mg / L.

[0012] More preferably, the composition B comprises components: AEO-9 150-200mg / L; Insulin 4-6 mg / L; IGF-I 8-12 mg / L; Transferrin 6-7 mg / L; Ethanolamine 2-4mg / L.

[0013] Preferably, the basic culture medium comprises sugars, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), amino acids, vitamins, and inorganic salts.

[0014] Preferably, the sugars include: D-glucose, galactose, fructose or a combination of them.

[0015] Preferably, the sugars include: D-glucose 3000-5000 mg / L, galactose 200-300 mg / L, fructose 200-300 mg / L.

[0016] Preferably, the HEPES concentration is 1000-1500 mg / L.

[0017] Preferably, the amino acids include: 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.

[0018] Preferably, the amino acids include: One or a combination of 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, glutamate 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, valine 50-80 mg / L.

[0019] Preferably, the vitamins include: 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.

[0020] Preferably, the vitamins include: One or a combination of 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.

[0021] Preferably, the inorganic salt comprises: 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, manganese chloride, or a combination of the two or more thereof.

[0022] Preferably, the additive further comprises an inorganic salt, and the inorganic salt comprises: 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 mg / L, zinc sulfate 0.5-2mg / L, sodium selenate 0.01-0.1mg / L, sodium metasilicate 0.003-0.006mg / L, copper sulfate 0.001-0.004mg / L, chromium sulfate 0.001-0.006mg / L, aluminum chloride 0.001-0.005mg / L, ammonium vanadate 0.001-0.005mg / L, barium acetate 0.001-0.003mg / L, nickel chloride 0.00005-0.0001mg / L, stannous chloride 0.00005-0.0001mg / L, manganese chloride 0.00001-0.00005mg / L or a combination of several thereof.

[0023] Preferably, the additive does not include cholesterol, or the cholesterol content does not exceed 0.005 mg / L.

[0024] Preferably, in the cell culturing step, The revived CHO cells were seeded at a density of 2-4×10 5 Each ml was added into the culture medium for cultivation.

[0025] More preferably, in the cell culturing step, The revived CHO cells were seeded at a density of 2.5-3×10 5 Each ml was added into the culture medium for cultivation.

[0026] Preferably, in the cell culture step, the temperature variation does not exceed 0.5°C / 6h.

[0027] Preferably, the CHO cell is CHO-K1.

[0028] Preferably, the expression vector may be one of pQkX2, pSV2, pEGFP-N1, and pCMVp-NEO-BAN. More preferably, the expression vector is pQKX2.

[0029] Preferably, in the cell culture step, when the cell density in the culture medium reaches 4-6×10 6 After that, 2-3% of the culture medium was fed every 24h.

[0030] Preferably, the protein purification comprises the steps of: a supernatant collection step, wherein cells are separated by centrifugation or filtration, and the supernatant is collected; affinity chromatography step, using an affinity chromatography column to load and elute the supernatant; 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.

[0031] Preferably, the affinity chromatography column is Blue Sepharose 6 FF (GE) 1.6×2.5cm 5ml.

[0032] Preferably, the hydrophobic chromatography column is HiTrap Butyl S FF (GE) 1ml.

[0033] Preferably, the hydrophobic chromatography step comprises the steps of: Sample pretreatment was performed by adjusting the affinity eluent with 4 M ammonium sulfate to obtain an affinity eluent containing 1.5 M ammonium sulfate, and then performing hydrophobic chromatography.

[0034] Preferably, the anion exchange chromatography column is DEAE FF (GE) 0.7×2.5cm 1ml.

[0035] 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.

[0036] In summary, this application has the following beneficial effects: 1. The long-acting erythropoietin preparation method provided in the present 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 additives of the culture medium.

[0037] 2. The long-acting erythropoietin preparation method provided in the present application achieves stable growth of CHO cells during culture by controlling the inoculation amount of CHO cells, thereby ensuring the expression of erythropoietin for a longer period of time.

