CN110042137B

CN110042137B - Method for producing human follicle-stimulating hormone by high-density perfusion culture of recombinant CHO cells, culture medium and application thereof

Info

Publication number: CN110042137B
Application number: CN201910398124.2A
Authority: CN
Inventors: 王延涛; 惠觅宙; 李国军; 赵喜红
Original assignee: Shanghai Saimei Biotechnology Co ltd
Current assignee: Shanghai Saimei Biotechnology Co ltd
Priority date: 2019-05-14
Filing date: 2019-05-14
Publication date: 2023-02-28
Anticipated expiration: 2039-05-14
Also published as: CN110042137A

Abstract

The invention discloses a method for producing human follicle stimulating hormone by high-density culture of recombinant CHO cells, a culture medium and application thereof, belonging to the field of biological pharmacy, wherein the preparation method comprises the following steps: after the CHO cells are recovered, performing cell density amplification by using a basic culture medium; when the viable cell density reaches a certain density, the production medium is used for perfusion culture: the fresh production culture medium is supplemented into the bioreactor tank body, and the CHO cells are intercepted by a hollow fiber intercepting device connected outside the bioreactor tank body, so that the cells can maintain high density and activity; when the cells in the reactor were in a steady state, CHO cell expression product collection was turned on. The invention can intercept cells, filter fragments and expressed protein through the stirring culture of the bioreactor and the external connection of a cell interception device, provides a high-density environment for the growth of the cells, reduces the fiber blockage by the back flushing of the hollow fiber column, prolongs the high-density culture time of the cells and obviously improves the batch yield.

Description

Method for producing human follicle stimulating hormone by high-density perfusion culture of recombinant CHO cells, culture medium and application thereof

Technical Field

The invention relates to the field of biological pharmacy, in particular to a method for producing human follicle-stimulating hormone by high-density perfusion culture of recombinant CHO cells, a culture medium and application thereof.

Background

Human follicle stimulating hormone (hFSH) is a glycoprotein gonadotropin synthesized and secreted by anterior pituitary basophilic cells, belongs to the family of heterodimeric glycoproteins, and consists of two non-covalently linked alpha and beta chains encoded by separate genes. Follicle Stimulating Hormone used in the first medicine was purified from urine of menopausal women, which is at risk of contamination with Human proteins, and the product difference between batches was large, and it was difficult to meet the market demand (previous R. Recombinant folliculture-Stimulating Hormone: new Biotechnology for improvement [ J ]. Human Reproduction Update,1998,18 (5): 1001-1010.). With the rapid development of molecular biology, recombinant human follicle stimulating hormone products appear in the market, which have higher purity, no human protein pollution, higher batch consistency and safety, and lower side effects (Santi D, simoni M. Biological recombinant viral stimulating hormones in genetic Therapy [ J ]. Expert Opinion on Biological Therapy,2014,14 (10): 1399-1409.).

Most FSH products are produced by recombinant Chinese Hamster Ovary Cells (CHO). In culture, spinner flasks or microcarrier paper adherent cultures (Recombinant viral stimulating hormone: reduction of the first biological technology Product for the project of infection. Recombinant Human FSH Product Development group. Hum Reproduction update.1998,4 (6): 862-881) were initially carried out using serum-containing medium, with the addition of serum being a potential risk for infection by animal viruses and detrimental to purification. With the advent of serum-free culture media, there are reports on the study of large-scale suspension culture processes in China (the study of large-scale serum-free culture processes for rhFSH expression by CHO cells, the jilin university, 2012), but the process still needs to add serum in the seed recovery stage, and although the serum is removed in the later stage, the expression level is not high.

Chinese patent CN 105462909A discloses a culture method of CHO cell for high-efficiency expression of human follicle stimulating hormone, which comprises recovering seed, culturing cells with commercial serum-free culture medium and supplemented culture, wherein the highest cell density can reach 4 × 10 ⁷ The expression amount of each cell per ml reaches 40mg/L, the batch production of a 5L reactor can reach 140mg, but the cell culture time is short, and the batch production is still not high.

Therefore, it is important to provide a culture method capable of improving batch yield and safely and efficiently expressing human follicle stimulating hormone.

Disclosure of Invention

The invention aims to provide a method for producing human follicle-stimulating hormone by culturing recombinant CHO cells with high Viable Cell Density (VCD) and high safety and efficiency, which can improve batch yield, and application thereof.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

in one aspect, the invention provides a method for producing human follicle stimulating hormone by high-density perfusion culture of recombinant CHO cells, which comprises the following steps:

1) After the CHO cells are revived and activated, a basic culture medium is used for culturing to obtain seed suspension;

2) When the seed culture solution reaches a certain density, performing perfusion culture by adopting a perfusion culture medium;

the perfusion culture method comprises the following steps: supplementing a fresh production culture medium into a tank body of a bioreactor, connecting a hollow fiber interception device outside the bioreactor, and intercepting and backwashing CHO cells to maintain higher density and activity of the CHO cells; performing perfusion and backwashing by using the perfusion culture medium;

3) When the cell density and activity in the reactor are in a stable state, starting the collection of CHO cell expression products, wherein the expression products are human follicle-stimulating hormone;

the perfusion culture medium is a cell culture medium which is serum-free, protein-free and clear in chemical components, the perfusion culture medium is formed by mixing a basic culture medium and a supplementary culture medium according to a certain proportion, and correspondingly, the basic culture medium is also a cell culture medium which is serum-free, protein-free and clear in chemical components. The CHO cells are trapped inside the bioreactor tank body by the hollow fiber trapping device connected outside the reactor tank body, and culture medium supernatant with the same weight can be discharged from the tank body through the hollow fiber device. The perfusion process removes the cell metabolic waste from the tank body in real time, and the fresh nutrient substances are supplemented into the tank body in real time, so that the CHO cells can maintain high density, activity and production time.

Further, the method comprises the following specific steps:

1) After the recombinant CHO cells are recovered and activated, basic cell culture medium with serum-free, protein-free and definite chemical components is used for basic suspension culture to obtain seed cell solution; wherein the basic cell culture medium without serum and protein and with definite chemical components contains 5000-8000 mg/mL glucose;

2) Inoculating the seed cell solution into a 5L seed reactor for culture, wherein the initial cell density in the bioreactor is 0.5-1.0 × 10 ⁶ Sampling every day for cell counting, and performing amplification culture by using a basal cell culture medium to a certain volume and density;

3) Inoculating a 50L bioreactor, and performing perfusion culture: pouring a certain proportion of a pouring culture medium every day, and starting CHO cell expression product collection when cells in the reactor are in a stable state;

4) Maintaining the highest cell density at 60-90X 10 ⁶ one/mL, preferably 60 to 85X 10 ⁶ And (4) stopping liquid collection when the cell viability is lower than 90%. One skilled in the art of cell culture can remove a portion of the cells from the reactor in a suitable manner to maintain the cell density in the reactor within a target range.

After the seed cells are recovered, after the seed cells are cultured to reach a certain density by a shake flask, the seed cells are inoculated to a bioreactor for suspension culture, after the seed cells are expanded to reach a certain volume and density, perfusion culture is started, a part of cells are not periodically removed, when the cells grow to reach a steady state, the culture temperature of the cells is reduced, the perfusion culture is started, and cell expression products are collected. The cell culture status is monitored by periodically measuring the culture parameters of the cells, such as cell density, cell viability, glucose level, lactate level, ammonia accumulation, pH, temperature, dissolved oxygen, etc.

