Serum-free culture medium for industrial production of human erythropoietin and culture method

Abstract

The invention discloses a serum-free culture medium for industrial production of human erythropoietin and a culture method, the serum-free culture medium comprises a basic culture medium and an additive, the basic culture medium is a DMEM / F12 culture medium, and the additive comprises the following components: sodium n-butyrate, NaHCO3, yeast hydrolysate, D-glucose, D-mannose and D-galactose; the DMEM / F12 culture medium contains a zwitterionic buffer agent HEPES, and the zwitterionic buffer agent HEPES and bicarbonate in the additive form a double-buffer system. The expression intensity of EPO can be effectively improved, and the in-vivo and in-vitro activity of EPO can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to a serum-free culture medium for industrial production of human erythropoietin and a culture method thereof. Background Art

[0002] The prevalence of chronic kidney disease (CKD) in my country has been increasing year by year. According to official statistics, the overall prevalence of CKD in China is 10.8%, and the number of patients with CKD is about 120 million. Among them, renal anemia is the most common complication that affects the quality of life of CKD patients. There are about 3.8 million new cases of cancer in the country each year, and chemotherapy is still the most important means of cancer treatment. About 50% of chemotherapy patients will experience anemia.

[0003] Human erythropoietin, also known as human erythropoietin (EPO), is a glycoprotein hormone that promotes the proliferation, differentiation and maturation of erythroid progenitor cells. It is produced in the kidneys, secreted into the blood circulation, and acts on erythroid progenitor cells in the bone marrow. Recombinant human erythropoietin (rhEPO) is mainly used to treat anemia caused by chronic renal failure, anemia caused by cancer and bone marrow failure, anemia caused by radiotherapy and chemotherapy, anemia associated with immune diseases (including AIDS, etc.), and blood reserves due to autologous blood transfusion. It also has certain therapeutic effects on myelodysplastic syndrome (MDS), aplastic anemia, anemia of premature infants, hematopoietic stem cell transplantation, etc.

[0004] EPO contains 165 amino acids, with a relative molecular weight of about 34KD, and has 4 glycosylation sites, including 3 N-glycosylation sites (Asn24, 38, 83) and 1 O-glycosylation site (Ser126), and is connected by 2 disulfide bonds (Cysteine6-161, Cysteine29-33). About 60% of it is protein (single polypeptide chain) and about 40% is carbohydrate. It has been reported that the sugar content in the EPO molecule is related to its biological activity in vivo. Glycosylation plays an important role in stabilizing the in vivo activity of EPO. If there is a lack of sugar chains, EPO will be rapidly hydrolyzed and inactivated in the body.

[0005] The gene of erythropoietin is constructed into Chinese hamster ovary cells (CHO cells) using DNA recombination technology to form CHO cells that efficiently express the recombinant human erythropoietin gene. After cell amplification and culture, the expression culture medium is replaced, the culture supernatant is collected, and the target glycoprotein-EPO is prepared by separation and purification. The use of culture medium has an important influence on the expression amount and glycosylation stability of EPO.

[0006] At present, in terms of culture medium application, recombinant human erythropoietin production cell lines are mainly expanded and cultured in culture medium containing 8%-10% bovine serum. However, serum culture medium has many shortcomings, such as complex serum components and difficulty in separation and purification; serum may contain mycoplasma, viruses or other unknown components, which has certain risks. The source of blood makes it difficult to maintain consistency in quality. Therefore, serum-free culture technology has begun to attract people's attention.

[0007] Serum-free medium is generally considered to be composed of two parts: one part is a basal medium containing a variety of nutrients, and the other part is a variety of supplementary factors that can replace serum. The basal medium should contain a variety of nutrients, and the rich nutrients can better maintain a series of life activities such as cell growth, development and reproduction. At present, MEM, DMEM, etc. are widely used. Supplementary factors should be a general term for various factors that can replace the role of serum in the culture medium to maintain cell growth, development and reproduction, which mainly include growth factors and hormones, binding proteins, enzyme inhibitors and various trace elements.

