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

A follicle-stimulating hormone and cell-producing technology, which is applied in the field of high-density perfusion culture of recombinant CHO cells to produce human follicle-stimulating hormone, can solve the problems of short cell culture time and low batch yield, improve product quality, and solve the problem of low expression , The effect of simplifying the purification pretreatment steps

Active Publication Date: 2019-07-23
上海赛迈生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Chinese patent CN 105462909 A mentioned a method for culturing CHO cells that highly express human follicle-stimulating hormone. Serum medium and feed culture, the highest cell density can reach 4×107 cells / ml, the expression level can reach 40mg / L, and the batch production of 5L reactor can reach 140mg, but the cell culture time is longer Short, batch yield is still not high

Method used

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  • Method and culture medium for producing human follicle stimulating hormone through high-density perfusion culture of recombinant CHO cells and application of human follicle stimulating hormone
  • Method and culture medium for producing human follicle stimulating hormone through high-density perfusion culture of recombinant CHO cells and application of human follicle stimulating hormone
  • Method and culture medium for producing human follicle stimulating hormone through high-density perfusion culture of recombinant CHO cells and application of human follicle stimulating hormone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] (1) Cell recovery to obtain seed cell fluid

[0050]Take out a cell from the liquid nitrogen tank, and after melting in a 37°C water bath, transfer the cells to a 15ml sterile centrifuge tube, add 5ml of 37°C preheated serum-free, protein-free, and chemically defined basal cell culture medium, gently Pipette, mix, 800rpm, centrifuge for 5min, discard the supernatant, add 5ml of fresh medium to resuspend, transfer the cell suspension to a 125ml ventilated shake flask, add 15ml of medium, at 37°C, 120rpm, 8% CO2 , Cultivated under 70% to 90% humidity. Sampling and counting every other day, replenishing the medium, and maintaining the cell density at 4-6×10 6 pieces / ml. The volume of the shaker flask is gradually enlarged from 125ml, 250ml, 500ml, 1000ml, and 2000ml. After 7-10 days of amplification, the seed volume reaches about 500ml, and the density is 3.6×10 6 pieces / ml.

[0051] In this example, the serum-free, protein-free, and chemically defined basal medium con...

Embodiment 2

[0063] (1) Cell recovery to obtain seed cell fluid

[0064] Take out a cell from the liquid nitrogen tank, and after melting in a 37°C water bath, transfer the cells to a 15ml sterile centrifuge tube, add 5ml of 37°C preheated serum-free, protein-free, and chemically defined basal cell culture medium, and blow gently , mix well, 800rpm, centrifuge for 5min, discard the supernatant, add 5ml of fresh medium to resuspend, transfer the cell suspension to a 125ml ventilated shake flask, add 15ml of medium, at 37°C, 120rpm, 8% CO2, Cultivate at 70% to 90% humidity. Sampling and counting every other day, replenishing the medium, and maintaining the cell density at 3-4×10 6 pieces / ml. The volume of the shaker flask is gradually enlarged from 125ml, 250ml, 500ml, 1000ml, and 2000ml. After 7-10 days of expansion, the seed volume reaches about 600ml, and the density is 3.8.×10 6 pieces / ml.

[0065]The serum-free, protein-free, chemically defined basal medium in this example is: 400.4...

Embodiment 3

[0079] (1) Cell recovery to obtain seed cell fluid

[0080] Take out a cell from the liquid nitrogen tank, and after melting in a 37°C water bath, transfer the cells to a 15ml sterile centrifuge tube, add 5ml of 37°C preheated serum-free, protein-free, and chemically defined basal cell culture medium, and blow gently , mix well, 800rpm, centrifuge for 5min, discard the supernatant, add 5ml of fresh medium to resuspend, transfer the cell suspension to a 125ml ventilated shake flask, add 15ml of medium, at 37°C, 120rpm, 8% CO2, Cultivate at 70% to 90% humidity. Sampling and counting every other day, replenishing the medium, and maintaining the cell density at 3-4×10 6 pieces / ml. The volume of the shaker flask is gradually enlarged from 125ml, 250ml, 500ml, 1000ml, and 2000ml. After 7-10 days of expansion, the seed volume reaches about 600ml, and the density is 3.5×10 6 pieces / ml.

[0081]In this example, the serum-free, protein-free, and chemically defined basal cell culture...

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Abstract

The invention discloses a method and culture medium for producing a human follicle stimulating hormone through high-density perfusion culture of recombinant CHO cells and an application of the human follicle stimulating hormone, and belongs to the field of biological pharmacy. The preparation method comprises the steps of after CHO cell recovery, performing cell density amplification with a basicculture medium; after the density of living cells achieves a certain density, performing perfusion culture with a production culture medium; additionally adding a fresh production culture medium to the inner part of a bioreaction tank, trapping the CHO cells with a hollow fiber trapping device connected to the outer part of the reaction tank, so that the cells can maintain high density and vitality; and when the cells in the reactor are in the stable state, starting expression product collection of the CHO cells. Through bioreactor spinner culture and a circumscription cell trapping device, the cells can be trapped, broken slices and expressive protein can be filtered, and high-density environment can be provided for cell growth. Besides, through backward flushing of a hollow fiber column,the fiber plugging is reduced, the high-density culture time of the cells can be prolonged, and the batch yield can be notably increased.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for producing human follicle-stimulating hormone by high-density perfusion culture of recombinant CHO cells, a culture medium and applications thereof. Background technique [0002] Human follicle 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 consists of non-covalently linked α and β chains. The follicle-stimulating hormone used in medicine was originally purified from the urine of postmenopausal women, and there was a risk of human protein contamination, and there were large product differences between batches, making it difficult to meet market demand (Prevost R R. Recombinant Follicle- Stimulating Hormone: New Biotechnology for Infertility [J]. Human Reproduction Update, 1998, 18(5):...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C12N5/071C12N5/02C12R1/91
CPCC07K14/59C12N5/0682C12N2500/32C12N2500/34C12N2500/36C12N2500/38C12N2500/20C12N2500/46C12N2500/12C12N2500/16C12N2500/22C12N2500/24C12N2501/999C12N2500/40
Inventor 王延涛惠觅宙李国军赵喜红
Owner 上海赛迈生物科技有限公司
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