The invention provides a culture method of recombinant human follicle-stimulating hormone, which adopts a mode of perfusion culture by adopting a serum-free, protein-free and chemical-component-clear culture medium and a suspension culture plus cell retention device, wherein a bioreactor is stirred for culture and is externally connected with the cell retention device, so that cells can be retained, fragments and expressed protein can be filtered, a high-density environment is provided for cell growth, meanwhile, the reverse flushing of a hollow fiber column reduces fiber blockage, the high-density culture time of the cells is prolonged, the batch yield is obviously improved, and the harvested cell sap does not contain the cells, thereby simplifying the purification pretreatment step, obviously reducing the cost and improving the product quality. Solves the problems of low expression of a serum-free culture medium, short expression time, low yield of batch products and the like in the prior art.

The perfusion culture refers to sampling and counting every day after the cells are inoculated into the reactor, discharging a part of culture medium after the cells reach a certain density, intercepting the cells by using a hollow fiber column, and feeding the cells back to the reactor by using the back flushing function of the column. Ensuring cell density and long-term replacement of culture medium, and maintaining high density and high activity of cells.

Generally, the cell seed fluid is obtained as: taking out a recombinant human follicle-stimulating hormone CHO cell from a liquid nitrogen tank, recovering the cell by a conventional method, and culturing the cell by using a basic cell culture medium which is serum-free, protein-free and clear in chemical components to reach a certain density to obtain the seed liquid.

Further, in the step 2), after inoculating the seed liquid cells into a 5L seed reactor, sampling and counting every day, supplementing a basic culture medium to a certain volume, inoculating a 50L bioreactor, and starting perfusion culture and expression product collection.

Further, the culture conditions of the reactor in the step 2) are as follows: the temperature is 36-37 ℃, the pH value is 6.8-7.1, the dissolved oxygen is 30-60 percent, and the linear velocity is 0.6-1.2 m/s. Preferably, the culture volume in the reactor in the step 2) is 4.0 to 4.5L. Preferably, the cell culture time in the reactor in the step 2) is 5 to 7 days.

Further, the conditions of perfusion culture in the step 3) are as follows: the temperature is 34 ℃, the pH value is 6.8-7.1, the dissolved oxygen is 30% -60%, and the linear velocity is 1.0m/s. The culture volume in the reactor is 40-45L. Preferably, the perfusion cell culture time of the step 3) is 30 to 60 days.

Further, the Perfusion amount of the medium increases with the Cell density, and the Cell Specific Perfusion Rate (CSPR) is 40 to 60pL (c. D) ^-1 。

Further, during perfusion culture, the glucose concentration was maintained at greater than 1000mg/mL.

Further, the hollow fiber membranes in the fiber column are made of hydrophilic polysulfone/polyvinyl chloride.

On the other hand, the perfusion medium for producing the human follicle stimulating hormone by the high-density perfusion culture recombinant CHO cells used in the perfusion culture is a perfusion medium which is serum-free, protein-free and has definite chemical components, consists of 70-90% of a basal medium and 10-30% of a supplement medium, and contains glucose, amino acids, vitamins, salts, trace elements and other components.

The basic culture medium comprises the following specific components: 300-500 mg/ml L-arginine hydrochloride, 100-300 mg/ml L-arginine, 100-200 mg/ml L-asparagine, 1200-1800 mg/ml L-asparagine monohydrate, 200-400 mg/ml L-aspartic acid sodium salt, 200-300 mg/ml L-cysteine, 200-400 mg/ml L-cysteine hydrochloride, 200-400 mg/ml L-2 cysteine hydrochloride, 200-400 mg/ml L-glutamic acid, 100-300 mg/ml L-monosodium glutamate, 200-300 mg/ml L-glutamine, 100-200 mg/ml glycine, 100-200 mg/ml L-histidine, 200-400 mg/ml L-histidine monohydrochloride, 200-400 mg/ml L-isoleucine, 300-500 mg/ml L-leucine, 400-600 mg/ml L-lysine hydrochloride, 100-300 mg/ml L-lysine, 100-300 mg/ml L-methionine, 200-400 mg/ml L-phenylalanine, 200-400 mg/ml L-proline, 300-500 mg/ml L-serine, 200-400 mg/ml L-threonine, 100-300 mg/ml L-tryptophan, 200-400 mg/ml L-2 water sodium tyrosine, 200-400 mg/ml L-valine, 0.1-0.3 mg/ml biotin, 50-70 mg/ml choline chloride, 2-4 mg/ml cyanocobalamine, 10-15 mg/ml folic acid, 100-150 mg/ml inositol, 12-18 mg/ml nicotinamide, 18-25 mg/ml D-pentadienoic acid calcium, 18-25 mg/ml pyridoxine hydrochloride, 1-3 mg/ml riboflavin, 12-15 mg/ml thiamine hydrochloride, 0.4-1 mg/ml DL-alpha-lipoic acid, 0.006-0.008 mg/ml ammonium metavanadate, 0.005-0.008 mg/ml ammonium molybdate tetrahydrate, 0.05-0.1 mg/ml copper sulfate pentahydrate, 0.001-0.003 mg/ml manganese chloride tetrahydrate, 0.004-0.006 mg/ml manganese sulfate monohydrate, 0.001-0.006 mg/ml nickel sulfate hexahydrate, 0.004-0.006 mg/ml potassium bromide, 0.004-0.006 mg/ml sodium metavanadate, 0.5-1.5 mg/ml sodium metasilicate nonahydrate, 0.001-0.003 stannous chloride dihydrate, 0.2-0.5 mg/ml ferrous sulfate heptahydrate, 20-40 mg/ml magnesium chloride, 40-60 mg/ml magnesium sulfate, 300-400 mg/ml potassium chloride, 0.03-0.05 mg/ml potassium nitrate, 50-80 mg/ml monobasic sodium phosphate monohydrate, 20-40 mg/ml sodium bicarbonate, 50-80 mg/ml putrescine, 100-140 mg/ml sodium pyruvate, 5000-8000 mg/ml glucose, 400-600 mg/ml dibasic sodium phosphate, 0.03-0.1 mg/ml sodium selenite, 2-10 mg/ml zinc sulfate heptahydrate, 1-5 mg/ml ethanolamine, 100-150 mg/ml calcium chloride, 1-10 mg/ml ferric citrate, 1000-5000 mg/ml sodium chloride, and 200-400 mg/ml dibasic sodium fumarate.