[0008] Patent document CN118389402A discloses a serum-free culture medium for CHO cells to efficiently express erythropoietin. The serum-free culture medium includes a growth medium and an additive. The growth medium is composed of DMEM culture medium and BPT6N culture medium in a volume ratio of 1:9, and contains D-galactose, D-mannose, N-acetylglucosamine and D-glucose. The culture method includes a 1:5 subculture and expansion in the square bottle culture stage, a subculture and expansion every 2 to 4 days, at least 3 subcultures, and then all are transferred to a rotating bottle for culture. After 4 to 6 days, 1×10 9 The cells / mL inoculation density was inoculated into a small reactor and the culture was terminated on the 60th day or when the liquid activity was harvested to be lower than 4000 IU / mL or the daily sugar consumption was lower than 40 g / day.

[0009] Patent document CN102268402A discloses a serum-free culture medium and a culture method for efficiently expressing erythropoietin in CHO cells. The basal culture medium is DMEM culture medium and CHO-S-SFMII culture medium, and the ratio of the number of parts is 1:9. The additives are D-glucose and sodium butyrate, and also contain one or more of the additives D-galactose, D-mannose, and N-acetylglucosamine. The culture method includes first culturing the activated seed cells with serum-containing culture medium, and then culturing them without serum with the serum-free culture medium. By using the culture medium and culture method of the present invention, it is possible to obtain recombinant human erythropoietin protein with high efficiency and stable expression, and to increase the degree of glycosylation of erythropoietin, that is, to increase the proportion of EPO sialic acid.

[0010] Patent document CN104130971A discloses a serum-free culture medium for recombinant human erythropoietin, its application and a method for preparing recombinant human erythropoietin. The basal culture medium in the serum-free culture medium is CHO SFM culture medium, and the additives are 4-hydroxyethylpiperazineethanesulfonic acid, NaHCO3 and soy protein hydrolyzate; it also contains sodium butyrate 3, D-glucose, D-mannose, D-galactose, and N-acetylglucosamine. The culture medium increases the expression of CHO cells in the serum-free culture stage, increases the proportion of CHO cells expressing highly active EPO in the serum-free culture stage, prolongs the survival time of cells in the serum-free state, and increases the yield of EPO.

[0011] Patent document CN103205478A discloses an industrial production method of recombinant human erythropoietin, including amplification culture and separation and purification, wherein the amplification culture adopts a serum-free culture medium, which is a culture medium formed by adding 2-4% Ultroser G, 250-300 mg / L sodium butyrate and 2-3 mg / L riboflavin to DMEM. The invention adopts serum-free culture throughout the cell culture process.

[0012] Ham's F-12 cell culture medium was originally used for serum-free culture of CHO cells. It contains some components that are not found in traditional basal culture medium, such as putrescine, hypoxanthine, thymidine, zinc, etc. F12 can also be mixed with DMEM in equal volumes to obtain a product that combines high concentration with diversified components. It is the basis of serum-free culture medium and is also suitable for mammalian cell culture with low serum content. It is currently widely used in the growth of various mammalian cells, including MDCK cells, glial cells, fibroblasts, human endothelial cells and rat fibroblasts, etc. After searching, no document has been found to disclose the use of DMEM / F12 as a basal culture medium for CHO cells to express erythropoietin.

[0013] In industrial production, long cell culture time, low cell activity and low expression rate mean long production cycle, high cost and waste of raw materials. Therefore, if the density of CHO cells can be quickly increased, the expression intensity of EPO and its in vivo and in vitro activity can be improved, it will not only bring great economic benefits to enterprises, but also meet the sustainable development requirements of conservation and environmental protection, and form good social benefits. Summary of the invention

[0014] In view of this, in order to solve the above technical problems, the purpose of the present invention is to research and develop a serum-free culture medium and culture method for industrial production of human erythropoietin, which is a fully serum-free culture medium, replacing the previous method of first culturing cells with serum and then transferring them to serum-free culture in a tank. It can not only reduce the components and simplify the operation, but also effectively improve the expression intensity of EPO and improve the in vivo and in vitro activity of EPO.

[0015] The technical solutions adopted are:

[0016] The invention discloses a serum-free culture medium for industrial production of human erythropoietin, comprising a basal culture medium and an additive, wherein the basal culture medium is a DMEM / F12 culture medium, and the additive is composed of the following components: sodium butyrate, NaHCO3, yeast hydrolyzate, D-glucose, D-mannose and D-galactose; the DMEM / F12 culture medium contains a zwitterionic buffer HEPES, which together with bicarbonate in the additive forms a double buffer system.

[0017] Furthermore, the DMEM / F12 culture medium also contains sodium hypoxanthine, thymidine and linoleic acid, and putrescine or putrescine hydrochloride.