[0038] 3. The method for preparing long-acting erythropoietin provided in the present application can fully exert the effect of the culture medium and maintain the stable growth of CHO cells by controlling the addition of cholesterol components to the culture medium additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a temperature fluctuation cycle based on 37°C and 34°C in Experimental Example 4; Figure 2 It is a temperature fluctuation cycle based on 37°C and 34°C in Experimental Example 5; Figure 3 The cell density after 7 days of culture of technical solution AJ in Experimental Example 7; Figure 4 The EPO expression level after 7 days of culture of technical solution AJ in Experimental Example 7; Figure 5 is the cell density of Example 1-6 and Comparative Example 1-2 (unit: 106 cells / ml); Figure 6 It is the EPO expression level of Example 1-6 and Comparative Example 1-2. DETAILED DESCRIPTION

[0040] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application. Instead, they are merely examples of devices and methods consistent with some aspects of the present application as detailed in the appended claims.

[0041] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0042] The present application will be described in detail below through examples.

[0043] 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.

[0044] In the existing technology, DNA recombination technology has been 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.

[0045] In recent production and testing, the applicant unexpectedly discovered that the constructed CHO cell line that can express rhEPO is sensitive to the external environment, which may affect the final expression of EPO. Specifically, the applicant found in production that when the culture temperature fluctuates drastically, the EPO expression of CHO cells in conventional culture medium will decrease significantly.

[0046] In view of this, the inventive concept of the present application is to provide a method for preparing long-acting erythropoietin, including the steps of: 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%; protein purification, purifying the EPO in the culture medium; wherein the CHO fermentation medium includes a basal medium and an additive, the additive includes a composition A, the composition A is composed of fatty acids and lecithin, and the fatty acid concentration is adjusted to 2-5 mg / L, and the lecithin concentration is adjusted to 1-3 mg / L. By adopting the above technical scheme, by adding an appropriate amount of fatty acids and lecithin components to the additives of the CHO fermentation medium, the survival rate of CHO cells in reactor culture and the stability of protein expression can be improved.

[0047] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with specific implementation methods. Those skilled in the art should also understand that the reaction time and drug feeding involved in this application cannot be absolutely accurate in the actual production process, but are all within the allowable error range. For example, if you expect to heat the sample for 30 minutes, the actual operation may be 1 second more or less than 1 second in 30 minutes; if you expect to weigh 30g of sample, you may actually weigh 30.001g or 29.998g.

[0048] Experimental Example 1 CHO cell line capable of expressing recombinant human erythropoietin

[0049] Unless otherwise specified, the CHO cells in the experimental examples and embodiments of the present application are the cell lines described in the experimental examples. The CHO cells are selected from CHO-K1 cells, which contain an expression vector pQKX2, and the expression vector has a gene sequence capable of expressing EPO units, and the gene sequence is SEQ ID NO.1 in the prior art CN111019907B disclosed by the applicant.

[0050] Specifically, the CHO cells are also prepared using the prior art CN111019907B method: Prepare a cloning vector containing the sequence SEQ ID NO.1 and add corresponding restriction sites; prepare an expression vector pQKX2; use restriction endonucleases SapI and EcoRI to simultaneously digest the cloning vector and the expression vector; after digestion of the cloning vector, generate a target sequence, the target sequence includes the sequence SEQ ID NO.1; connect the target sequence to the expression vector after digestion to obtain a recombinant expression vector; The recombinant expression vector was cut with PvuI to obtain a linearized recombinant plasmid, and the linearized recombinant plasmid was transfected into CHO cells using a Bio-Rad electroporator to obtain CHO cells capable of expressing recombinant EPO.

[0051] Experimental Example 2 Blank Control CHO Cell Line CHO-K1 cells were purchased from ATCC (58995535).