The feed medium comprises the following specific components: <xnotran> 20 ～ 30mg/L L- ,2000 ～ 2500mg/L L- ,4000 ～ 5000mg/L L- ,1200 ～ 1500mg/L L- ,7000 ～ 8000mg/L L- ,30 ～ 40mg/L ,1200 ～ 1500mg/L L- ,200 ～ 300mg/L L- ,5000 ～ 6000mg/L L- ,4000 ～ 5000mg/L L- ,800 ～ 1000mg/L L- ,2000 ～ 3000mg/L L- ,1000 ～ 2000mg/L L- ,3000 ～ 4000mg/L L- ,2000 ～ 3000mg/L L- ,1500 ～ 2000mg/L L- ,2000 ～ 3000mg/L L- ,3000 ～ 4000mg/L L- ,10 ～ 20mg/L C,4 ～ 8mg/L H,2000 ～ 3000mg/L ,20 ～ 30mg/L ,200 ～ 300mg/L ,20 ～ 30mg/L ,5 ～ 10mg/L ,20 ～ 30mg/L ,2 ～ 5mg/L B12, 20 ～ 30mg/L ,30 ～ 50mg/L ,400 ～ 500mg/L ,0.02 ～ 0.05mg/L ,20 ～ 30mg/L ,0.00005 ～ 0.0001mg/L ,0.1 ～ 0.2mg/L ,0.0005 ～ 0.001mg/L ,0.0001 ～ 0.0002mg/L , </xnotran> 0.001-0.003 mg/L aluminum chloride, 0.001-0.003 mg/L silver nitrate, 0.0001-0.0002 mg/L barium chloride, 0.0001-0.0003 mg/L potassium chloride, 0.003-0.005 mg/L sodium bromide, 0.0001-0.0003 mg/L sodium iodide, 0.001-0.002 mg/L rubidium chloride, 0.003-0.005 mg/L zirconium chloride, 0.0001-0.0003 mg/L chromium chloride, 0.002-0.005 mg/L cobalt chloride, 0.003-0.005 mg/L sodium fluoride, 20-30 mg/L ferric citrate, 800-1200 mg/L F-68, 10000-30000 mg/L glucose, 2-5 mg/L DL-alpha-fatty acid, 200-300 mg/L sodium acetone, 5-10 mg/L adenosine, 10-10 mg/L uridine.

Preferably, the perfusion medium without serum, protein and specific chemical components consists of 80-90% of basal medium and 10-20% of supplemented medium, and contains glucose, amino acids, vitamins, salts, trace elements and other components.

The basic culture medium comprises the following specific components: 350-450 mg/ml L-arginine hydrochloride, 120-200 mg/ml L-arginine, 100-180 mg/ml L-asparagine, 1300-1500 mg/ml L-asparagine monohydrate, 250-350 mg/ml L-aspartic acid, 300-350 mg/ml L-aspartic acid sodium salt, 200-250 mg/ml L-cysteine, 250-350 mg/ml L-cysteine hydrochloride, 250-350 mg/ml L-2 cysteine hydrochloride, 350-400 mg/ml L-glutamic acid, 200-250 mg/ml L-monosodium glutamate, 200-250 mg/ml L-glutamine, 100-150 mg/ml glycine, 150-200 mg/ml L-histidine, 250-350 mg/ml L-histidine monohydrochloride, 300-400 mg/ml L-isoleucine, 400-500 mg/ml L-leucine, 500-600 mg/ml L-lysine hydrochloride, 200-300 mg/ml L-lysine, 200-300 mg/ml L-methionine, 280-350 mg/ml L-phenylalanine, 300-380 mg/ml L-proline, 400-450 mg/ml L-serine, 300-350 mg/ml L-threonine, 200-280 mg/ml L-tryptophan, 250-320 mg/ml L-2 water sodium tyrosine, 300-350 mg/ml L-valine, 0.1-0.2 mg/ml biotin, 60-70 mg/ml choline chloride, 3-4 mg/ml cyanocobalamine, 10-12 mg/ml folic acid, 100 to 120mg/ml inositol, 13 to 17mg/ml nicotinamide, 19 to 22mg/ml D-pentadienoic acid calcium, 18 to 23mg/ml pyridoxine hydrochloride, 1.2 to 2mg/ml riboflavin, 13 to 14mg/ml thiamine hydrochloride, 0.6 to 0.8mg/ml DL-alpha-lipoic acid, 0.007 to 0.008mg/ml ammonium metavanadate, 0.006 to 0.007mg/ml ammonium molybdate tetrahydrate, 0.08 to 0.1mg/ml copper sulfate pentahydrate, 0.001 to 0.002mg/ml manganese chloride tetrahydrate, 0.005 to 0.006mg/ml manganese sulfate monohydrate, 0.001 to 0.002mg/ml nickel sulfate hexahydrate, 0.005 to 0.006mg/ml potassium bromide, 0.005 to 0.006mg/ml sodium metavanadate, 0.8 to 1.2mg/ml sodium silicate, 0.0015mg/ml stannous chloride nonahydrate, 0.4-0.5 mg/ml ferrous sulfate heptahydrate, 28-32 mg/ml magnesium chloride, 48-55 mg/ml magnesium sulfate, 320-350 mg/ml potassium chloride, 0.035-0.045 mg/ml potassium nitrate, 60-65 mg/ml sodium dihydrogen phosphate monohydrate, 30-35 mg/ml sodium bicarbonate, 72-76 mg/ml putrescine, 110-120 mg/ml sodium pyruvate, 6000-7000 mg/ml glucose, 450-500 mg/ml disodium hydrogen phosphate, 0.035-0.05 mg/ml sodium selenite, 5-8 mg/ml zinc sulfate heptahydrate, 3-4 mg/ml ethanolamine, 100-120 mg/ml calcium chloride, 5-8 mg/ml ferric citrate, 2000-3000 mg/ml sodium chloride, 300-350 mg/ml disodium fumarate.

The feed medium comprises the following specific components: <xnotran> 24 ～ 28mg/L L- ,2200 ～ 2400mg/L L- ,4500 ～ 4800mg/L L- ,1300 ～ 1400mg/L L- ,7500 ～ 7800mg/L L- ,35 ～ 38mg/L ,1200 ～ 1300mg/L L- ,260 ～ 290mg/L L- ,5600 ～ 5800mg/L L- ,4500 ～ 4800mg/L L- ,900 ～ 1000mg/L L- ,2400 ～ 2800mg/L L- ,1500 ～ 1800mg/L L- ,3300 ～ 3800mg/L L- ,2600 ～ 2900mg/L L- ,1500 ～ 1800mg/L L- ,2300 ～ 2600mg/L L- ,3500 ～ 3800mg/L L- ,12 ～ 15mg/L C,5 ～ 7mg/L H,2300 ～ 2500mg/L ,24 ～ 28mg/L ,220 ～ 280mg/L ,25 ～ 29mg/L ,6 ～ 8mg/L ,24 ～ 26mg/L ,3 ～ 5mg/L B12, 25 ～ 27mg/L ,35 ～ 45mg/L ,420 ～ 480mg/L ,0.03 ～ 0.045mg/L ,25 ～ 27mg/L ,0.000055 ～ 0.000095mg/L ,0.15 ～ 0.18mg/L ,0.0008 ～ 0.001mg/L ,0.00012 ～ 0.00016mg/L , </xnotran> 0.0012-0.0025 mg/L aluminum chloride, 0.0015-0.0025 mg/L silver nitrate, 0.00012-0.00014 mg/L barium chloride, 0.00016-0.00025 mg/L potassium chloride, 0.004-0.0045 mg/L sodium bromide, 0.00012-0.00024 mg/L sodium iodide, 0.0012-0.0015 mg/L rubidium chloride, 0.0035-0.0045 mg/L zirconium chloride, 0.00015-0.0002 mg/L chromium chloride, 0.0025-0.0045 mg/L cobalt chloride, 0.004-0.0045 mg/L sodium fluoride, 26-28 mg/L ferric citrate, 900-1000 mg/L F-68, 15000-25000 mg/L glucose, 3-4 mg/L DL, 260-260 mg/L fatty acid, 9-6 mg/L acetone, 9-6 mg/L sodium adenosine, 9-9 mg/L acetone.

Further preferably, the serum-free, protein-free and chemically defined perfusion medium is composed of 90% of a basal medium and 10% of a feed medium, and contains glucose, amino acids, vitamins, salts, trace elements and other components.

