阅读说明：本技术 一种高比活性重组人促卵泡激素的制备方法 (Preparation method of high specific activity recombinant human follicle stimulating hormone ) 是由 曹玉锋 常东英 陈子杨 张健锋 刘国瑞 贾媛 李铮 迟祥 孟多佳 唐剑光 王金敏 于 2021-01-20 设计创作，主要内容包括：本发明涉及促卵泡激素的技术领域,特别是涉及一种高比活性重组人促卵泡激素的制备方法,包括以下步骤：(1)取rhFSH/CHO-S工作库细胞株接种于装有CD Forti CHO培养基(含8mM谷氨酰胺)的摇瓶中进行复苏,振摇培养2-4天,待活细胞浓度达到2-3×106/ml,活度不低于80%时,按2-4×105/ml的密度转接下一级摇瓶,按同样方法逐级放大完成种子液制备；(2)将上述所述种子液按活细胞4-6×105/ml的密度接种生物反应器,溶氧为30-60%,转速为180-220rpm(7L生物反应器)、100-140rpm(14L生物反应器),基础通气速率为0.001-0.01vvm,分别于接种后第4、6、8、10天按2-5%比例(V/V)流加CellTurbo Feed2补料培养基。(The invention relates to the technical field of follicle stimulating hormone, in particular to a preparation method of high specific activity recombinant human follicle stimulating hormone, which comprises the following steps: (1) inoculating rhFSH/CHO-S working library cell strain into a shake flask filled with a CD Forti CHO medium (containing 8mM glutamine) for resuscitation, carrying out shake culture for 2-4 days, transferring to the next-stage shake flask according to the density of 2-4X 105/ml when the concentration of living cells reaches 2-3X 106/ml and the activity is not lower than 80%, and amplifying step by step according to the same method to complete the preparation of seed liquid; (2) inoculating the seed liquid into a bioreactor according to the density of 4-6 multiplied by 105/ml of living cells, wherein the dissolved oxygen is 30-60%, the rotating speed is 180-.)

1. A preparation method of recombinant human follicle stimulating hormone with high specific activity is characterized by comprising the following steps:

(1) inoculating rhFSH/CHO-S working bank cell strain into shake flask containing CD Forti CHO medium (containing 8mM glutamine) for resuscitation, and shakingCulturing for 2-4 days until the concentration of viable cells reaches 2-3 × 10⁶Per ml, when the activity is not less than 80%, the ratio is 2-4 × 10⁵Transferring the density of/ml to the next-stage shake flask, amplifying step by the same method to complete the preparation of seed liquid, and ensuring that the cell density is not lower than 3 × 10 before entering the bioreactor⁶Per ml, the activity is not lower than 95%;

(2) mixing the above seed liquid according to living cell 4-6 × 10⁵A density inoculation bioreactor of/ml, dissolved oxygen is 30-60%, the rotating speed is 180-220rpm (7L bioreactor), 100-140rpm (14L bioreactor), the basic aeration rate is 0.001-0.01vvm, the Cellturbo Feed2 feeding medium is fed according to the proportion of 2-5% (V/V) on the 4 th day, 6 th day, 8 th day and 10 th day after inoculation, glucose and glutamine are fed in a proper flow mode, the glucose concentration is controlled to be 2-4g/L, the glutamine concentration is controlled to be 2-4mM, the cell density and activity are monitored at fixed time, and the concentration change of glucose, glutamine, lactic acid and ammonia is monitored; controlling the culture temperature at 36-37 deg.C in cell proliferation stage after inoculation, and allowing viable cell density to reach 1.5-1.7 × 10⁷When the cell activity is reduced to 75-85%, stopping culturing, and collecting the cell; detecting rhFSH terminal expression quantity by a quantitative ELISA method to be not less than 200 mg/L;

(3) clarifying the cell culture solution, ultrafiltering with 5KD membrane, concentrating by 5-10 times, replacing with 20mM PBS buffer solution (pH5.0-5.5), filtering with 0.45 μm membrane, and purifying by subsequent chromatography;

(4) DEAE sephaseff chromatography: equilibrating DEAE chromatography column with 20mM PBS (pH 5.0-5.5) for 3-5 Column Volumes (CV), loading with 2-3 CV samples, further equilibrating with 20mM PBS (pH 5.0-5.5) to baseline, collecting flow-through peak number 2 for subsequent purification;