[0018] Furthermore, the concentration of the DMEM / F12 culture medium is 15-25 g / L, and the dosage of the additives is 0.06-0.15 g / L sodium butyrate, 0.50-2.00 g / L NaHCO3, 0.1-1.0 g / L yeast hydrolyzate, 1.0-3.0 g / L D-glucose, 0.1-0.5 g / L D-mannose and 0.1-0.5 g / L D-galactose.

[0019] Furthermore, the pH of the serum-free culture medium is 6.6-7.6, the temperature is 35-39° C., and the dissolved oxygen content is 30%-90%.

[0020] Furthermore, the concentration of the DMEM / F12 culture medium is 16-18 g / L.

[0021] Furthermore, sodium butyrate 0.05-0.12 g / L, NaHCO3 0.80-1.60 g / L, yeast hydrolyzate 0.15-0.35 g / L.

[0022] Furthermore, the concentration of D-glucose is 1.4-2.6 g / L, D-mannose is 0.15-0.25 g / L, and D-galactose is 0.15-0.25 g / L.

[0023] A method for culturing CHO cells to efficiently express erythropoietin of the present invention comprises the following steps:

[0024] First, serum-free culture is performed using the serum-free medium described in any of the above schemes. During the square bottle culture stage, the culture is subcultured and expanded at a ratio of 1:6, and the culture is subcultured and expanded every 2 to 4 days, at least 3 times and no more than 5 generations;

[0025] Then all cells were transferred to roller bottles for culture. After 4 to 6 days, the number of cells was 1 to 2 × 10 9The cells in the spinner flask were transferred to a cell tank for continuous perfusion culture. When the perfusion acceleration of the serum-free medium was less than 20 ml / h / L or the extracellular activity of the harvested liquid was less than 3000 IU / mL, the perfusion culture was stopped.

[0026] Furthermore, the culture conditions of the continuous perfusion culture in the cell tank are: pH 6.6-7.6, temperature 35-39° C., and dissolved oxygen content 30%-90%.

[0027] Furthermore, the culture conditions of the continuous perfusion culture in the cell tank are: pH 7.0-7.4, temperature 36-38° C., and dissolved oxygen content 40%-80%.

[0028] Increasing the expression level of EPO can usually be achieved through a variety of methods, among which developing a new culture medium that efficiently expresses EPO is one of the ways.

[0029] Cell culture is an important part of producing recombinant proteins, which directly affects the level and quality of protein expression. In the process of recombinant protein production, the culture medium provides nutrients for cell growth and protein expression and a suitable culture environment. Therefore, the selection of a suitable basal culture medium is crucial for industrial-grade large-scale production. The present invention develops a novel serum-free serum-free culture medium for industrial production of human erythropoietin, which replaces the existing staged culture, merges the serum-containing and serum-free culture stages, and resuscitates, passages, and tanks CHO without adding newborn calf serum, while increasing the expression of EPO.

[0030] The basal medium of the present invention adopts DMEM / F12 culture medium. It is found through experiments that the serum-free culture medium composed of the basal medium and additives can not only quickly increase the density of CHO cells, but also increase the expression of EPO, prolong the cell growth cycle, and finally add glycosylation reagents (such as D-glucose, D-mannose and D-galactose) to increase the glycosylation degree of EPO. The interaction of each component of the serum-free culture medium can play a role in increasing the density of CHO cells, increasing the expression intensity of EPO and increasing the in vivo and in vitro activity of EPO, and can also increase the volume of harvested liquid. At the same time, the purified stock solution of the serum-free culture medium harvested liquid has the advantages of high stability and stable quality, which can ensure the quality of recombinant EPO biological products.

[0031] The present invention adopts serum-free culture in the whole process of cell culture, thus avoiding the disadvantages caused by serum culture, and at the same time prolonging the harvest period, increasing the harvest amount and improving the yield.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The DMEM / F12 culture medium of the present invention contains a zwitterionic buffer HEPES, which forms a dual buffer system with the bicarbonate in the additive. HEPES is an excellent biological buffer that has no toxic effect on cells. The culture medium containing the dual buffer system composed of HEPES and the bicarbonate in the additive can maintain a constant pH range for a long time, which can effectively prevent the pH fluctuation of the culture solution from having an adverse effect on cell growth.