The cell basic culture medium comprises: 400.45mg/ml L-arginine hydrochloride, 150.66mg/ml L-arginine, 130.89mg/ml L-asparagine, 1498.68mg/ml L-asparagine monohydrate, 345.65mg/ml L-aspartic acid, 300.12mg/ml L-aspartic acid sodium salt, 246.54mg/ml L-cysteine, 345.45mg/ml L-cysteine monohydrochloride, 300.00mg/ml L-2 cysteine hydrochloride, 371.78mg/ml L-glutamic acid, 213.54mg/ml L-monosodium glutamate, 245.56mg/ml L-glutamine, 125.34mg/ml glycine, 192.62mg/ml L-histidine, 300.12mg/ml L-histidine monohydrochloride, 380.00mg/ml L-isoleucine, 460.43mg/ml L-leucine, 556.76mg/ml L-lysine hydrochloride, 234.54mg/ml L-lysine, 235.57mg/ml L-methionine, 294.11mg/ml L-phenylalanine, 308.61mg/ml L-proline, 435.65mg/ml L-serine, 332.45mg/ml L-threonine, 207.12mg/ml L-tryptophan, 278.34mg/ml L-2 sodium tyrosinate hydrate, 300.32mg/ml L-valine, 0.12mg/ml biotin, 65.67mg/ml choline chloride, 3.65mg/ml cyanocobalamin, 11.23mg/ml folic acid, 116.43mg/ml inositol, 15.55mg/ml nicotinamide, 20.12mg/ml calcium D-pentadienoate, 19.65mg/ml pyridoxine hydrochloride, 1.55mg/ml riboflavin, 13.43mg/ml thiamine hydrochloride, 0.72mg/ml DL-alpha-lipoic acid, 0.00786mg/ml ammonium metavanadate, 0.00653mg/ml ammonium molybdate tetrahydrate, 0.0906mg/ml copper sulfate pentahydrate, 0.00186mg/ml manganese chloride tetrahydrate, 0.00554mg/ml manganese sulfate monohydrate, 0.00120mg/ml nickel sulfate hexahydrate, 0.00540mg/ml potassium bromide, 0.00529mg/ml sodium metavanadate, 1.03mg/ml sodium metasilicate nonahydrate, 0.00101mg/ml stannous chloride dihydrate, 0.43mg/ml ferrous sulfate heptahydrate, 30.03mg/ml magnesium chloride, 50.75mg/ml magnesium sulfate, 341.75mg/ml potassium chloride, 0.04mg/ml potassium nitrate, 64.93mg/ml monobasic sodium phosphate monohydrate, 34.56mg/ml sodium bicarbonate, 75.6mg/ml putrescine, 113.91mg/ml sodium pyruvate, 6543.68mg/ml glucose, 489.75mg/ml dibasic sodium phosphate, 0.04mg/ml sodium selenite, 5.58mg/ml zinc sulfate heptahydrate, 3.67mg/ml ethanolamine, 103.82mg/ml calcium chloride, 6.87mg/ml ferric citrate, 2065.43mg/ml sodium chloride, 320mg/ml dibasic sodium fumarate.

The feed medium comprises: <xnotran> 25mg/L L- ,2300mg/L L- ,4600mg/L L- ,1350mg/L L- ,7600mg/L L- ,36mg/L ,1250mg/L L- ,280mg/L L- ,5650mg/L L- ,4600mg/L L- ,950mg/L L- ,2500mg/L L- ,1600mg/L L- ,3500mg/L L- ,2800mg/L L- ,1600mg/L L- ,2500mg/L L- ,3700mg/L L- ,14.5mg/L C,6.5mg/L H,2380mg/L ,25mg/L ,260mg/L ,28mg/L ,6.5mg/L ,25.5mg/L ,4.5mg/L B12, 26.5mg/L ,40mg/L ,450mg/L ,0.038mg/L ,25.5mg/L ,0.000075mg/L ,0.165mg/L ,0.0009mg/L ,0.00015mg/L ,0.0024mg/L ,0.002mg/L ,0.00013mg/L ,00018mg/L ,0.0042mg/L ,0.00018mg/L ,0.0014mg/L , </xnotran> 0.004mg/L zirconium chloride, 0.00018mg/L chromium chloride, 0.0035mg/L cobalt chloride, 0.0042mg/L sodium fluoride, 27.5mg/L ferric citrate, 950mg/L F-68, 18000mg/L glucose, 3.5mg/L DL-alpha-fatty acid, 275mg/L sodium pyruvate, 8.5mg/L adenosine, 8.5mg/L uridine, 8.5mg/L cytidine, 8.5mg/L guanosine.

On the other hand, the application of the human follicle stimulating hormone is provided, and the human follicle stimulating hormone is prepared by the method for producing the human follicle stimulating hormone by the high-density perfusion culture recombinant CHO cells.

After adopting such design, the invention has at least the following advantages:

(1) The invention provides a culture method of recombinant human follicle-stimulating hormone, which adopts a culture medium without serum, protein or definite chemical components and a mode of perfusion culture by a suspension culture and an additional cell retention device, wherein a bioreactor is used for stirring culture and is externally connected with the cell retention device (namely a hollow fiber retention device) to retain cells, filter fragments and expressed protein, provide a high-density environment for cell growth, and simultaneously, the back flushing of a hollow fiber column reduces fiber blockage, prolongs the high-density culture time of the cells, obviously improves the batch yield, and the harvested cell sap does not contain cells, simplifies the purification pretreatment steps, and can obviously reduce the cost and improve the product quality. Solves the problems of low expression of a serum-free culture medium, short expression time, low yield of batch products and the like in the prior art.

(2) After seed cells are recovered, after the seed cells are cultured to reach a certain density through a shake flask, the seed cells are inoculated to a bioreactor for suspension culture, when glucose in a culture solution is consumed to a certain level, perfusion culture is started, a part of cells are not periodically removed, when the cells grow to a steady state, the culture temperature of the cells is reduced, the perfusion culture is started, culture parameters of the cells, such as cell density, cell viability, glucose level, lactic acid level, ammonia accumulation amount, pH value, temperature, dissolved oxygen and the like, are periodically detected, and the cell culture state is monitored.

Drawings

The foregoing is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and the detailed description.

FIG. 1 is a graph of cell density and viability of recombinant human FSH of example 1 of the present invention as a function of time;

FIG. 2 is a graph showing the cell density and the survival rate of recombinant human FSH according to example 2 of the present invention as a function of time;

FIG. 3 is a graph showing the cell density and the survival rate of recombinant human FSH according to example 3 of the present invention as a function of time.

Detailed Description

The present invention is further illustrated by the following examples, but the present invention is not limited to the following examples, and any products similar or equivalent to the present invention, which are obtained by the teaching of the present invention, are within the scope of protection.

The instruments, consumables and reagents used in the following examples are all conventional instruments, consumables and reagents in the field, and are commercially available to those skilled in the art.

Example 1

(1) Recovering cells to obtain seed cell liquid

Taking out a cell from a liquid nitrogen tank, thawing in 37 deg.C water bath, transferring the cell into 15ml sterile centrifugal tube, adding 5ml 37 deg.C preheated serum-free, protein-free and chemical component-defined basic cell culture medium, lightly blowing, mixing, centrifuging at 800rpm for 5min, discardingThe supernatant was removed, resuspended in 5ml fresh medium, the cell suspension transferred to a 125ml gas-permeable shake flask, supplemented with 15ml medium, and incubated at 37 ℃ at 120rpm,8% CO2, 70% to 90% humidity. Sampling and counting every other day, supplementing culture medium and maintaining cell density at 4-6X 10 ⁶ One per ml. The volume of the shake flask is gradually enlarged from 125ml,250ml,500ml,1000ml and 2000ml, through the enlargement for 7-10 days, the volume of the seed reaches about 500ml, and the density is 3.6 multiplied by 10 ⁶ Each/ml.