(5) affinity chromatography: the CaptureSelect FSH affinity column is equilibrated for 5-10 CVs with a 20mM PBS (pH 7.2-7.4) equilibration solution, DEAE sephaseff flow is taken to pass through a sample, the pH value is adjusted to 7.2-7.4, the sample is loaded for 3-5 CVs, the equilibration is continued until the baseline is leveled, 3-5 CVs are eluted by a 20mM PBS 2M MgCl2 (pH 7.2-7.4) eluent, and the target elution front is collected for subsequent purification;

(6) hydrophobic interaction chromatography: a phenyl sepharose 6FF chromatographic column balances 3-5 CVs with 2M NaCl solution, an affinity chromatography elution sample is mixed with 4M NaCl solution according to the equal volume of 1:1, samples are loaded according to 0.5-2 CVs, 3-5 CVs are continuously balanced until a base line is leveled, the elution is carried out by physiological saline, a target elution peak is collected, a 0.22 mu M filter membrane is used for sterilization, and the sample is stock solution which is frozen and stored for standby at minus 20 ℃;

(7) the rhFSH stock solution is proved to reach the standards of protein structure, physicochemical property, biological activity, impurity residue and the like through comprehensive detection and structure confirmation, and the specific activity of the pure rhFSH is not lower than 10000IU/mg through the detection of a rat ovary weight increasing method (Steelman-Pohley method).

2. The method according to claim 1, wherein the clarification method of step (3) is a step centrifugation method (4000 rpm/8000 rpm) or a deep filtration method (5 μm/0.2 μm).

3. The method for preparing recombinant human fsh of claim 2, wherein the shaking conditions in step (1) are 37 ℃, 120rpm, and 8% CO₂Under the conditions.

4. The method for preparing recombinant human FSH with high specific activity according to claim 3, wherein CO2/NaHCO is added after the bioreactor is inoculated in step (2)₃The pH value is controlled to be 7.0 +/-0.2.

Technical Field

The invention relates to the technical field of follicle stimulating hormone, in particular to a preparation method of high specific activity recombinant human follicle stimulating hormone.

Background

Follicle-Stimulating Hormone (FSH), a glycoprotein gonadotropin secreted by anterior pituitary basophils, is a central Hormone regulating mammalian growth, sexual maturation and reproduction. FSH is of great interest in the treatment of ovulation disorders, in promoting the normal growth of follicles, in maturation and production of gonadal steroids, in activating and maintaining the number and quality of normal sperm, in vitro fertilization and embryo transfer assisted pregnancy techniques.

Currently, commercially available FSH preparations mainly include urinary FSH (ufsh) and recombinant human follicle stimulating hormone (rhFSH), wherein rhFSH is mostly expressed in an exocrine form by using CHO cells as an expression host, and has a glycosylation configuration similar to that of natural FSH, and the product has the advantages of high specific activity, controllable quality and easy industrialization. The FSH has a complex structure containing two alpha and beta subunits, 5 and 6 pairs of intrachain disulfide bonds, respectively, and 2N-sugar glycosylation sites each. Saccharides are indispensable important components of FSH, sialic acid distributed at the end of a sugar chain can directly influence the activity, metabolism, half-life and the like of the FSH, the number of glycosylated side chains and the content of sialic acid have important effects on the biological activity and half-life of the FSH, and the structural properties of a product determine a more severe cell culture and purification process. The adherent cell culture mode based on the traditional serum-containing culture medium has the disadvantages of potential animal-derived safety risk, large batch difference, high cost, unfavorable industrialization and the like. The serum-free full-suspension culture process for mammalian cells developed in recent years adopts a serum-free culture medium, particularly a Chemical Defined Media (CDM), and has the advantages of clear components, no animal-derived components, high safety, easy amplification and the like. In addition, how to obtain rhFSH with high purity and high specific activity from the cultured cell culture solution is also a key technical problem which restricts the industrialization of the product.