[0052] Experimental Example 3 EPO protein purification method The EPO protein purification steps include: 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; wherein the affinity chromatography column is Blue Sepharose 6 FF (GE) 1.6×2.5cm 5ml; In the hydrophobic chromatography step, the affinity eluent is adjusted with 4M ammonium sulfate to obtain an affinity eluent containing 1.5M ammonium sulfate, and the affinity eluent is eluted through a hydrophobic chromatography column; wherein the hydrophobic chromatography column is HiTrap Butyl S FF (GE) 1ml; In the anion exchange chromatography step, the eluate obtained in the previous step is eluted through an anion exchange chromatography column; wherein the anion exchange chromatography column is DEAE FF (GE) 0.7×2.5cm 1ml.

[0053] Experimental Example 4 Temperature Fluctuation Simulation Test 1 The revived CHO cells were seeded at a density of 2.5×10 5 Each / ml was 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 60-70r / min, and the culture was carried out for 7 days.

[0054] The initial culture temperature was 37°C. 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.

[0055] according to Figure 1 Example of a temperature fluctuation cycle between 37°C and 34°C.

[0056] Experimental Example 5 Temperature Fluctuation Simulation Test 2 The revived CHO cells were seeded at a density of 2.5×10 5Each / ml was 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 60-70r / min, and the culture was carried out for 7 days.

[0057] The initial culture temperature was 37°C. 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.

[0058] according to Figure 2 Example of a temperature fluctuation cycle between 37°C and 34°C.

[0059] Experimental Example 6 Conventional culture test The revived CHO cells were seeded at a density of 2.5×10 5 Each cell / ml was added to CHO fermentation medium for cultivation. The temperature was controlled at 37±0.1℃, pH was 7.0±0.1, dissolved oxygen content was 50±5%, stirring speed was set at 60-70r / min, and cultivation was carried out for 7 days.

[0060] When the cell density in the culture medium reached 5×10 6 After that, 2% culture medium was supplemented every 24 h.

[0061] Experimental Example 7 CHO cell culture test in different culture media

[0062] Technical Solution A CHO fermentation medium OPM-CHO CD07 was purchased from Shanghai Aopuma Biotechnology Co., Ltd.

[0063] Technical Solution B CHO fermentation medium DMEM / F12 was purchased from GIBCO.

[0064] Technical Solution C CHO fermentation medium, comprising: Basic culture medium, including: sugars, HEPES, amino acids, vitamins, inorganic salts; Wherein, the sugars include: D-glucose 4000 mg / L, galactose 250 mg / L, and fructose 250 mg / L.

[0065] The HEPES concentration is 1000 mg / L.

[0066] The amino acids include: 30 mg / L glycine, 80 mg / L alanine, 30 mg / L isoleucine, 150 mg / L leucine, 300 mg / L arginine, 20 mg / L asparagine, 30 mg / L aspartic acid, 50 mg / L cysteine, 10 mg / L glutamic acid, 200 mg / L glutamine, 30 mg / L histidine, 200 mg / L lysine, 30 mg / L methionine, 30 mg / L phenylalanine, 5 mg / L proline, 10 mg / L serine, 60 mg / L threonine, 8 mg / L tryptophan, 20 mg / L tyrosine, and 60 mg / L valine.

[0067] 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.

[0068] 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.

[0069] Technical Solution D CHO fermentation medium, comprising: Basic culture medium, the same as that of technical solution C; Additives, including composition B; 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.

[0070] Technical Solution E CHO fermentation medium, comprising: Basic culture medium, the same as that of technical solution C; Additives, including composition A and composition B; The composition A comprises: 1 mg / L oleic acid, 0.5 mg / L arachidonic acid, and 1 mg / L linolenic acid; 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.

[0071] Technical Solution F CHO fermentation medium, comprising: Basic culture medium, the same as that of technical solution C; Additives, including composition A and composition B; The composition A comprises: 1 mg / L oleic acid, 1 mg / L linolenic acid, and 2 mg / L lecithin; 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.