The basic culture medium without serum, protein and clear chemical components in the embodiment comprises the following components: 380.86mg/ml L-arginine hydrochloride, 124.68mg/ml L-arginine, 110.09mg/ml L-asparagine, 1413.54mg/ml L-asparagine monohydrate, 350.78mg/ml L-aspartic acid, 322.55mg/ml L-aspartic acid sodium salt, 256.51mg/ml L-cysteine, 300.08mg/ml L-cysteine hydrochloride, 350.06mg/ml L-2 cysteine hydrochloride, 354.78mg/ml L-glutamic acid, 200.00mg/ml L-monosodium glutamate, 232.12mg/ml L-glutamine, 124.35mg/ml glycine, 200.12mg/ml L-histidine, 323.43mg/ml L-histidine monohydrochloride, 371, 78mg/ml L-isoleucine, 438.45mg/ml L-leucine, 550.60mg/ml L-lysine hydrochloride, 221.26mg/ml L-lysine, 230.00mg/ml L-methionine, 311.54mg/ml L-phenylalanine, 300.89mg/ml L-proline, 422.65mg/ml L-serine, 327.98mg/ml L-threonine, 200.00mg/ml L-tryptophan, 261.44mg/ml L-2 sodium tyrosinate, 300.56mg/ml L-valine, 0.14mg/ml biotin, 61.13mg/ml choline chloride, 3.56mg/ml cyanocobalamin, 10.34mg/ml folic acid, 110.56mg/ml inositol, 14.58mg/ml nicotinamide, 19.57mg/ml D-calcium pentadienoate, 20.55mg/ml pyridoxine hydrochloride, 1.45mg/ml riboflavin, 13.85mg/ml thiamine hydrochloride, 0.69mg/ml DL-alpha-lipoic acid, 0.00754mg/ml ammonium metavanadate, 0.00601mg/ml ammonium molybdate tetrahydrate, 0.0832mg/ml copper sulfate pentahydrate, 0.00178mg/ml manganese chloride tetrahydrate, 0.00544mg/ml manganese sulfate monohydrate, 0.00108mg/ml nickel sulfate hexahydrate, 0.005mg/ml potassium bromide, 0.00518mg/ml sodium metavanadate, 0.886mg/ml sodium metasilicate nonahydrate, 0.001mg/ml stannous chloride dihydrate, 0.40mg/ml ferrous sulfate heptahydrate, 28.50mg/ml magnesium chloride, 49.90mg/ml magnesium sulfate, 320.76mg/ml potassium chloride, 0.038mg/ml potassium nitrate, 63.65mg/ml monobasic sodium phosphate monohydrate, 34.55mg/ml sodium bicarbonate, 75.5mg/ml putrescine, 112.65mg/ml sodium pyruvate, 6478.98mg/ml glucose, 476.57mg/ml dibasic sodium phosphate, 0.035mg/ml sodium selenite, 6.73mg/ml zinc sulfate heptahydrate, 3.45mg/ml ethanolamine, 100.56mg/ml calcium chloride, 5.78mg/ml ferric citrate, 2000.23mg/ml sodium chloride, 311.54mg/ml dibasic sodium fumarate.

The serum-free, protein-free, chemically defined feed medium of this example consisted of: <xnotran> 25mg/L L- ,2300mg/L L- ,4600mg/L L- ,1350mg/L L- ,7600mg/L L- ,36mg/L ,1250mg/L L- ,280mg/L L- ,5650mg/L L- ,4600mg/L L- ,950mg/L L- ,2500mg/L L- ,1600mg/L L- ,3500mg/L L- ,2800mg/L L- ,1600mg/L L- ,2500mg/L L- ,3700mg/L L- ,14.5mg/L C,6.5mg/L H,2380mg/L ,25mg/L ,260mg/L ,28mg/L ,6.5mg/L ,25.5mg/L ,4.5mg/L B12, 26.5mg/L ,40mg/L ,450mg/L ,0.038mg/L ,25.5mg/L ,0.000075mg/L ,0.165mg/L ,0.0009mg/L ,0.00015mg/L ,0.0024mg/L ,0.002mg/L ,0.00013mg/L ,00018mg/L ,0.0042mg/L ,0.00018mg/L ,0.0014mg/L , </xnotran> 0.004mg/L zirconium chloride, 0.00018mg/L chromium chloride, 0.0035mg/L cobalt chloride, 0.0042mg/L sodium fluoride, 27.5mg/L ferric citrate, 950mg/L F-68, 18000mg/L glucose, 3.5mg/L DL-alpha-fatty acid, 275mg/L sodium pyruvate, 8.5mg/L adenosine, 8.5mg/L uridine, 8.5mg/L cytidine, 8.5mg/L guanosine.

In this example, the perfusion medium without serum, protein and defined chemical components consists of: 80% of basal medium and 20% of feed medium.

(2) Inoculation 5L seed reactor

The volume of the inoculated seed reactor is 4L, and the cell density is 4.5X 10 ⁵ The culture temperature is 37 ℃, the linear velocity is 1.0m/s, the dissolved oxygen is 30-60 percent, and the pH value is 6.8-7.1. Samples were taken daily and counted.

(3) Perfusion culture

When the cell density reaches 5X 10 ⁶ And when the seeds are cultured per ml, inoculating a 50L bioreactor, inoculating the seeds with the volume of 40L, starting perfusion culture, opening a liquid changing pipeline and a clear liquid collecting pipeline of the hollow fiber interception device, closing a cleaning pipeline and a reverse flushing pipeline, finishing clear liquid collection, starting reverse flushing, closing the pipelines after flushing is finished, and operating the cleaning pipeline to continuously clean the main body. When the cells are in a stable state, reducing the culture temperature to 34 ℃, the linear velocity to 1.0m/s, the dissolved oxygen to 30-60 percent and the pH value to 6.8-7.1, continuously filling, collecting liquid, maintaining the glucose content to be more than 1g/L until the cell viability is lower than 90 percent, and stopping collecting the liquid. The whole perfusion culture is maintained for 35 days from the inoculation reactor, 2100L of liquid is collected, the volume of the liquid is 52.5 times of the culture volume, the expression amount is 15mg/L, and the batch yield is 31500mg.

The adjustment strategy of the perfusion amount of the culture medium in the embodiment is as follows: CSPR is 40-60 pL (c, d) ^-1 Maintaining the cell density at 60-85 × 10 ⁶ About one/mL, excess cells were removed using peristaltic pumping. The cell density and cell viability curves of this example are shown in FIG. 1, which are maintained at higher cell density and cell viability levels at all times.

The quality of the harvested stock solution is detected according to the detection method in pharmacopoeia of the people's republic of China (2015 edition), the quality standard of the detection standard is referred to the quality standard of the levamisole imported injection, and the detection result is shown in table 1.

TABLE 1 detection results of recombinant human follicle-stimulating hormone stock solution

Example 2

(1) Cell recovery to obtain seed cell liquid

Taking one cell out of a liquid nitrogen tank, thawing in a 37 ℃ water bath, transferring the cell into a 15ml sterile centrifuge tube, adding 5ml of 37 ℃ preheated serum-free, protein-free and chemically defined basic cell culture medium, lightly blowing, uniformly mixing, centrifuging at 800rpm for 5min, discarding supernatant, adding 5ml of fresh culture medium again for heavy suspension, transferring the cell suspension into a 125ml air-permeable shake flask, supplementing 15ml of culture medium, and culturing at 37 ℃,120rpm,8% CO2 and 70% -90% humidity. Sampling and counting every other day, supplementing culture medium and maintaining cell density at 3-4X 10 ⁶ Each/ml. The volume of the shake flask is gradually enlarged from 125ml,250ml,500ml,1000ml and 2000ml, after 7-10 days of amplification, the volume of the seeds reaches about 600ml, and the density is 3.8. Times.10 ⁶ One per ml.