Currently marketed FSH preparations are mainly classified into the following two groups according to their origin and preparation process: the first is urinary fsh (ufsh) extracted from the urine of postmenopausal women, but this follicle stimulating hormone contains contamination with other human proteins, and there are differences in the glycosyl structure between batches, and the demand for urinary sources is enormous, and cannot meet the rapidly increasing market demand. The other type is recombinant human follicle stimulating hormone (rhFSH) expressed by mammalian cells represented by CHO cells, wherein the rhFSH is not only expressed in an exocrine form, but also has the characteristic of post-translational glycosylation modification similar to that of natural FSH and can generate FSH with high biological activity, so that the FSH has the advantages of high safety, good specific activity, controllable quality and easy industrialization. The traditional serum-containing culture mode has the disadvantages of large batch difference, high safety risk, only adherent culture, difficult amplification and the like. Compared with the traditional adherent culture process containing serum, the serum-free full-suspension culture process has the following advantages: (1) the difference between serum batches is overcome, and the cell culture is more stable; (2) removing animal-derived components such as serum and pancreatin, and reducing animal-derived pollution caused by the components such as virus or mycoplasma; (3) the CDM culture medium does not contain macromolecules such as protein and the like, does not contain a mixture such as protein hydrolysate and the like, and is beneficial to downstream purification design; (4) the culture medium has definite components, and is beneficial to researching the aspects of growth, metabolism, gene regulation and the like of cells. The serum-free suspension culture technology is easy to culture in a bioreactor, has high automation degree, is convenient for observing the number and the state of cells on line or off line, has simple and controllable process and is easy to amplify; the steps of traditional adherent cell pancreatin digestion and the like are not needed, the operation links are few, the pollution rate is low, and the product is safer and has more stable quality. In addition, the purification process also becomes the rate-limiting step of industrial production, and how to obtain rhFSH from the cell culture solution with high efficiency and ensure that the structure is correct and the specific activity is high is another key technical problem. At present, the rhFSH purification process mostly comprises a metal chelating or dye affinity chromatography step, but the metal chelating based affinity chromatography process has potential adverse effects on the activity of a target protein, and the dye affinity chromatography has the problems of poor affinity specificity and the like.

Initially, rhFSH is cultured in a culture medium containing serum for adherent culture of spinner flasks or microcarriers, and the adherent cell culture method based on the conventional culture medium containing serum, such as spinner flask culture, cell factory culture and culture process based on sheet carriers or microcarriers, has high safety risk, large difference between serum batches, high cost and unfavorable industrial amplification due to the animal-derived components such as serum, and greatly limits the development of the animal cell culture process, so it is important to develop a serum-free full suspension culture process for mammalian cells to replace the conventional culture process. Compared with a cell culture medium containing serum, the serum-free culture medium, particularly CDM, has the advantages of definite components, small batch difference, no animal-derived components, high safety, easiness in bioreactor culture, high automation degree, convenience in cell sampling, observation and counting, simple and controllable process, easiness in amplification, no need of the steps of traditional adherent cell pancreatin digestion and the like, few operation links and low pollution rate. In recent years, Shixinxin and the like report that a cell bioreactor is used for large-scale culture of CHO cells to express rhFSH, a serum-free and microcarrier perfusion culture technology is adopted in the process, the process belongs to an adherent cell-based culture technology and is not a full suspension culture, and the expression amount is about 20 mg/L. Patent CN 105462909B reports a CHO cell culture method for efficiently expressing human follicle stimulating hormone, which adopts commercial serum-free and fed-batch culture medium to culture from seed recovery to cell culture in the whole process, obtains cells with high density and high activity, and the batch yield reaches 40 mg/L. Another method for producing human follicle stimulating hormone by high-density perfusion culture of recombinant CHO cells is reported in patent CN 110042137A, the process adopts a serum-free and protein-free culture medium, the culture time is maintained for 35-52 days from an inoculation reactor to the end of the whole perfusion culture, the expression amount is different from 15 mg/L to 25mg/L, the yield of single batch of protein is more than 30g, the yield is considerable, but the too long culture time also greatly increases the risk of contamination, and the production cost of culture, purification, storage and the like is high.