[0072] Technical Solution G CHO fermentation medium, comprising: Basic culture medium, the same as that of technical solution C; Additives, including composition A and composition B; 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; 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.

[0073] Technical Solution H CHO fermentation medium, comprising: Basic culture medium, the same as that of technical solution C; Additives, including composition A and composition B; The composition A comprises: 1 mg / L oleic acid, 0.5 mg / L arachidonic acid, 1 mg / L linolenic acid, 2 mg / L lecithin, and 1 mg / L cholesterol; 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.

[0074] Technical Solution I CHO fermentation medium, comprising: Basic culture medium, the same as that of technical solution C; Additives, including composition A and composition B; The composition A comprises: 1 mg / L oleic acid, 0.5 mg / L arachidonic acid, 1 mg / L linolenic acid, 2 mg / L lecithin, and 0.005 mg / L cholesterol; 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.

[0075] Technical Solution J It is basically the same as technical solution G, except that: The revived CHO cells were seeded at a density of 6×10 5 Each ml was added into CHO fermentation medium for cultivation.

[0076] The CHO fermentation medium provided by Technical Scheme A to Technical Scheme J was applied to the culture conditions in Experimental Example 4, Example 5, and Example 6, and the method of Experimental Example 3 was used to purify the EPO protein, and the amount of the purified protein was calculated.

[0077] The cell density of technical scheme A-technical scheme J after 7 days of culture is shown in Figure 3 shown.

[0078] The EPO expression levels of technical solutions A to J after 7 days of culture are shown in Figure 4 shown.

[0079] The research basis of this application is that the applicant found that the temperature fluctuated greatly due to unexpected circumstances during the fermentation of the strain, and after the temperature fluctuation, the production of EPO 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.

[0080] according to Figure 3-Figure 4 Those skilled in the art will understand that: First, under the temperature fluctuation environment, the cell growth state and EPO expression level of the experimental CHO cells under the culture conditions of commercially available culture medium (technical solution A, technical solution B) are significantly lower than those of the culture medium provided in this application (technical solution CJ), indicating that the culture medium and preparation method provided in this application are more tolerant to external environment fluctuations, especially temperature fluctuations; 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); 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. If there is no special requirement, 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 be higher than 0.005 mg / L.

[0081] 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 certain adverse effects. 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 be higher than 6×10 5 / ml, or more preferably not more than 6×10 5 Pieces / ml.

[0082] Example 1 Cell activation: Take the CHO cells in Experimental Example 1 and resuscitate them; Cell culture: The revived CHO cells were inoculated at a density of 2×10 5 The cells were added into CHO fermentation medium for cultivation at a controlled temperature of 36±0.1℃, a pH of 6.8±0.1, a dissolved oxygen content of 45±5%, and a stirring speed of 60r / min for 13 days. When the cell density in the medium reached 4×10 6 After reaching 100 μg / ml, feed 2-3% of the culture medium every 24 hours; Protein purification: purify the EPO in the culture medium according to the method of Experimental Example 3; Wherein, the CHO fermentation medium includes a basal medium and additives; The additive comprises composition A, which is composed of fatty acids and lecithin, and the fatty acid concentration is adjusted to 2 mg / L and the lecithin concentration is adjusted to 1 mg / L; the fatty acid is composed of oleic acid, arachidonic acid and linolenic acid, wherein the ratio of the three is 2:0.2:1.

[0083] The additive comprises composition B, and the composition B comprises 100 mg / L AEO-9, 2 mg / L insulin, 5 mg / L IGF-I, 5 mg / L transferrin, and 1 mg / L ethanolamine.

[0084] The basic culture medium comprises sugars, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), amino acids, vitamins and inorganic salts.

[0085] Wherein, the sugars include: D-glucose 3000 mg / L, galactose 200 mg / L, and fructose 200 mg / L.

[0086] The HEPES concentration is 1000 mg / L.