The basic culture medium without serum, protein and specific chemical components in the embodiment is as follows: 400.45mg/ml L-arginine hydrochloride, 150.66mg/ml L-arginine, 130.89mg/ml L-asparagine, 1498.68mg/ml L-asparagine monohydrate, 345.65mg/ml L-aspartic acid, 300.12mg/ml L-aspartic acid sodium salt, 246.54mg/ml L-cysteine, 345.45mg/ml L-cysteine monohydrochloride, 300.00mg/ml L-2 cysteine hydrochloride, 371.78mg/ml L-glutamic acid, 213.54mg/ml L-glutamic acid sodium glutamate monohydrate, 245.56mg/ml L-glutamine, 125.34mg/ml glycine, 192.62mg/ml L-histidine, 300.12mg/ml L-histidine monohydrochloride, 380.00mg/ml L-isoleucine, 460.43mg/ml L-leucine, 556.76mg/ml L-lysine hydrochloride, 234.54mg/ml L-lysine, 235.57mg/ml L-methionine, 294.11mg/ml L-phenylalanine, 308.61mg/ml L-proline, 435.65mg/ml L-serine, 332.45mg/ml L-threonine, 207.12mg/ml L-tryptophan, 278.34mg/ml L-2 sodium tyrosinate hydrate, 300.32mg/ml L-valine, 0.12mg/ml biotin, 65.67mg/ml choline chloride, 3.65mg/ml cyanocobalamin, 11.23mg/ml folic acid, 116.43mg/ml inositol, 15.55mg/ml nicotinamide, 20.12mg/ml calcium D-pentadienoate, 19.65mg/ml pyridoxine hydrochloride, 1.55mg/ml riboflavin, 13.43mg/ml thiamine hydrochloride, 0.72mg/ml DL-alpha-lipoic acid, 0.00786mg/ml ammonium metavanadate, 0.00653mg/ml ammonium molybdate tetrahydrate, 0.0906mg/ml copper sulfate pentahydrate, 0.00186mg/ml manganese chloride tetrahydrate, 0.00554mg/ml manganese sulfate monohydrate, 0.00120mg/ml nickel sulfate hexahydrate, 0.00540mg/ml potassium bromide, 0.00529mg/ml sodium metavanadate, 1.03mg/ml sodium metasilicate nonahydrate, 0.00101mg/ml stannous chloride dihydrate, 0.43mg/ml ferrous sulfate heptahydrate, 30.03mg/ml magnesium chloride, 50.75mg/ml magnesium sulfate, 341.75mg/ml potassium chloride, 0.04mg/ml potassium nitrate, 64.93mg/ml monobasic sodium phosphate monohydrate, 34.56mg/ml sodium bicarbonate, 75.6mg/ml putrescine, 113.91mg/ml sodium pyruvate, 6543.68mg/ml glucose, 489.75mg/ml disodium hydrogen phosphate, 0.04mg/ml sodium selenite, 5.58mg/ml zinc sulfate heptahydrate, 3.67mg/ml ethanolamine, 103.82mg/ml calcium chloride, 6.87mg/ml ferric citrate, 2065.43mg/ml sodium chloride, 320mg/ml disodium fumarate.

The serum-free, protein-free and chemically defined feed medium of this example consisted of: <xnotran> 25mg/L L- ,2300mg/L L- ,4600mg/L L- ,1350mg/L L- ,7600mg/L L- ,36mg/L ,1250mg/L L- ,280mg/L L- ,5650mg/L L- ,4600mg/L L- ,950mg/L L- ,2500mg/L L- ,1600mg/L L- ,3500mg/L L- ,2800mg/L L- ,1600mg/L L- ,2500mg/L L- ,3700mg/L L- ,14.5mg/L C,6.5mg/L H,2380mg/L ,25mg/L ,260mg/L ,28mg/L ,6.5mg/L ,25.5mg/L ,4.5mg/L B12, 26.5mg/L ,40mg/L ,450mg/L ,0.038mg/L ,25.5mg/L ,0.000075mg/L ,0.165mg/L ,0.0009mg/L ,0.00015mg/L ,0.0024mg/L ,0.002mg/L ,0.00013mg/L ,00018mg/L ,0.0042mg/L ,0.00018mg/L ,0.0014mg/L , </xnotran> 0.004mg/L zirconium chloride, 0.00018mg/L chromium chloride, 0.0035mg/L cobalt chloride, 0.0042mg/L sodium fluoride, 27.5mg/L ferric citrate, 950mg/L F-68, 18000mg/L glucose, 3.5mg/L DL-alpha-fatty acid, 275mg/L sodium pyruvate, 8.5mg/L adenosine, 8.5mg/L uridine, 8.5mg/L cytidine, 8.5mg/L guanosine.

The perfusion medium without serum, protein and chemical components in this example was composed of: 85% of basal medium and 15% of feed medium.

(2) Inoculation 5L seed reactor

After inoculation, the volume of the 5L seed reactor is 4.3L, and the cell density is 5.3 multiplied by 10 ⁵ The culture temperature is 37 ℃, the linear velocity is 1.0m/s, the dissolved oxygen is 30-60 percent, and the pH value is 6.8-7.1. Samples were taken daily and counted.

(3) Perfusion culture

When the cell density reaches 5X 10 ⁶ And when the seeds are cultured per ml, inoculating a 50L bioreactor, inoculating the seeds with the volume of 40L, starting perfusion culture, opening a liquid changing pipeline and a clear liquid collecting pipeline of the hollow fiber interception device, closing a cleaning pipeline and a reverse flushing pipeline, finishing clear liquid collection, starting reverse flushing, closing the pipelines after flushing is finished, and operating the cleaning pipeline to continuously clean the main body. When the cells are in a stable state, reducing the culture temperature to 34 ℃, the linear velocity to 1.0m/s, the dissolved oxygen to 30-60 percent and the pH value to 6.8-7.1, continuously perfusing, maintaining the glucose content to be more than 1g/L until the cell viability is lower than 90 percent, and stopping liquid collection. The whole perfusion culture is maintained for 42 days from the inoculation reactor, 2520L of liquid is collected in total, the volume of the liquid is 63 times of the culture volume, the expression amount is 22mg/L, and the batch yield is 55440mg.

The adjustment strategy of the perfusion amount of the culture medium in the embodiment is as follows:

CSPR 40-60 pL (c, d) ^-1 Maintaining the cell density at 60-80X 10 ⁶ About one/mL, excess cells were removed using peristaltic pumping.

The cell density and cell viability curves of this example are shown in FIG. 2, which are maintained at high cell density and cell viability levels at all times.

The quality of the harvested stock solution is detected according to the detection method in pharmacopoeia of the people's republic of China (2015 edition), the quality standard of the detection standard is referred to the quality standard of the levamisole imported injection, and the detection result is shown in table 2.