In addition, the purification process also becomes the rate-limiting step of rhFSH industrial production, and how to obtain rhFSH pure product from cell culture solution with high efficiency and ensure that the structure and function of the rhFSH are not affected is another key technical problem. Patent CN 102295695A reports purification process routes of ultrafiltration concentration, anion exchange, hydrophobicity, anion exchange, molecular exclusion and anion exchange, and the process steps are too complicated and the yield is low. In patent CN100554277C, FSH was purified by anion exchange chromatography and immobilized metal ion chromatography (i.e. metal chelate chromatography) and hydrophobic chromatography, but the metal chelate affinity chromatography based process has a potentially adverse effect on the activity of the target protein. Patents CN 103570820 a and CN 102448979 a disclose dye affinity chromatography processes based on Blue Sepharose CL-6B chromatography media and Cibacron Blue F3G-a chromatography media, respectively, which have the advantages of low cost and easy control and amplification of the process, while dye affinity chromatography has the problems of poor affinity specificity, low affinity efficiency, and the like.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of recombinant human follicle-stimulating hormone with high specific activity.

The invention relates to a preparation method of recombinant human follicle-stimulating hormone with high specific activity, which comprises the following steps:

(1) inoculating rhFSH/CHO-S working bank cell strain into shake flask containing CD Forti CHO medium (containing 8mM glutamine) for resuscitation, shake culturing for 2-4 days until living cell concentration reaches 2-3 × 10⁶Per ml, when the activity is not less than 80%, the ratio is 2-4 × 10⁵Transferring the density of/ml to the next-stage shake flask, amplifying step by the same method to complete the preparation of seed liquid, and ensuring that the cell density is not lower than 3 × 10 before entering the bioreactor⁶Per ml, the activity is not lower than 95%;

(2) mixing the above seed liquid according to living cell 4-6 × 10⁵A density inoculation bioreactor of/ml, dissolved oxygen is 30-60%, the rotating speed is 180-220rpm (7L bioreactor), 100-140rpm (14L bioreactor), the basic aeration rate is 0.001-0.01vvm, the Cellturbo Feed2 feeding medium is fed according to the proportion of 2-5% (V/V) on the 4 th day, 6 th day, 8 th day and 10 th day after inoculation, glucose and glutamine are fed in a proper flow mode, the glucose concentration is controlled to be 2-4g/L, the glutamine concentration is controlled to be 2-4mM, the cell density and activity are monitored at fixed time, and the concentration change of glucose, glutamine, lactic acid and ammonia is monitored; controlling the culture temperature at 36-37 deg.C in cell proliferation stage after inoculation, and allowing viable cell density to reach 1.5-1.7 × 10⁷When the cell activity is reduced to 75-85%, stopping culturing, and collecting the cell; detecting rhFSH terminal expression quantity by a quantitative ELISA method to be not less than 200 mg/L;

(3) clarifying the cell culture solution, ultrafiltering with 5KD membrane, concentrating by 5-10 times, replacing with 20mM PBS buffer solution (pH5.0-5.5), filtering with 0.45 μm membrane, and purifying by subsequent chromatography;

(4) DEAE sephaseff chromatography: equilibrating DEAE chromatography column with 20mM PBS (pH 5.0-5.5) for 3-5 Column Volumes (CV), loading with 2-3 CV samples, further equilibrating with 20mM PBS (pH 5.0-5.5) to baseline, collecting flow-through peak number 2 for subsequent purification;

(5) affinity chromatography: the CaptureSelect FSH affinity column is equilibrated for 5-10 CVs with a 20mM PBS (pH 7.2-7.4) equilibration solution, DEAE sephaseff flow is taken to pass through a sample, the pH value is adjusted to 7.2-7.4, the sample is loaded for 3-5 CVs, the equilibration is continued until the baseline is leveled, 3-5 CVs are eluted by a 20mM PBS 2M MgCl2 (pH 7.2-7.4) eluent, and the target elution front is collected for subsequent purification;

(6) hydrophobic interaction chromatography: a phenyl sepharose 6FF chromatographic column balances 3-5 CVs with 2M NaCl solution, an affinity chromatography elution sample is mixed with 4M NaCl solution according to the equal volume of 1:1, samples are loaded according to 0.5-2 CVs, 3-5 CVs are continuously balanced until a base line is leveled, the elution is carried out by physiological saline, a target elution peak is collected, a 0.22 mu M filter membrane is used for sterilization, and the sample is stock solution which is frozen and stored for standby at minus 20 ℃;

(7) the rhFSH stock solution is proved to reach the standards of protein structure, physicochemical property, biological activity, impurity residue and the like through comprehensive detection and structure confirmation, and the specific activity of the pure rhFSH is not lower than 10000IU/mg through the detection of a rat ovary weight increasing method (Steelman-Pohley method).

