Protein expression method

An antibody and PD-L1 technology, applied in the biological field, can solve problems affecting the therapeutic effect of protein drugs and side effects of protein drugs

Inactive Publication Date: 2021-07-16
SHANGHAI BIOMABS PHARMA +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Chinese Hamster Ovary (CHO) is a commonly used cultured host cell in the large-scale production of protein drugs. The CHO cell expression system can express protein drugs similar to human protein structures. The mechanism of glycosylation of CHO cells It is similar to the glycosylation mechanism of human beings, but the expression of CHO cells still has non-human glycosylation expression, and these non-human glycosylation will make the expressed protein drugs immunogenic, which is harmful to The application of protein drugs produces side effects, which affect the therapeutic effect of the protein drugs expressed

Method used

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1, the screening of culture medium kind

[0038] Purchase CHOM-B series products and CHOM-S series products of serum-free, animal protein-free, animal-derived component-free medium from Shanghai Maitai Junao Biotechnology Co., Ltd., select CHOM-B01, CHOM-B02, CHOM-B03 and CHOM-B01 S01, CHOM-S03, CHOM-S04 products.

[0039] According to the test results of using CHOM-B series medium and CHOM-S series medium in the early stage, the basic medium and supplementary medium were further screened. According to the type of basal medium and supplementary culture and the ratio of each component, the basal medium is divided into CHOM-B01:CHOM-B02 as 1:1, 1:3, 1:5 and CHOM-B01:CHOM-B03 as 1:0.1, 1:0.5, 1:1, divide the supplementary medium into CHOM-S01:CHOM-S03 as 1:0.3, 1:0.6, 1:1 and CHOM-S01:CHOM-S04 as 1:1, 1:4, 1:7.

[0040] CHO cells were cultured using the above-mentioned different proportions of medium, and after basic culture and supplementary culture, the cel...

Embodiment 2

[0047] Embodiment 2, protein expression method

[0048] Select the basal medium and the supplementary medium CHOM-B01:CHOM-B02 after the screening of Example 1 to be 1:3, CHOM-B01:CHOM-B03 to be 1:1, and the supplementary medium CHOM-S01:CHOM-S03 to be 1:0.3, CHOM-S01:CHOM-S04 is 1:4, large-scale production and expression of the target protein.

[0049] The large-scale production and expression of proteins mainly includes the stages of cell expansion and perfusion culture, and the stage of supplementary culture.

[0050] 1. Cell expansion:

[0051] 1) Recovery of working cells: Take out 1 working cell from the working cell bank, put it in a 37°C water bath, and melt it; use 5ml of recovery culture medium, centrifuge to change the medium, inoculate into a 100ml~150ml shake flask, add 10~ 15ml; the shaker flask is placed in a carbon dioxide shaker for shaking culture, the temperature is 37°C, 70~170rpm, and the carbon dioxide concentration is 2~10%.

[0052] 2) First-class se...

Embodiment 3

[0061] Embodiment 3, the expression of Pan-P antibody

[0062] Taking the Pan-P antibody as an example, use the above-mentioned different basal medium and supplementary medium to culture and express it in CHO cells, and detect the glycoform of the Pan-P antibody obtained by culturing and expressing it.

[0063] Pan-P is the fully human epidermal growth factor receptor EGFR monoclonal antibody disclosed in the patent application number CN201410724477.4. According to the method disclosed in the patent CN201410724477.4, the expression vector containing the Pan-P gene sequence was obtained, and transfected into CHO cells for expression.

[0064]For large-scale production and expression of Pan-P antibody, CHO host cells containing Pan-P antibody gene sequence are cultivated and expressed by basal culture and supplementary culture. The basal medium is based on CHOM-B01:CHOM-B02 at 1:3 or CHOM-B01:CHOM-B03 at 1:1; the supplementary medium is: CHOM-S01:CHOM-S03 at 1:0.3 or CHOM-S01 ...

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Abstract

The invention provides a protein expression method. A serum-free animal-origin-free culture medium is used, a basic culture medium and a supplementary culture medium are adopted for large-scale production and expression of protein drugs, the basic culture medium is suitable for cell amplification and perfusion culture, and the supplementary culture medium is suitable for supplementary batch culture. The protein drug expressed by the method does not contain non-human glycosylation.

Description

technical field [0001] The present invention is a divisional application, and the original application number is 2019114049586. [0002] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a protein expression method, and more specifically, the present invention relates to a CHO cell expression method of a protein drug. The expressed protein drug does not contain non-human glycosylation. Background technique [0003] Among the post-translational modifications of proteins, glycosylation modification is one of the most important and complex modifications, and it is also one of the key attributes for evaluating the quality of protein drugs, such as antibody drugs. [0004] The realization of the function of protein drug is closely related to its glycosylation modification, which will affect the performance of protein drug, such as conformation, stability, solubility, pharmacokinetics, activity and immunogenicity, etc. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12P21/02C12N5/071
CPCC07K14/70503C07K14/70532C07K16/2827C12N5/0682C12N2500/92C12P21/02
Inventor 侯盛郭怀祖聂丽戴建新寇庚徐进郭清城张存超黄卫红
Owner SHANGHAI BIOMABS PHARMA
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