TABLE 2 detection results of recombinant human follicle stimulating hormone stock solution

Example 3

(1) Recovering cells to obtain seed cell liquid

Taking out a cell from a liquid nitrogen tank, thawing in a water bath at 37 ℃, transferring the cell into a 15ml sterile centrifugation tube, adding 5ml of a 37 ℃ preheated serum-free, protein-free and chemically-defined basic cell culture medium, gently blowing and beating, uniformly mixing, centrifuging at 800rpm for 5min, discarding supernatant, adding 5ml of fresh culture medium again for resuspension, transferring the cell suspension into a 125ml air-permeable shake flask, supplementing 15ml of culture medium, and culturing at 37 ℃,120rpm,8% CO2 and 70% -90% humidity. Sampling and counting every other day, supplementing culture medium and maintaining cell density at 3-4X 10 ⁶ One per ml. The volume of the shake flask is gradually enlarged from 125ml,250ml,500ml,1000ml and 2000ml, through the enlargement for 7-10 days, the volume of the seed reaches about 600ml, and the density is 3.5 multiplied by 10 ⁶ One per ml.

The basic cell culture medium without serum, protein and clear chemical components in the embodiment comprises the following components: 430.54mg/ml L-arginine hydrochloride, 172.78mg/ml L-arginine, 166.70mg/ml L-asparagine, 1499.76mg/ml L-asparagine monohydrate, 347.88mg/ml L-aspartic acid, 335.54mg/ml L-aspartic acid sodium salt, 248.80mg/ml L-cysteine, 347.85mg/ml L-cysteine monohydrate, 321.65mg/ml L-2 cysteine hydrochloride, 389.91mg/ml L-glutamic acid, 245.60mg/ml L-glutamic acid sodium monohydrate, 244.67mg/ml L-glutamine, 138.86mg/ml glycine, 195.67mg/ml L-histidine, 322.08mg/ml L-histidine monohydrochloride, 394.62mg/ml L-isoleucine, 489.65mg/ml L-leucine, 556.76mg/ml L-lysine hydrochloride, 235.54mg/ml L-lysine, 243.87mg/ml L-methionine, 311.76mg/ml L-phenylalanine, 316.76mg/ml L-proline, 443.55mg/ml L-serine, 340.00mg/ml L-threonine, 210.07mg/ml L-tryptophan, 280.87mg/ml L-2 sodium tyrosinate hydrate, 300.32mg/ml L-valine, 0.15mg/ml biotin, 65.67mg/ml choline chloride, 3.80mg/ml cyanocobalamin, 11.54mg/ml folic acid, 115.52mg/ml inositol, 15.06mg/ml nicotinamide, 20.00mg/ml calcium D-pentadienoate, 19.65mg/ml pyridoxine hydrochloride, 1.55mg/ml riboflavin, 13.67mg/ml thiamine hydrochloride, 0.65mg/ml DL-alpha-lipoic acid, 0.00785mg/ml ammonium metavanadate, 0.00655mg/ml ammonium molybdate tetrahydrate, 0.091mg/ml copper sulfate pentahydrate, 0.00185mg/ml manganese chloride tetrahydrate, 0.0055mg/ml manganese sulfate monohydrate, 0.0015mg/ml nickel sulfate hexahydrate, 0.0055mg/ml potassium bromide, 0.00530mg/ml sodium metavanadate, 1.00mg/ml sodium metasilicate nonahydrate, 0.00101mg/ml stannous chloride dihydrate, 0.45mg/ml ferrous sulfate heptahydrate, 30.05mg/ml magnesium chloride, 50.45mg/ml magnesium sulfate, 345.83mg/ml potassium chloride, 0.04mg/ml potassium nitrate, 65.00mg/ml monobasic sodium phosphate monohydrate, 34.12mg/ml sodium bicarbonate, 75.5mg/ml putrescine, 115.0mg/ml sodium pyruvate, 6500.0mg/ml glucose, 485.45mg/ml dibasic sodium phosphate, 0.04mg/ml sodium selenite, 5.60mg/ml zinc sulfate heptahydrate, 3.55mg/ml ethanolamine, 105.40mg/ml calcium chloride, 5.65mg/ml ferric citrate, 2124.5mg/ml sodium chloride, 320.50mg/ml dibasic sodium fumarate.

The perfusion medium without serum, protein and chemical components in this example was composed of: 90% of basal medium and 10% of feed medium.

(2) Inoculation 5L seed reactor

Volume after inoculation of seed reactor is 42L, cell density is 5.0X 10 ⁵ The culture temperature is 37 ℃, the linear velocity is 1.0m/s, the dissolved oxygen is 30-60 percent, and the pH value is 6.8-7.1. Samples were taken daily and counted.

(3) Perfusion culture

When the cell density reaches 5X 10 ⁶ When the culture medium is used for culture, a 50L bioreactor is inoculated, the inoculation volume is 40L, perfusion culture is started, a liquid changing pipeline and a clear liquid collecting pipeline of the hollow fiber interception device are opened, a cleaning pipeline and a back flushing pipeline are closed, clear liquid collection is finished, back flushing is started, after flushing is finished, the pipelines are closed, and the main body is continuously cleaned by running the cleaning pipeline. When the cells are in a stable state, reducing the culture temperature to 34 ℃, the linear velocity to 1.0m, the dissolved oxygen to 30-60 percent and the pH value to 6.8-7.1, continuously perfusing, maintaining the glucose content to be more than 1g/L until the cell viability is lower than 90 percent, and stopping liquid collection. Whole perfusion culture from inoculated reactorThe culture medium is maintained for 52 days totally, 3500L of liquid is collected totally, the culture volume is 87.5 times of the culture volume, the expression quantity is 25mg/L, and the batch yield is 87500mg.

The cell density and cell viability curves of this example are shown in FIG. 2, which are maintained at higher cell density and cell viability levels at all times.

The quality of the harvested stock solution is detected according to the detection method in pharmacopoeia of the people's republic of China (2015 edition), the quality standard of the detection standard is referred to the quality standard of the levamisole imported injection, and the detection result is shown in table 3.

TABLE 3 detection results of recombinant human follicle stimulating hormone stock solution

The inventor of the present invention, through continuous research, finds that when the serum-free protein-free cell culture medium of the present invention is used in combination with a perfusion culture process, the batch expression amount is not only influenced by the formulation of the cell culture medium, for example, in example 1, 80% of basal medium +20% of feed medium is used as the perfusion culture medium, the batch yield of recombinant human follicle-stimulating hormone is 31.5g, while the batch yield of example 2 is 55.44g, and the batch yield of example 3 is 87.5g, which indicates that under the same perfusion culture process, the culture medium has a great influence on the product yield, and different culture medium components have a great influence on the quality of the product. In the previous small-scale research, we also find that the temperature reduction culture in perfusion culture has a great influence on both perfusion culture time and batch expression, and the inventors continuously research and determine the final culture medium formula and culture conditions.

In conclusion, the cell culture method of the invention combines the adjustment of the culture medium and the perfusion culture process, so that one period of perfusion culture can reach 30-60 days, even longer, the recombinant CHO cell can express the human follicle-stimulating hormone at a higher level for a long time, the batch yield is more than 30g, even more than 87.5g, compared with the traditional suspension culture, the batch yield is improved by dozens of times, the invention is beneficial to industrialized production and application, and the serum-free and protein-free cell culture medium is adopted, so that the obtained human follicle-stimulating hormone reduces the infection risk, and the culture medium has low cost and is suitable for popularization and application.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention in any way, and it will be apparent to those skilled in the art that the above description of the present invention can be applied to various modifications, equivalent variations or modifications without departing from the spirit and scope of the present invention.