[0087] 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.

[0088] 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.

[0089] The inorganic salts include: 2000 mg / L sodium chloride, 800 mg / L sodium bicarbonate, 100 mg / L disodium hydrogen phosphate, 25 mg / L magnesium chloride, 5 mg / L calcium chloride, 0.25 mg / L ferric nitrate, 0.5 mg / L zinc sulfate, 0.01 mg / L sodium selenate, 0.003 mg / L sodium metasilicate, 0.001 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, 0.00005 mg / L nickel chloride, 0.00005 mg / L stannous chloride, and 0.00001 mg / L manganese chloride.

[0090] Example 2 Cell activation: Take the CHO cells in Experimental Example 1 and resuscitate them; Cell culture: The revived CHO cells were inoculated at a density of 2.5×10 5 The cells were added into CHO fermentation medium for cultivation at a controlled temperature of 37±0.1℃, pH of 7.0±0.1, dissolved oxygen content of 50±5%, stirring speed of 65r / min, and cultured for 13 days. When the cell density in the culture medium reached 5×10 6 After reaching 100 μg / ml, feed 2-3% of the culture medium every 24 hours; Protein purification: purify the EPO in the culture medium according to the method of Experimental Example 3; Wherein, the CHO fermentation medium includes a basal medium and additives; The additive comprises 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 adjusted to 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.

[0091] The additive comprises composition B, and the composition B comprises 150 mg / L AEO-9, 4 mg / L insulin, 8 mg / L IGF-I, 6 mg / L transferrin, and 2 mg / L ethanolamine.

[0092] The basic culture medium comprises sugars, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), amino acids, vitamins and inorganic salts.

[0093] Wherein, the sugars include: D-glucose 4000 mg / L, galactose 250 mg / L, and fructose 250 mg / L.

[0094] The HEPES concentration is 1000 mg / L.

[0095] The amino acids include: 30 mg / L glycine, 80 mg / L alanine, 30 mg / L isoleucine, 150 mg / L leucine, 300 mg / L arginine, 20 mg / L asparagine, 30 mg / L aspartic acid, 50 mg / L cysteine, 10 mg / L glutamic acid, 200 mg / L glutamine, 30 mg / L histidine, 200 mg / L lysine, 30 mg / L methionine, 30 mg / L phenylalanine, 5 mg / L proline, 10 mg / L serine, 60 mg / L threonine, 8 mg / L tryptophan, 20 mg / L tyrosine, and 60 mg / L valine.

[0096] 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.

[0097] 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.

[0098] Example 3 Cell activation: Take the CHO cells in Experimental Example 1 and resuscitate them; Cell culture: CHO cells were revived and inoculated at a density of 4 × 10 5 The 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 medium reached 6 × 10 6 After reaching 100 μg / ml, feed 2-3% of the culture medium every 24 hours; Protein purification: purify the EPO in the culture medium according to the method of Experimental Example 3; Wherein, the CHO fermentation medium includes a basal medium and additives; 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 adjusted to 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.

[0099] The additive comprises composition B, and the composition B comprises 300 mg / L AEO-9, 8 mg / L insulin, 15 mg / L IGF-I, 8 mg / L transferrin, and 5 mg / L ethanolamine.

[0100] The basic culture medium comprises sugars, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), amino acids, vitamins and inorganic salts.

[0101] Wherein, the sugars include: D-glucose 5000 mg / L, galactose 300 mg / L, and fructose 300 mg / L.

[0102] The HEPES concentration is 1000 mg / L.

[0103] The amino acids include: 40 mg / L glycine, 100 mg / L alanine, 50 mg / L isoleucine, 200 mg / L leucine, 400 mg / L arginine, 30 mg / L asparagine, 50 mg / L aspartic acid, 100 mg / L cysteine, 20 mg / L glutamic acid, 300 mg / L glutamine, 50 mg / L histidine, 350 mg / L lysine, 50 mg / L methionine, 60 mg / L phenylalanine, 10 mg / L proline, 50 mg / L serine, 100 mg / L threonine, 10 mg / L tryptophan, 30 mg / L tyrosine, and 80 mg / L valine.

