Screening culture method of cells capable of efficiently expressing anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody

A high-efficiency expression technology of monoclonal antibodies, which is applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, animal cells, tissue culture, etc., can solve the problems of antibody protein modification and low expression, and achieve The effect of simple process

Inactive Publication Date: 2021-02-23
BEIJING SL PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] For this reason, the embodiment of the present invention provides a method for screening and culturing highly efficient anti-EGFR monoclonal antibody cells to solve the problem of low expression level of recombinant anti-EGFR monoclonal antibody cells and the presence of fucoidan in the expressed antibody protein in the prior art. sugar modification problem

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  • Screening culture method of cells capable of efficiently expressing anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody
  • Screening culture method of cells capable of efficiently expressing anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody
  • Screening culture method of cells capable of efficiently expressing anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody

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Embodiment 1

[0037] Example 1. Screening method for high-efficiency expression of fucose-modified anti-EGFR monoclonal antibody cells

[0038] 1. Recovery of seeded cells with recombinant anti-EGFR monoclonal antibody

[0039] Take out the seeded cells of the recombinant anti-EGFR monoclonal antibody from the liquid nitrogen tank, melt in a 37°C water bath, take the cell suspension into a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant. Resuspend with fresh Actipro serum-free basal medium pre-warmed at 37°C. Cultivate in a 125ml ventilated shake flask with a culture volume of 20ml. The culture conditions are as follows: the rotation speed of the incubator is 100rpm, the culture temperature is 37°C, the culture humidity is 75%, and the CO in the air in the incubator 2 The volume concentration is 5%. Seed cell expansion of recombinant anti-EGFR monoclonal antibody is passed step by step in the order of 250ml-1000ml shake flasks. After two expansions, the c...

Embodiment 2

[0044] Example 2. Screening method for high-efficiency expression of fucose-modified anti-EGFR monoclonal antibody cells

[0045] This embodiment provides a method for screening and culturing CHO cells highly expressing recombinant anti-EGFR monoclonal antibodies, which includes the following steps:

[0046] 1. Recovery of seeded cells with recombinant anti-EGFR monoclonal antibody

[0047] Take out the seed cells of the recombinant anti-EGFR monoclonal antibody from the liquid nitrogen tank, melt the seed cells in a 37°C water bath, take the cell suspension into a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant. Resuspend with fresh Actipro serum-free basal medium pre-warmed at 37°C. Cultivate in a 125ml ventilated shake flask, the culture volume is 20ml, and the culture conditions are: the speed of the incubator is 100rpm, the temperature is 37°C, the humidity is 75%, and the CO in the air 2 The volume concentration is 5%. Seed expansion ...

Embodiment 3

[0052] Example 3. Screening method for high-efficiency expression of fucose-modified anti-EGFR monoclonal antibody cells

[0053] The screening and culturing method of CHO cells highly expressing recombinant anti-EGFR monoclonal antibody provided in this embodiment comprises the following steps:

[0054] 1. Recovery of seeded cells with recombinant anti-EGFR monoclonal antibody

[0055] Take out the seeded cells from the liquid nitrogen tank, melt them in a water bath at 37°C, put the cell suspension in a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant. Resuspend with fresh Actipro serum-free basal medium pre-warmed at 37°C. Cultivate in a 125ml ventilated shake flask, the culture volume is 20ml, the culture condition is 100rpm, the temperature is 37°C, the humidity is 75%, and the CO in the air 2 The volume concentration is 5%. Seed expansion is subcultured step by step in the order of 250ml-1000ml shake flasks. After two expansions, the c...

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Abstract

The embodiment of the invention discloses a screening culture method of cells capable of efficiently expressing an anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody. The method comprises the following steps: resuscitating a seed cell of a recombinant anti-EGFR monoclonal antibody, carrying out scale-up culture in an Actipro basal medium to obtain a seed cell culture suspension,inoculating the seed cell culture suspension, carrying out fermentation culture, periodically feeding a fed-batch culture medium and a glucose solution, performing culture until the cell viability islower than 80%, and harvesting a fermentation culture solution to obtain a culture solution for the cells capable of efficiently expressing the anti-EGFR monoclonal antibody. According to the methoddisclosed by the invention, a fucosyltransferase gene knocked-out CHO cell line capable of efficiently secreting and expressing the anti-EGFR antibody is screened, so that expressed protein is free from fucosyl modification, the antibody-dependent cell-mediated cytotoxicity (ADCC) effect of the recombinant anti-EGFR monoclonal antibody is enhanced, the anti-tumor effect of the recombinant anti-EGFR monoclonal antibody is enhanced, and the applicable population is expanded.

Description

technical field [0001] The embodiment of the present invention relates to the field of biotechnology, in particular to a method for screening and culturing cells highly expressing anti-EGFR monoclonal antibodies. Background technique [0002] EGFR is a transmembrane glycoprotein with a relative molecular weight of 170kDa, which belongs to the tyrosine kinase receptor of the HER / ErbB receptor family. EGFR can bind to a variety of ligands such as epidermal growth factor (EGF), tissue growth factor (TGF-α), etc., and the combination of the two will cause EGFR to dimerize to form homologous or heterologous dimers. A cross-linked phosphorylation reaction occurs in the body, that is, one receptor phosphorylates a specific tyrosine residue on another receptor, thereby activating the tyrosine kinase subregion in the intracellular region, and then stimulating the next level of signal transduction. EGFR signal transduction is related to cell proliferation, differentiation, apoptosis,...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/071C07K16/28
CPCC07K16/2863C12N5/0682C12N2510/02
Inventor于红阳申阳阳王红丽胡桂珍杨仲璠徐明波
OwnerBEIJING SL PHARMA