The preparation method of the recombinant human follicle stimulating hormone with high specific activity comprises the step (3) of clarifying by a sectional centrifugation method (4000 rpm/8000 rpm) or a deep filtration method (5 mu m/0.2 mu m).

The invention relates to a preparation method of high specific activity recombinant human follicle stimulating hormone, wherein the shaking condition in the step (1) is 37 ℃, 120rpm and 8 percent CO₂Under the conditions.

The invention relates to a preparation method of high specific activity recombinant human follicle stimulating hormone, which comprises the following steps of inoculating CO2/NaHCO into a bioreactor in the step (2)₃The pH value is controlled to be 7.0 +/-0.2.

Compared with the prior art, the invention has the beneficial effects that: the invention adopts an rhFSH/CHO-S engineering cell line as an independently constructed stable high-yield cell line, clones the genes of human follicle-stimulating hormone F alpha and F beta into a pCHO1.0 high-expression vector, transfects CHO-S suspension cells with independent property right, and screens to obtain the stable high-yield cell line; the cell strain is suitable for a serum-free CD culture medium suspension culture process, has the advantages of high expression quantity, good stability, high specific activity and easy industrialization, and has higher commercial value;

the invention provides a culture method for efficiently expressing human follicle stimulating hormone rhFSH/CHO-S, wherein a basic culture medium CD Forti CHO suitable for the cell line is screened to be combined with a supplementary culture medium Cellturbo Feed2, and a two-stage full suspension culture process is established, wherein the temperature is controlled to be 36-37 ℃ in a cell proliferation stage, 30-33 ℃ in a product expression stage, and proper pH, dissolved oxygen, rotating speed and aeration speed are controlled simultaneously, and a proper amount of culture medium is fed in a flow manner to obtain ideal cell density and expression quantity; the whole process of the process adopts a serum-free culture medium, and no serum or animal-derived components exist, so that the product quality risk caused by exogenous factor pollution and serum batch difference due to serum addition is overcome; in addition, the full suspension culture technology is easy to culture and amplify in a bioreactor, has high automation degree, overcomes the problems of complicated operation links, high pollution rate and the like of the traditional adherent cells, and has gradually become the mainstream trend of cell culture in the field of biological pharmacy as an advanced cell culture technology;

the invention establishes an efficient rhFSH purification process, the purification process firstly clarifies, ultrafilters, concentrates and replaces cell culture supernatant, and then sequentially passes through an ion exchange chromatographic column, an affinity chromatographic column and a hydrophobic chromatographic column, and the process has the advantages of simple process, high yield and easy amplification; the chromatographic process of the CaptureSelect FSH affinity filler based on the camel source VHH nano antibody has the advantages of strong specificity, high affinity efficiency and high loading capacity, the filler has the characteristics of good stability and strong selectivity, and only selectively combines rhFSH complete molecules, but not free alpha and beta single subunits, so that the specific activity of the obtained purified product is higher, and the industrial application is facilitated.

Drawings

FIG. 1 is a flow chart of the present invention;

FIG. 2 is a graph showing the trend of cell activity and cell number change in two stages of culture at 37 ℃→ 33 ℃;

FIG. 3 is a graph showing the trend of rhFSH expression level change in two stages of culture at 37 ℃ → 33 ℃;

FIG. 4 is a DEAE sephaseFF chromatogram;

FIG. 5 is a CaptureSelect FSH affinity chromatogram;

FIG. 6 is phenyl sepharose 6FF hydrophobic chromatogram;

FIG. 7 is a graph showing the purity of rhFSH detected by SDS-PAGE;

FIG. 8 is a schematic diagram showing the measurement of an isoelectric point of rhFSH by a flat-plate focusing electrophoresis method.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1:

(1) taking 1 cell of rhFSH/CHO-S working seed bank from a liquid nitrogen tank, rapidly thawing in 37 deg.C water bath, suspending the cell suspension as soon as possible in a 125ml triangular flask containing 25ml of 37 deg.C preheated CD Forti CHO medium (containing 8mM glutamine), shaking and culturing at 120rpm, 37 deg.C and 8% CO2 for 3-4 days, detecting cell density and activity, and determining cell density and activity according to 3.0 × 10⁵Performing continuous passage of each strain, amplifying step by step according to the sequence of 125ml shake flask, 500ml shake flask and 1000ml shake flask, amplifying for 3-4 generations, determining shake flask number according to the scale of the bioreactor, completing preparation of seed solution, and ensuring that the cell density before the bioreactor is not lower than 3 × 10⁶Per ml, the activity is not lower than 95%;

(2) injecting 2L CD Forti CHO culture medium into a 7L bioreactor sterilized by moist heat and high pressure, adding glutamine with final concentration of 8mM, F68 with final concentration of 1.0g/L and anti-caking agent with final concentration of 1% (V/V), and mixing the above cell seed solution at living cell density of 4.6 × 10⁵The culture medium is inoculated with/ml of the culture medium, a microbubble gas distributor is adopted, the basic aeration rate is adjusted to be 0.001vvm, the rotating speed is 200rpm, the temperature is 37 ℃, and CO is added₂/NaHCO₃Controlling pH value to 7.0 + -0.2, feeding Cellturbo Feed2 supplemented medium at a ratio of 2-5% (V/V) on days 4, 6, 8, and 10, simultaneously feeding glucose and glutamine, controlling glucose concentration to 2-4g/L and glutamine concentration to 2-4mM, and monitoring in real timeMonitoring biochemical parameters such as glucose, glutamine, lactic acid and ammonia; controlling the culture temperature at 37 deg.C in cell proliferation stage after inoculation, and allowing viable cell density to reach 1.7 × 10⁷When the cell activity is reduced to 80%, stopping culturing, and collecting the cells; the final expression quantity of rhFSH detected by adopting an FSH ELISA quantitative detection kit method of DRG company is 208.3 mg/L;

(3) 2.2L of the cell culture solution is clarified by a fractional centrifugation method: centrifuging at 4000rpm for 30min, sucking supernatant, centrifuging at 8000rpm for 30min, ultrafiltering the supernatant with 5KD membrane, concentrating with 20mM PBS buffer solution (pH 5.0), replacing to 400ml, and filtering with 0.45 μm membrane for subsequent chromatographic purification;

(4) DEAE sephaseff chromatography: loading 200ml DEAE sepharoseFF chromatographic column, balancing 5 CVs of DEAE chromatographic column with 20mM PBS (pH 5.0), loading the 400ml ultrafiltration concentrated solution at 150cm/h, balancing with 20mM PBS (pH 5.0), collecting about 200ml number 2 flow-through peak for subsequent affinity chromatography purification;

(5) affinity chromatography: loading 50ml CaptureSelect FSH affinity column, equilibrating with 20mM PBS (pH 7.2) for 5 CVs, flowing 200ml DEAE sephaseFF, adjusting pH to 7.2 with 1N NaOH, loading at 200cm/h, equilibrating with 5 CVs, passing through 20mM PBS-2M MgCl₂(pH 7.2) eluting the eluent, and collecting about 90ml of target elution peak for subsequent purification;

(6) hydrophobic interaction chromatography: loading phenyl sepharose 6FF chromatographic column 170ml, balancing 5 CVs with 2M NaCl solution, mixing affinity chromatography elution samples with 4M NaCl solution in a volume of 1:1, loading 90ml at a flow rate of 200cm/h, continuously balancing 3-5 CVs until a base line is leveled, eluting with normal saline, collecting a target elution peak, sterilizing with a 0.22 mu M filter membrane to obtain a stock solution, and freezing at-20 ℃ for later use;

(7) the rhFSH stock solution is shown to reach the standards of protein structure, physicochemical properties, biological activity, impurity residue and the like through comprehensive detection and structure confirmation, and the specific activity of the pure rhFSH product detected by a rat ovary weight increasing method (Steelman-Pohley method) is not less than 10000IU/mg, thereby reaching the advanced level at home and abroad.