[0034] (2) Compared with the patent documents CN118389402A, CN102268402A, CN104130971A, and CN103205478A, DMEM / F12 culture medium is supplemented with sodium hypoxanthine, thymidine, linoleic acid, and putrescine (or putrescine hydrochloride) and other additives to replace serum and protein components, promote rapid cell growth, and rapidly increase the density of CHO cells.

[0035] (3) Use of non-animal source protein hydrolysate: Yeast hydrolysate replaces animal tissue hydrolysate to overcome the shortcomings of animal tissue protein hydrolysate and plant protein hydrolysate, which have uncertain chemical composition and affect the purification of target protein. Yeast hydrolysate is added to the culture medium of the present invention as a non-animal source protein hydrolysate, which can support cell growth and promote protein expression.

[0036] (4) Based on the combined effects of the various components in the serum-free culture medium of the present invention, the CHO cell culture medium with clear chemical composition of the present invention is more conducive to cell growth, easy to purify and downstream process, and increases the expression of EPO, which has significant technical advantages.

[0037] (5) The buffer system is also an important component of cell culture medium. Although amino acids and multivalent ions can provide a certain buffering capacity, cell culture medium still requires a strong buffering agent to maintain the pH of the culture medium. Many traditional cell culture media, such as DMEM and RPMI-1640, use a bicarbonate buffer system (CO2 / NaHCO3). In addition, the phosphate ions contained in the culture medium are also an important source of buffering capacity. However, in order to improve the stability of the pH value, DMEM / F12 culture medium also contains zwitterionic buffers, such as HEPES, which provides strong buffering capacity in the pH range of 7.0-7.4. The buffer system of DMEM / F12 culture medium contains the zwitterionic buffer HEPES, which forms a dual buffer system with bicarbonate, which can improve cell activity and maintain a more stable growth cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the curve diagram of CHO cell density change in different serum-free culture media;

[0039] Figure 2 This is a comparison chart of protein content in different serum-free culture media;

[0040] Figure 3 IEF+Western-blot images of supernatants in different serum-free culture media;

[0041] Figure 4 It is the curve diagram of in vitro activity change in different serum-free culture media;

[0042] Figure 5 It is the curve diagram of CHO cell density change in different buffer systems;

[0043] Figure 6 This is the result of EPO imaging capillary isoelectric focusing electrophoresis in XP-1 culture medium;

[0044] Figure 7 This is the result of EPO imaging capillary isoelectric focusing electrophoresis in XP-2 culture medium;

[0045] Figure 8 This is the result of EPO imaging capillary isoelectric focusing electrophoresis in XP-3 culture medium;

[0046] Fig. 9 This is the result of EPO imaging capillary isoelectric focusing electrophoresis in DC-1 culture medium. DETAILED DESCRIPTION

[0047] The composition of the DMEM / F12 medium used in the present invention is as shown in Table 6:

[0048]

[0049] Continuation of Table 6:

[0050]

[0051] Putrescine 2HCl is putrescine hydrochloride, also known as 1,4-butanediamine dihydrochloride. HEPES is 4-hydroxyethylpiperazineethanesulfonic acid.

[0052] The percentage of dissolved oxygen used in the present invention is a volume percentage, which indicates the volume of dissolved oxygen per 1 volume of culture medium.

[0053] The L in “perfusion acceleration is less than 20 ml / h / L” is the effective volume in the cell tank.

[0054] Tris-HCl buffer is tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0055] Basic culture media such as DMEM, CHO S-SFMII, RPMI-1640, etc. can be obtained through commercial means or conventional technical means in the field.

[0056] The present invention is further described below through the description of embodiments, comparative examples and experimental tests, but this is not a limitation of the present invention. Any non-substantial changes and substitutions made by those skilled in the art on the basis of the present invention shall fall within the scope of protection claimed by the present invention.

[0057] Among them, experimental methods without specifying specific conditions are usually carried out according to conventional conditions in the art or according to conditions recommended by the manufacturer; materials and reagents involved in the present invention, unless otherwise specified, can be obtained through commercial means or conventional technical means in the art.

[0058] Example 1

[0059] A serum-free culture medium for industrial production of human erythropoietin in this embodiment comprises a basal culture medium and additives, wherein the basal culture medium is a DMEM / F12 culture medium with a concentration of 17 g / L;

[0060] The additives are composed of the following ingredients and their contents:

[0061] D-glucose 2.00g / L, D-mannose 0.21g / L, D-galactose 0.21g / L, sodium butyrate 0.08g / L, yeast hydrolysate 0.25g / L, NaHCO3 1.30g / L.

