Culture method for high-efficiency human follicle stimulating hormone expression CHO cells

A high-efficiency expression technology of human follicle-stimulating hormone, applied in the field of biopharmaceuticals, can solve the problem of low expression of human FSH, and achieve the effects of stable and controllable culture process, stable quality and simple process flow

Active Publication Date: 2016-04-06
哈药集团股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the above problems, the present invention provides a method for using recombinant CHO cells containing human FSH gene to overcome the problem of low human FSH expression caused by existing culture methods

Method used

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  • Culture method for high-efficiency human follicle stimulating hormone expression CHO cells
  • Culture method for high-efficiency human follicle stimulating hormone expression CHO cells
  • Culture method for high-efficiency human follicle stimulating hormone expression CHO cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] This embodiment provides a method for culturing CHO cells highly expressing human follicle-stimulating hormone, the steps are as follows:

[0042] After taking out the seed cells from the liquid nitrogen tank, melt them in a water bath at 37°C, take the cell suspension into a 15ml centrifuge tube, centrifuge at 1000rpm for 4min, and discard the supernatant. After resuspension with fresh basal medium preheated at 37°C, the basal medium consists of 18g / L CD-CHO medium, 0.2mM methionine, 0.3g / L sodium bicarbonate and 1.0g / L Composition of F68 of L. Cultivate in a 250ml air-permeable shake flask with a culture volume of 30ml, and the culture conditions are 120rpm rotation speed, 37°C temperature, 75% humidity, and 5% CO2. Seed expansion is passed step by step in the order of 250ml shake flask-500ml shake flask-1000ml shake flask-2000ml shake flask. After 7 days of expansion, the cell culture volume reaches 500ml, and the density is 3.0×10 6 pieces / ml.

[0043] Take the s...

Embodiment 2

[0046] This embodiment provides a method for culturing CHO cells highly expressing human follicle-stimulating hormone, the steps are as follows:

[0047] After taking out the seed cells from the liquid nitrogen tank, melt them in a water bath at 37°C, take the cell suspension into a 15ml centrifuge tube, centrifuge at 1000rpm for 4min, and discard the supernatant. After resuspending with fresh basal medium preheated at 37°C, the basal medium consists of 18g / L CDOptiCHO medium, 0.2mM methionine, 0.3g / L sodium bicarbonate and 1.0g / L Composition of F68. Cultivate in a 250ml air-permeable shake flask with a culture volume of 30ml, and the culture conditions are 120rpm rotation speed, 37°C temperature, 75% humidity, and 5% CO2. Seed expansion is passed step by step in the order of 250ml shake flask-500ml shake flask-1000ml shake flask-2000ml shake flask. After 8 days of expansion, the cell culture volume reaches 500ml and the density is 3.5×10 6 pieces / ml.

[0048] Take the seed...

Embodiment 3

[0051] This embodiment provides a method for culturing CHO cells highly expressing human follicle-stimulating hormone, the steps are as follows:

[0052] After taking out the seed cells from the liquid nitrogen tank, melt them in a water bath at 37°C, take the cell suspension into a 15ml centrifuge tube, centrifuge at 1000rpm for 4min, and discard the supernatant. After resuspension with fresh basal medium preheated at 37°C, the basal medium consists of 20g / L Ex-cell302 medium, 0.2mM methionine, 0.3g / L sodium bicarbonate and 1.0g / L Composition of F68 of L. Cultivate in a 250ml air-permeable shake flask with a culture volume of 30ml, and the culture conditions are 120rpm rotation speed, 37°C temperature, 75% humidity, and 5% CO2. Seed expansion is passed step by step in the order of 250ml shake flask-500ml shake flask-1000ml shake flask-2000ml shake flask. After 8 days of expansion, the cell culture volume reaches 500ml and the density is 3.1×10 6 pieces / ml.

[0053] Take th...

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Abstract

The invention discloses a culture method for high-efficiency human follicle stimulating hormone expression CHO cells and belongs to the technical field of bio-pharmaceuticals. According to the culture method, seed cells are prepared into cell suspension, and subculture is performed after resuspension through a serum-free basal culture medium to obtain seed suspension; the obtained seed suspension is inoculated into a bioreactor to be cultured, the serum-free basal culture medium is fed at regular intervals in the culture process, growth situations of the cells are monitored, glucose solution is supplemented, and the cells are harvested after culture. According to the method, the serum-free basal culture medium is used throughout the process, culture in a fermentation tank is performed after reviving and amplification through bottle shaking, the process flow is simple, the cell culture density is high and can be as high as 4*107/mL , the cell survival rate reaches more than 98%, maintaining time under high density and a high survival rate is long, the yield of end products can reach 40mg/L, and the method is applicable to industrial production.

Description

technical field [0001] The invention relates to a method for cultivating CHO cells highly expressing human follicle-stimulating hormone, and belongs to the technical field of biopharmaceuticals. Background technique [0002] Human follicle stimulating hormone (humanfollicle stimulating hormone, hFSH) is a glycoprotein gonadotropin synthesized and secreted by basophilic cells in the anterior pituitary gland. It belongs to the heterodimeric glycoprotein family and is encoded by two independent genes. It is composed of α-chain and β-chain connected by valence. The two potential asparagine-linked glycosylation sites of the FSH α chain are at positions 52 and 78, respectively, while the two potential asparagine-linked glycosylation sites of the β chain are at 7 and the position of 24 (Olijve W, de Boer W, Mulders JW et al (1996) Molecular biology and biochemistry of human recombinant follicles stimulating hormone (Puregon). Mol. Hum. Reprod. 2 (5): 371-382). For women, its main...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/071C12P21/02C12R1/91
Inventor 袁淑杰李郑武李国军王莹袁媛张雪亭王锐孟庆勇张磊关录凡姜媛媛吕中华高红梅高晶赵华南
Owner 哈药集团股份有限公司
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