[0104] 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.

[0105] 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.

[0106] Example 4 It is basically the same as Example 2, except that: The sugar does not contain galactose or fructose components; The amino acids do not contain proline or tryptophan components; The vitamins do not contain an inositol component; The inorganic salt does not contain sodium metasilicate and nickel chloride components.

[0107] Example 5 It is basically the same as Example 2, except that: The sugar does not contain a fructose component; The amino acids do not contain a serine component; The vitamins do not contain a vitamin B12 component; The inorganic salt does not contain stannous chloride components.

[0108] Example 6 It is basically the same as Example 2, except that: The sugar does not contain a fructose component; The amino acids do not contain a tyrosine component; The vitamins do not contain vitamin B2 components; The inorganic salt does not contain a copper sulfate component.

[0109] Comparative Example 1 The CHO fermentation medium was OPM-CHO CD07 medium, which was purchased from Shanghai Aopuma Biotechnology Co., Ltd.

[0110] Comparative Example 2 It is basically the same as Example 2, except that: The CHO fermentation medium does not contain the composition A proposed in the present application.

[0111] Example 7 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 Figure 5 , Figure 6 shown.

[0112] according to Figure 5-Figure 6 Those skilled in the art will understand that: First, compared with Comparative Example 1, the technical solution of the embodiment of the present application shows that under normal culture conditions, the cell density and mechanical change trends are relatively normal, and there is no significant difference from the commercial culture medium; Second, compared with 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; 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; Fourth, referring to Example 2 and Comparative Example 2, in the culture medium without composition A, the expression of EPO was reduced, indicating that composition A in the additive in the present application also has a certain positive effect on the expression of EPO.

[0113] In summary, the method for preparing long-acting erythropoietin provided in the present 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.

[0114] 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.

[0115] 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 protection scope of the claims of the present application.

Claims

1. A method for preparing a long-acting erythropoietin, characterized in that: Includes steps: Cell activation, reviving CHO cell lines that can express recombinant human erythropoietin; Cell culture: adding the revived CHO cells to CHO fermentation medium for culture, controlling the temperature at 36-38°C, pH at 6.8-7.2, and dissolved oxygen content at 40-60%; Protein purification: purify EPO in the culture medium; 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.

2. The preparation method according to claim 1, characterized in that: 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, characterized in that: 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.

4. The preparation method according to claim 1, characterized in that: The additive also includes a composition B, which includes components: AEO-9 100-300mg / L; Insulin 2-8 mg / L; IGF-I 5-15 mg / L; Transferrin 5-8 mg / L; Ethanolamine 1-5mg / L.

5. The preparation method according to claim 1, characterized in that: The basic culture medium comprises sugars, HEPES, amino acids, vitamins and inorganic salts.

6. The preparation method according to claim 1, characterized in that: The additive does not include cholesterol, or the cholesterol content does not exceed 0.005 mg / L.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The revived CHO cells were seeded at a density of 2-4×10 5 Each ml was added into the culture medium for cultivation.

8. The preparation method according to claim 1, characterized in that: The protein purification comprises the steps of: a supernatant collection step, wherein cells are separated by centrifugation or filtration, and the supernatant is collected; affinity chromatography step, using affinity chromatography column to load and elute the supernatant; 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.

9. The preparation method according to claim 8, characterized in that: The hydrophobic chromatography step comprises the steps of: Sample pretreatment was performed by adjusting the affinity eluent with 4 M ammonium sulfate to obtain an affinity eluent containing 1.5 M ammonium sulfate, and then performing hydrophobic chromatography.

10. Use of the long-acting erythropoietin according to any one of claims 1 to 9 in treating anemia symptoms.

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