[0062] Example 2

[0063] A serum-free culture medium for industrial production of human erythropoietin in this embodiment comprises a basal culture medium and additives, wherein the basal culture medium is a DMEM / F12 culture medium with a concentration of 17 g / L;

[0064] The additives are composed of the following ingredients and their contents:

[0065] D-glucose 1.50g / L, D-mannose 0.23g / L, D-galactose 0.23g / L, sodium butyrate 0.10g / L, yeast hydrolysate 0.30g / L, NaHCO3 1.50g / L.

[0066] Example 3

[0067] A serum-free culture medium for industrial production of human erythropoietin in this embodiment comprises a basal culture medium and additives, wherein the basal culture medium is a DMEM / F12 culture medium with a concentration of 17 g / L;

[0068] The additives are composed of the following ingredients and their contents:

[0069] D-glucose 1.75g / L, D-mannose 0.22g / L, D-galactose 0.22g / L, sodium butyrate 0.09g / L, yeast hydrolysate 0.28g / L, NaHCO3 1.40g / L.

[0070] Example 4

[0071] A serum-free culture medium for industrial production of human erythropoietin in this embodiment comprises a basal culture medium and additives, wherein the basal culture medium is a DMEM / F12 culture medium with a concentration of 17 g / L;

[0072] The additives are composed of the following ingredients and their contents:

[0073] D-glucose 2.25g / L, D-mannose 0.20g / L, D-galactose 0.20g / L, sodium butyrate 0.07g / L, yeast hydrolysate 0.23g / L, NaHCO3 1.20g / L.

[0074] Example 5

[0075] A serum-free culture medium for industrial production of human erythropoietin in this embodiment comprises a basal culture medium and additives, wherein the basal culture medium is a DMEM / F12 culture medium with a concentration of 17 g / L;

[0076] The additives are composed of the following ingredients and their contents:

[0077] D-glucose 2.5g / L, D-mannose 0.19g / L, D-galactose 0.19g / L, sodium butyrate 0.06g / L, yeast hydrolysate 0.20g / L, NaHCO3 1.10g / L.

[0078] Comparative Example 1

[0079] Compared with Example 1, the only difference of this comparative example is that the basic culture medium of this comparative example is DMEM, and DMEM / F12 culture medium is not used.

[0080] Comparative Example 2

[0081] Compared with Example 1, the only difference of this comparative example is that the basic culture medium of this comparative example is CHO S-SFMII, and DMEM / F12 culture medium is not used.

[0082] Comparative Example 3

[0083] Compared with Example 1, the only difference of this comparative example is that the basal culture medium of this comparative example is a culture medium prepared by combining DMEM and CHO S-SFMII in a ratio of 1:9, and DMEM / F12 culture medium is not used.

[0084] Comparative Example 4

[0085] Compared with Example 1, the difference of this comparative example is that the buffer system of the DMEM / F12 medium used does not contain HEPES, and only NaHCO3 in the additive is used as the buffer system, and the NaHCO3 content is 18.30 g / L.

[0086] Comparative Example 5

[0087] Compared with Example 1, the only difference of this comparative example is that 1.30 g / L HEPES is added in the additive instead of NaHCO3.

[0088] Experimental Test:

[0089] Experimental Example 1: Effect of serum-free medium on CHO cell culture performance

[0090] 1. Test materials:

[0091] A serum-free culture medium for industrial production of human erythropoietin prepared in Example 1, Example 2, Example 3, Example 4 and Example 5; the basic culture medium of Comparative Example 1, Comparative Example 2 and Comparative Example 3 is DMEM, CHO S-SFM II and DMEM / CHO S-SFM II 1:9 combination, and the additive consists of the following components and their contents:

[0092] D-glucose 2.00g / L, D-mannose 0.21g / L, D-galactose 0.21g / L, sodium butyrate 0.08g / L, yeast hydrolysate 0.25g / L, NaHCO3 1.30g / L.

