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Cell culture method and medium for preparing human interferon betala and application of cell culture medium

A technology of interferon beta and culture medium, applied in the field of biomedicine, can solve the problems of less research on interferon beta 1a and low efficiency of culture medium formula

Active Publication Date: 2012-07-04
深圳未名新鹏生物医药有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few studies on interferon β1a in China, and the efficiency of the medium formula for producing interferon IFN-β1a (expressed in CHO cells) is low. It is urgent to develop a recombinant human interferon β1a that is suitable for industrial production with low cost and high yield. Cell Culture Media and Culture Methods

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  • Cell culture method and medium for preparing human interferon betala and application of cell culture medium
  • Cell culture method and medium for preparing human interferon betala and application of cell culture medium
  • Cell culture method and medium for preparing human interferon betala and application of cell culture medium

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Embodiment 1

[0037] The configuration of the cell culture medium of human interferon beta 1a of the present invention, its component comprises basal medium and supplement, and described supplement is D-galactose, D-mannose, N-acetylglucosamine, D-glucose, so The additives also include one or more of sodium n-butyrate, potassium dihydrogen phosphate, and sodium chloride.

[0038] The basal medium of the present invention is SFM medium (serum-free medium). The basal medium in this example is CHO-S-SFM II medium from GIBCO Company and SFM4CHO medium from HYCLONE Company.

[0039] The serum-free medium of the present invention comprises the following components: basal medium: 12-18g / L, D-galactose: 0.05-0.45g / L, D-mannose: 0.3-0.7g / L, N - Acetylglucosamine: 0.10-0.50g / L, D-glucose: 1.0-5.0g / L, Sodium n-butyrate: 0.05-1mM, Potassium dihydrogen phosphate: 0.5-3.5mM, Sodium chloride: 10-60mM.

[0040] The configuration of the medium: add the above substances to the following concentration in the...

Embodiment 2

[0052] Comparative Experimental Analysis of Each Component of the Culture Medium of Embodiment 2 Human Interferon β1a

[0053] Experimental Materials:

[0054] 1. Cell line: The cell line comes from Shenzhen Vocational and Technical College. The plasmid expression vector expressing recombinant human interferon β1a is transferred into CHO-DHFR-cells to construct engineered cells. The cell line with high expression is obtained by screening. After amplification, prepare cell cryopreservation tubes. When in use, take the cell cryopreservation tubes and thaw them quickly at 37°C and inoculate them in warmed serum-containing medium for culture.

[0055] 2. The serum-containing medium is the high-sugar DMEM medium of GIBCO Company; the serum-containing medium is 10% newborn bovine serum and 90% high-sugar DMEM medium.

[0056] Basal medium: CHO-S-SFM II medium from GIBCO, SFM4CHO medium from HYCLONE.

[0057] 3. Reagents: D-galactose, D-mannose, N-acetylglucosamine, and D-glucose a...

Embodiment 3

[0129] Embodiment 3 prepares the culture method of the cell of human interferon beta 1a

[0130] a. Take the Chinese hamster ovary cell cryopreservation tube carrying the human interferon β1a expression gene plasmid and resuscitate, pass through a 25cm 2 、75cm 2 、175cm 2 The square bottle was subcultured for 15 days, the subculture was 5-7 generations, the 3L spinner bottle was cultured for 7-12 days, and the amount of seed cells obtained was 5.0×10 7 -1×10 9 cells;

[0131] b. 5L cell tank culture data

[0132] 1. Serum-containing culture stage: 5L cell tank culture initial conditions: press 2×10 5 The cells / ml inoculum was inserted into the digested cells in the spinner bottle, and the initial conditions were set at 37°C, pH 7.2, dissolved oxygen greater than 30% (+3), and the rotation speed was 40rpm for culture. After inserting the cells for 4 hours, observe the cells on the microcarriers On the adherence situation, the cells adhered to the wall normally, and the cel...

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Abstract

The invention relates to the field of biological medicine, in particular to a cell culture method and medium for preparing human interferon betala and application of the cell culture medium. According to the technical scheme of the invention, the cell culture medium for preparing the human interferon betala is provided, and the ingredients of the cell culture medium consist of a basal culture medium and additives, wherein the concentration of the basal culture medium is 12-18 g / L, and the additives contain the following ingredients of: D-galactose with the concentration of 0.05-0.45 g / L, D-mannitol with the concentration of 0.3-0.7 g / L, N-acetyl glucosamine with the concentration of 0.10-0.50 g / L, and D-glucose with the concentration of 1.0-5.0 g / L. According to the serum-free culture medium and method, the problems of low yield and low activity in the cell culture of the recombinant human interferon betala, are solved, the biological activity of the interferon betala in the cell collection liquid finally obtained by the cell culture method reaches 1*10<6> IU / ml, and the content of the interferon betala reaches over 0.8 g / L through the detection of an ELISA method.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a cell culture method for preparing human interferon beta 1a, a cell culture medium and an application of the cell culture medium. Background technique [0002] Human interferon β (IFN-β) is an important cytokine with anti-virus, anti-tumor, and immune regulation effects, mainly produced by fibroblasts and some epithelial cells, common interferon-inducing agents, such as viruses, double-stranded Both RNA and some microorganisms can induce the production of IFN-β. Natural IFN-β is a glycoprotein (sugar content accounts for about 20%), with a relative molecular mass of about 20,000, consisting of 166 amino acids, and a signal peptide consisting of 21 amino acids, cut between S-M. The 17th, 31st, and 141st positions of the molecule are Cys, and the disulfide bond formed between the 31st and 141st positions is necessary for its biological activity. The hIFN-β gene is 777bp long, located ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12R1/91
Inventor 王军张慧娟王妍朱安稳黄志斌
Owner 深圳未名新鹏生物医药有限公司
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