Example 2

The embodiment provides a preparation method of human follicle stimulating hormone with high specific activity, which comprises the following steps:

(1) taking 1 cell of rhFSH/CHO-S working seed bank from a liquid nitrogen tank, rapidly thawing in 37 deg.C water bath, suspending the cell suspension as soon as possible in a 125ml triangular flask containing 25ml of 37 deg.C preheated CD Forti CHO medium (containing 8mM glutamine), shaking and culturing at 120rpm, 37 deg.C and 8% CO2 for 3-4 days, detecting cell density and activity, and determining cell density and activity according to 3.0 × 10⁵Performing continuous passage of each strain, amplifying step by step according to the sequence of 125ml shake flask, 500ml shake flask and 1000ml shake flask, amplifying for 3-4 generations, determining shake flask number according to the scale of the bioreactor, completing preparation of seed solution, and ensuring that the cell density before the bioreactor is not lower than 3 × 10⁶Per ml, the activity is not lower than 95%;

(2) injecting 8L CD Forti CHO culture medium into a 14L bioreactor sterilized by moist heat and high pressure, adding glutamine with final concentration of 8mM, F68 with final concentration of 1.0g/L and anti-caking agent with final concentration of 0.1% (V/V), and mixing the above cell seed solution at living cell density of 5.4 × 10⁵The culture medium is inoculated with/ml of the culture medium, a large bubble gas distributor is adopted, the basic aeration rate is adjusted to be 0.01vvm, the rotating speed is 110rpm, the temperature is 37 ℃, and CO is added₂/NaHCO₃Controlling the pH value to be 7.0 +/-0.2, feeding Cellturbo Feed2 supplemented medium according to the proportion of 2-5% (V/V) on the 4 th day, the 6 th day, the 8 th day and the 10 th day respectively, feeding glucose and glutamine simultaneously, controlling the glucose concentration to be 2-4g/L and the glutamine concentration to be 2-4mM, monitoring the cell density and activity in real time, and monitoring biochemical parameters such as glucose, glutamine, lactic acid, ammonia and the like; controlling the culture temperature to 36 ℃ in the cell proliferation stage after inoculation until the living cell density reaches 1.5 multiplied by 10⁷When the cell activity is reduced to 77%, the cell activity is stopped to be cultured, and the cell is collected; the final expression quantity of rhFSH is detected to be 226.2mg/L by adopting an FSH ELISA quantitative detection kit method of DRG company;

(3) clarifying 8.5L of the cell culture solution with disposable depth filtration membrane (5 μm/0.2 μm), ultrafiltering the supernatant with 5KD membrane package, concentrating with 20mM PBS buffer solution (pH5.2), replacing to 1.1L, filtering with 0.45 μm membrane, and purifying by subsequent chromatography;

(4) DEAE sephaseff chromatography: loading 600ml DEAE sepharoseFF chromatographic column, balancing 5 CVs of DEAE chromatographic column with 20mM PBS (pH 5.2), loading 1L of the ultrafiltration concentrate at 150cm/h, balancing with 20mM PBS (pH 5.2), collecting about 650ml of No. 2 flow-through peak, and purifying by subsequent affinity chromatography;

(5) affinity chromatography: loading 150ml CaptureSelect FSH affinity column, equilibrating with 20mM PBS (pH 7.2) for 5 CVs, flowing DEAE sephaseFF through 600ml sample, adjusting pH to 7.2 with 1N NaOH, loading at 200cm/h, continuously equilibrating with equilibration solution for 5 CVs, passing through 20mM PBS-2M MgCl₂(pH 7.2) eluting the eluate, collecting about 250ml of the desired eluate peak for subsequent purification;

(6) hydrophobic interaction chromatography: loading phenyl sepharose 6FF chromatographic column 400ml, balancing 5 CVs with 2M NaCl solution, mixing affinity chromatography elution samples with 4M NaCl solution in a volume of 1:1, loading 250ml at a flow rate of 200cm/h, continuously balancing 3-5 CVs until a base line is leveled, eluting with normal saline, collecting a target elution peak, sterilizing with a 0.22 mu M filter membrane to obtain a stock solution, and freezing at-20 ℃ for later use;

(7) the rhFSH stock solution is shown to reach the standards of protein structure, physicochemical properties, biological activity, impurity residue and the like through comprehensive detection and structure confirmation, and the specific activity of the pure rhFSH product detected by a rat ovary weight increasing method (Steelman-Pohley method) is not less than 10000IU/mg, thereby reaching the advanced level at home and abroad.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.