[0093] 2. Test methods:

[0094] The subcultured and expanded cells were inoculated into a serum-free culture medium for industrial production of human erythropoietin prepared in Examples 1, 2, 3, 4, 5, and Comparative Examples 1, 2, and 3 for roller bottle culture. The culture medium was recorded as DF-1, DF-2, DF-3, DF-4, DF-5, and DM-1, CH-1, and DC-1, respectively. The inoculation density was 1.6×10 9 cells / mL, and label the spinner bottles. Tubes 1 to 5 are labeled DF-1, DF-2, DF-3, DF-4, and DF-5, and tubes 6, 7, and 8 are labeled DM-1, CH-1, and DC-1 as control culture media.

[0095] The cell culture conditions were pH 7.2±0.2, temperature 37±1°C, dissolved oxygen 40%-80%, and rotation speed 15rpm. After 9 days of continuous culture, the CHO cell density, in vitro activity, and EPO expression during cell growth were determined. In vitro activity was detected by enzyme-linked immunosorbent assay, and EPO expression was detected by ELISA.

[0096] 3. Test results:

[0097] The test results are shown in Table 1 and Table 2. Figure 1 to Figure 4 As shown, where: Figure 1 CHO cell density change curve in different serum-free culture media; Figure 2 The figure is a comparison of protein content in different serum-free culture media. Figure 3 These are IEF+Western-blot images of supernatants from different serum-free culture media. Figure 4 : The in vitro activity change curve in different serum-free culture media.

[0098] Table 1: CHO cell density change curve in different serum-free culture media

[0099] <![CDATA[Cell density (1×10 6 cells / ml)]]> Day 0 1 day 3 days 5 days 7 days 9 days DF-1 0.19 0.25 0.87 1.65 1.57 1.52 DF-2 0.19 0.21 0.80 1.49 1.37 1.29 DF-3 0.19 0.23 0.84 1.59 1.51 1.47 DF-4 0.19 0.23 0.81 1.56 1.56 1.51 DF-5 0.19 0.22 0.80 1.51 1.46 1.44 DM-1 0.19 0.21 0.56 1.05 1.00 0.90 CH-1 0.19 0.21 0.57 1.07 1.02 0.97 DC-1 0.19 0.22 0.59 1.13 1.05 1.00

[0100] Table 2: In vitro activity and protein content in different serum-free media

[0101]

[0102] From Tables 1 to 2, Figure 1 to Figure 4 The results show that the growth, metabolism and EPO glycosylation degree of CHO cells in DF-1, DF-2, DF-3, DF-4 and DF-5 culture media are not much different, but the cell expression level is higher than that in the control culture medium. Therefore, Example 1 is the best example.

[0103] Experimental Example 2: Effect of the buffer system in serum-free culture medium on CHO cell culture performance

[0104] 1. Test materials:

[0105] A serum-free culture medium for industrial production of human erythropoietin prepared in Example 1;

[0106] Comparative Example 4 was used as a control culture medium.

[0107] 2. Test methods:

[0108] The subcultured cells were inoculated into the culture medium of Example 1 and the control medium for roller bottle culture. The culture medium was recorded as DF-1 and DF'-1 respectively. The inoculation density was 1.6×10 9cells / mL, marked the spinner bottles, one tube was marked as DF-1, and the other two tubes were marked as DF'-1, as control culture medium. The cell culture conditions were pH 7.2±0.2, temperature 37±1℃, dissolved oxygen content 40%-80%, and rotation speed 15rpm. After 7 days of continuous culture, the CHO cell density, in vitro activity, and EPO expression during cell growth were determined. In vitro activity was detected by enzyme-linked immunosorbent assay, and EPO expression was detected by ELISA.

[0109] 3. Test results:

[0110] The test results are as follows Figure 5 , Table 3, Table 4. Among them: Figure 5 :CHO cell density change curve in different buffer systems;

[0111] Table 3: CHO cell density in different buffer systems

[0112]

[0113] Table 4: In vitro activity and protein content

[0114] serial number DF-1 DF'-1 <![CDATA[In vitro activity (1×10 5 IU / ml)]]> 3.95 3.28 Protein content (mg / ml) 3.97 3.43

[0115] Depend on Figure 5 From the results of Table 3 and Table 4, it can be seen that the cell expression level, in vitro activity and protein content of CHO cells in DF-1 and DF'-1 medium are higher than those in the control medium DF'-1. Therefore, Example 1 is the best example.