Claims

1. A method for producing human follicle stimulating hormone by recombinant CHO cells through high-density perfusion culture is characterized by comprising the following steps:

the perfusion culture method comprises the following steps: supplementing a fresh production culture medium into a tank body of a bioreactor, connecting a hollow fiber interception device outside the bioreactor, and intercepting and back flushing CHO cells to maintain higher density and activity of the CHO cells; performing perfusion and backwashing by using the perfusion culture medium;

3) When the cell density and the cell activity in the reactor are in a stable state, starting the collection of CHO cell expression products, wherein the expression products are human follicle stimulating hormone;

the perfusion culture medium is a cell culture medium which is serum-free, protein-free and clear in chemical components, the perfusion culture medium is formed by mixing a basic culture medium and a supplementary culture medium according to a certain proportion, and correspondingly, the basic culture medium is also a cell culture medium which is serum-free, protein-free and clear in chemical components;

the perfusion culture medium without serum, protein and definite chemical components consists of 90 percent of basal culture medium and 10 percent of supplemented culture medium, and comprises the following specific components:

1) The basic culture medium comprises the following specific components: 400.45mg/ml L-arginine hydrochloride, 150.66mg/ml L-arginine, 130.89mg/ml L-asparagine, 1498.68mg/ml L-asparagine monohydrate, 345.65mg/ml L-aspartic acid, 300.12mg/ml L-aspartic acid sodium salt, 246.54mg/ml L-cysteine, 345.45mg/ml L-cysteine monohydrochloride, 300.00mg/ml L-2 cysteine hydrochloride, 371.78mg/ml L-glutamic acid, 213.54mg/ml L-monosodium glutamate, 245.56mg/ml L-glutamine, 125.34mg/ml glycine, 192.62mg/ml L-histidine, 300.12mg/ml L-histidine monohydrochloride, 380.00mg/ml L-isoleucine, 460.43mg/ml L-leucine, 556.76mg/ml L-lysine hydrochloride, 234.54mg/ml L-lysine, 235.57mg/ml L-methionine, 294.11mg/ml L-phenylalanine, 308.61mg/ml L-proline, 435.65mg/ml L-serine, 332.45mg/ml L-threonine, 207.12mg/ml L-tryptophan, 278.34mg/ml sodium L-2-tyrosinate, 300.32mg/ml L-valine, 0.12mg/ml biotin, 65.67mg/ml choline chloride, 3.65mg/ml cyanocobalamin, 11.23mg/ml folic acid, 116.43mg/ml inositol, 15.55mg/ml nicotinamide, 20.12mg/ml D-calcium pentadienoate, 19.65mg/ml pyridoxine hydrochloride, 1.55mg/ml riboflavin, 13.43mg/ml thiamine hydrochloride, 0.72mg/ml DL-alpha-lipoic acid, 0.00786mg/ml ammonium metavanadate, 0.00653mg/ml ammonium molybdate tetrahydrate, 0.0906mg/ml copper sulfate pentahydrate, 0.00186mg/ml manganese chloride tetrahydrate, 0.00554mg/ml manganese sulfate monohydrate, 0.00120mg/ml nickel sulfate hexahydrate, 0.00540mg/ml potassium bromide, 0.00529mg/ml sodium metavanadate, 1.03mg/ml sodium metasilicate nonahydrate, 0.00101mg/ml stannous chloride dihydrate, 0.43mg/ml ferrous sulfate heptahydrate, 30.03mg/ml magnesium chloride, 50.75mg/ml magnesium sulfate, 341.75mg/ml potassium chloride, 0.04mg/ml potassium nitrate, 64.93mg/ml monobasic sodium phosphate monohydrate, 34.56mg/ml sodium bicarbonate, 75.6mg/ml putrescine, 113.91mg/ml sodium pyruvate, 6543.68mg/ml glucose, 489.75mg/ml dibasic sodium phosphate, 0.04mg/ml sodium selenite, 5.58mg/ml zinc sulfate heptahydrate, 3.67mg/ml ethanolamine, 103.82mg/ml calcium chloride, 6.87mg/ml ferric citrate, 2065.43mg/ml sodium chloride, 320mg/ml dibasic sodium fumarate;

2) The feed medium comprises the following specific components: <xnotran> 25mg/L L- ,2300mg/L L- ,4600mg/L L- ,1350mg/L L- ,7600mg/L L- ,36mg/L ,1250mg/L L- ,280mg/L L- ,5650mg/L L- ,4600mg/L L- ,950mg/L L- ,2500mg/L L- ,1600mg/L L- ,3500mg/L L- ,2800mg/L L- ,1600mg/L L- ,2500mg/L L- ,3700mg/L L- ,14.5mg/L C,6.5mg/L H,2380mg/L ,25mg/L ,260mg/L ,28mg/L ,6.5mg/L ,25.5mg/L ,4.5mg/L B12, 26.5mg/L ,40mg/L ,450mg/L ,0.038mg/L ,25.5mg/L ,0.000075mg/L ,0.165mg/L ,0.0009mg/L ,0.00015mg/L ,0.0024mg/L ,0.002mg/L ,0.00013mg/L ,00018mg/L ,0.0042mg/L ,0.00018mg/L ,0.0014mg/L , </xnotran> 0.004mg/L zirconium chloride, 0.00018mg/L chromium chloride, 0.0035mg/L cobalt chloride, 0.0042mg/L sodium fluoride, 27.5mg/L ferric citrate, 950mg/L F-68, 18000mg/L glucose, 3.5mg/L DL-alpha-fatty acid, 275mg/L sodium pyruvate, 8.5mg/L adenosine, 8.5mg/L uridine, 8.5mg/L cytidine, 8.5mg/L guanosine.

2. The method for producing human FSH (follicle stimulating hormone) by using recombinant CHO cells cultured by high-density perfusion culture according to claim 1, comprising the steps of;

1) The culture method comprises the steps of resuscitating and activating the recombinant CHO cells, and then carrying out resuspension culture by using a basal medium to obtain a seed cell solution; wherein the basic culture medium contains 5000-8000 mg/mL glucose;

2) Inoculating the seed cell solution into a 5L seed reactor for culture, wherein the cell density in the bioreactor is 0.5-1.0 × 10 ⁶ cell/mL, sampling daily for cell counting;

3) After the seed liquid reaches a certain density, inoculating a 50L bioreactor, and performing perfusion culture: pouring a certain proportion of pouring culture medium every day, and starting CHO cell expression product collection when cells in the reactor are in a stable state;

4) Maintaining the highest cell density at 60-85 × 10 ⁶ And (4) stopping liquid collection when the cell viability is lower than 90%.

3. The method for producing human fsh by high-density perfusion culture of recombinant CHO cells as claimed in any one of claims 1 to 2, wherein the culture conditions of the reactor in step 3) are as follows: the temperature is 33-37 ℃, the pH value is 6.8-7.1, the dissolved oxygen is 30-60 percent, and the linear velocity is 0.6-1.2 m/s;

and/or, the conditions of perfusion culture in the step 4) are as follows: the temperature is 34 ℃, the pH value is 6.8-7.1, the dissolved oxygen is 30% -60%, and the stirring linear velocity is 1.0m/s;

and/or, in the step 4): the perfusion culture process is started after the next day of inoculation, and the cell specific perfusion rate is 40-60 pL (c.d) ^-1 。

4. The method for producing human FSH from CHO cells cultured by high-density perfusion culture according to any one of claims 1-2, wherein the glucose concentration is maintained at greater than 1000mg/mL during perfusion culture.