[0116] Test Example 3: Large-scale cell culture validation test

[0117] 1. Test materials:

[0118] A serum-free culture medium for industrial production of human erythropoietin prepared in Example 1.

[0119] Comparative Example 5 was used as a control culture medium.

[0120] 2. Test methods:

[0121] 2.1. Large-scale cell culture validation:

[0122] All cells in the inoculated bottle were inoculated into a cell tank that passed the sterility test and cultured in serum-free medium. The medium was DF-1, and the culture batches were XP-1, XP-2 and XP-3 respectively; the control medium was DC-1 medium. The culture conditions in the cell tank were: pH 7.2±0.2, dissolved oxygen was 60%±20%, culture temperature was 37℃±1℃, and glucose concentration was maintained at 0.5-2.5g / L. Monitor the consumption of glucose in the tank and start to add fresh perfusion medium, perfusion culture, control the stirring speed at 60rpm~150rpm, keep the working volume at about 11L, perform continuous flow culture, and collect cell harvest fluid. When the ratio of perfusion speed: effective tank volume is less than 20ml / h / L or the in vitro activity of the cell harvest fluid is less than 3000IU / ml, end the cell culture.

[0123] 2.2 Protein purification:

[0124] 2.2.1 Blue Sepharose 6 Fast Flow Chromatography (Blue Sepharose Chromatography) The filtered cell collection fluid was loaded onto the equilibrated blue gel column, and then eluted with Tris-HCl (pH 7.0 ± 0.1) equilibration buffer to the baseline, and then eluted with NaCl / Tris-HCl (pH 7.0 ± 0.1) buffer and the EPO peak was collected.

[0125] 2.2.2 Ultrafiltration concentration

[0126] The blue gel eluate was ultrafiltered and concentrated using Tris-HCl buffer (pH 7.0±0.1) until the conductivity of the concentrate differed from that of the buffer by no more than 150 μS / cm.

[0127] 2.2.3 DEAE-Sepharose Fast Flow Chromatography (Ion Exchange Chromatography I)

[0128] The ultrafiltration concentrate was loaded onto a DEAE-FF column equilibrated with Tris-HCl buffer (pH 7.0 ± 0.1). The column was eluted again with the equilibration buffer to the baseline, then washed with an equilibration buffer containing Tris-HCl (pH 7.0 ± 0.1) to the baseline, and then eluted with an equilibration buffer containing NaCl / Tris-HCl (pH 7.0 ± 0.1) and the EPO peak was collected.

[0129] 2.2.4 C4 reverse phase chromatography

[0130] After equilibrating the Vydac C4 column with Tris-HCl (pH 7.0 ± 0.1) buffer to the baseline, the ion exchange chromatography I collected in 2.2.3 above was loaded onto the Vydac C4 column, and then eluted with the above buffer until the baseline was flat, and then eluted with Tris-HCl (pH 7.0 ± 0.1) buffer containing ethanol, and the elution peak was collected.

[0131] 2.2.5 DEAE-Sepharose Fast Flow Chromatography (Ion Exchange Chromatography II)

[0132] The collected elution from the C4 column was loaded onto a DEAE-FF column equilibrated with Tris-HCl buffer (pH 7.0 ± 0.1), and the equilibration buffer was eluted to the baseline. The column was first washed with urea / glycine buffer, and after washing to the baseline, 12 column volumes were eluted with Tris-HCl (pH 7.0 ± 0.1) buffer. Then, the column was eluted with a buffer containing NaCl / Tris-HCl (pH 7.0 ± 0.1) and the EPO peak sample was collected. The sialic acid content of the collected solution should be no less than 10.5 mol / mol EPO.

[0133] 2.2.6 S-200 molecular sieve chromatography

[0134] The Sephacryl S-200 column was equilibrated with citric acid-sodium citrate buffer (pH 6.9±0.1), and the collected solution obtained from ion exchange chromatography II was loaded and eluted, and the elution peak was collected and sterilized and filtered through a 0.22 μm filter membrane to obtain the EPO stock solution. Samples were taken for stock solution testing.

[0135] 3. Test results:

[0136] 3.1. The quality test results are shown in Table 5.

[0137] (1) The results of the quality test of large-scale cell culture stock solution are shown in Table 5:

[0138] Table 5: Quality test results of large-scale cell culture stock solution

[0139]

[0140] (2) Glycosylation can produce charge heterogeneity. The evaluation of charge heterogeneity is carried out by imaging capillary isoelectric focusing electrophoresis detection technology. The imaging capillary isoelectric focusing electrophoresis pattern of large-scale cell culture stock solution is shown in Figure 2. Figure 6 , Figure 7 , Figure 8 , Fig. 9 As shown. Among them: Figure 6 :EPO imaging capillary isoelectric focusing electrophoresis results of XP-1 culture medium; Figure 7: EPO imaging capillary isoelectric focusing electrophoresis results of XP-2 culture medium; Figure 8 : EPO imaging capillary isoelectric focusing electrophoresis results of XP-3 culture medium; Fig. 9 : EPO imaging capillary isoelectric focusing electrophoresis results of DC-1 culture medium.

[0141] From the above results, it can be seen that the quality of large-scale cell culture stock solution samples was inspected, and each inspection item met the relevant standards, and the overall yield of DF-1 culture medium was significantly improved, indicating that the serum-free culture medium for industrial production of human erythropoietin provided by the present invention is expected to be used for large-scale CHO cell culture.

[0142] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A serum-free culture medium for industrial production of human erythropoietin, characterized in that: The invention comprises a basic culture medium and an additive, wherein the basic culture medium is a DMEM / F12 culture medium, and the additive is composed of the following components: sodium butyrate, NaHCO3, yeast hydrolyzate, D-glucose, D-mannose and D-galactose; the DMEM / F12 culture medium contains a zwitterionic buffer HEPES, which together with the bicarbonate in the additive forms a double buffer system.

2. The serum-free culture medium for industrial production of human erythropoietin according to claim 1, characterized in that: The DMEM / F12 culture medium further contains sodium hypoxanthine, thymidine and linoleic acid, and putrescine or putrescine hydrochloride.

3. The serum-free culture medium for industrial production of human erythropoietin according to claim 1, characterized in that: The concentration of the DMEM / F12 culture medium is 15-25 g / L, and the dosage of the additives is 0.06-0.15 g / L sodium butyrate, 0.50-2.00 g / L NaHCO3, 0.1-1.0 g / L yeast hydrolyzate, 1.0-3.0 g / L D-glucose, 0.1-0.5 g / L D-mannose and 0.1-0.5 g / L D-galactose.

4. The serum-free culture medium for industrial production of human erythropoietin according to claim 3, characterized in that: The pH of the serum-free culture medium is 6.6-7.6, the temperature is 35-39° C., and the dissolved oxygen content is 30%-90%.

5. The serum-free culture medium for industrial production of human erythropoietin according to claim 3, characterized in that: The concentration of the DMEM / F12 culture medium is 16-18 g / L.

6. The serum-free culture medium for industrial production of human erythropoietin according to claim 3, characterized in that: Sodium butyrate 0.05~0.12g / L, NaHCO3 0.80~1.60g / L, yeast hydrolysate 0.15~0.35g / L.

7. The serum-free culture medium for industrial production of human erythropoietin according to claim 3, characterized in that: The concentration of D-glucose is 1.4~2.6g / L, D-mannose is 0.15~0.25g / L, and D-galactose is 0.15~0.25g / L.

8. A method for culturing CHO cells to efficiently express erythropoietin, characterized in that: The steps include: First, serum-free culture is performed using the serum-free medium described in any one of claims 1 to 7, and the culture is subcultured and expanded at a ratio of 1:6 during the square bottle culture stage, and the subculture and expansion are performed every 2 to 4 days, at least 3 times and no more than 5 generations; Then all cells were transferred to roller bottles for culture. After 4 to 6 days, the number of cells was 1 to 2 × 10 9 The cells in the spinner flask were transferred to a cell tank for continuous perfusion culture. When the perfusion acceleration of the serum-free medium was less than 20 ml / h / L or the extracellular activity of the harvested liquid was less than 3000 IU / mL, the perfusion culture was stopped.

9. The method for culturing CHO cells for efficiently expressing erythropoietin according to claim 8, characterized in that: The culture conditions of continuous perfusion culture in the cell tank are: pH 6.6-7.6, temperature 35-39° C., and dissolved oxygen content 30%-90%.

10. The method for culturing HO cells to efficiently express erythropoietin according to claim 9, characterized in that: The culture conditions of continuous perfusion culture in the cell tank are: pH 7.0-7.4, temperature 36-38°C, and dissolved oxygen content 40%-80%.

Images