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Method for expressing and preparing double factor antibodies by utilizing serum-free culture of high-density CHO-S cells

A CHO-S, serum-free culture technology, applied in artificial cell constructs, biochemical equipment and methods, animal cells, etc., can solve the problems of long culture time and harsh fluid rehydration conditions

Pending Publication Date: 2021-09-03
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] When the existing CHO-S cells are prepared, the conditions for rehydration are relatively harsh, and the culture time is also relatively long

Method used

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  • Method for expressing and preparing double factor antibodies by utilizing serum-free culture of high-density CHO-S cells
  • Method for expressing and preparing double factor antibodies by utilizing serum-free culture of high-density CHO-S cells
  • Method for expressing and preparing double factor antibodies by utilizing serum-free culture of high-density CHO-S cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 of the present invention, a method for preparing a dual-factor antibody by using high-density CHO-S cells for serum-free culture expression, comprising the following steps:

[0031] S1: First take out the centrifuge tube containing CHO-S cells from liquid nitrogen, and thaw quickly in a 37°C water bath;

[0032] S2: After the CHO-S cell centrifuge tube is completely thawed, clean the outer wall of the CHO-S cell centrifuge tube with 70% ethanol, and then pour all the contents of the CHO-S cell centrifuge tube into 30ml preheated CHO-S In a disposable 125ml sterile Erlenmeyer shaker flask of expression medium;

[0033] S3: Then put the disposable 125ml sterile Erlenmeyer flask into the cell culture incubator for cultivation, and calculate the total number of cultured cells and the number of living cells every day;

[0034] S4: When the culture concentration does not exceed 5×106 viable cells / ml, transfer the cell suspension to a larger volume disposable ste...

Embodiment 2

[0046] Embodiment 2 of the present invention, a method for preparing a dual-factor antibody by using high-density CHO-S cells for serum-free culture expression, comprising the following steps:

[0047] S1: First take out the centrifuge tube containing CHO-S cells from liquid nitrogen, and thaw quickly in a 37°C water bath;

[0048] S2: After the CHO-S cell centrifuge tube is completely thawed, clean the outer wall of the CHO-S cell centrifuge tube with 72% ethanol, and then pour all the contents of the CHO-S cell centrifuge tube into 30ml preheated CHO-S In a disposable 125ml sterile Erlenmeyer shaker flask of expression medium;

[0049] S3: Then put the disposable 125ml sterile Erlenmeyer flask into the cell culture incubator for cultivation, and calculate the total number of cultured cells and the number of living cells every day;

[0050]S4: When the culture concentration does not exceed 5×106 viable cells / ml, transfer the cell suspension to a larger volume disposable ster...

Embodiment 3

[0062] Embodiment 3 of the present invention, a method for preparing a dual-factor antibody by using high-density CHO-S cells for serum-free culture expression, comprising the following steps:

[0063] S1: First take out the centrifuge tube containing CHO-S cells from liquid nitrogen, and thaw quickly in a 37°C water bath;

[0064] S2: After the CHO-S cell centrifuge tube is completely thawed, clean the outer wall of the CHO-S cell centrifuge tube with 74% ethanol, and then pour all the contents of the CHO-S cell centrifuge tube into 30ml preheated CHO-S In a disposable 125ml sterile Erlenmeyer shaker flask of expression medium;

[0065] S3: Then put the disposable 125ml sterile Erlenmeyer flask into the cell culture incubator for cultivation, and calculate the total number of cultured cells and the number of living cells every day;

[0066] S4: When the culture concentration does not exceed 5×106 viable cells / ml, transfer the cell suspension to a larger volume disposable ste...

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Abstract

The invention discloses a method for expressing and preparing double factor antibodies by utilizing serum-free culture of high-density CHO-S cells. The method includes the following steps: S1, firstly taking out a centrifuge tube containing CHO-S cells from liquid nitrogen, and performing quick thawing in a water bath with 37 DEG C; S2, after the CHO-S cell centrifuge tube is completely thawed, using 70% ethanol to clean the outer wall of the CHO-S cell centrifuge tube, then pouring all content in the CHO-S cell centrifuge tube into a 125 ml disposable sterile conical shake flask containing 30 ml of preheated CHO-S expression medium; and S3, then placing the 125 ml disposable sterile conical shake flask into a cell incubator to cultivate, and counting the total number of cultured cells and the number of living cells every day. The method using the serum-free culture of high-density CHO-S cells expresses and prepares bispecific antibodies to achieve optimization; and through the optimization of methods of plasmid extraction, endotoxin removal, concentration quantification and weight proportioning performed on antibody heavy chain and light chain plasmids used by transfected cells, higher expression products are obtained.

Description

technical field [0001] The invention relates to an antipyretic medicated bath pack, in particular to a method for preparing a dual-factor antibody by using high-density CHO-S cells for serum-free culture and expression. Background technique [0002] Mammalian cells have complex post-translational modifications similar to humans, and are often used to express active biomacromolecular proteins, and the functions of these biomacromolecules depend on these post-translational modifications to a certain extent. 3T3, BHK, 293, CHO, NS0, Hela and other cells have been studied and used for the expression of biological macromolecular proteins, but nearly 70% of the therapeutic proteins that have been marketed are expressed and produced by CHO cells, and CHO cells have become complex A major tool for the production of post-translationally modified therapeutic proteins. [0003] When the existing CHO-S cells are prepared, the fluid supplementation conditions are relatively harsh, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C07K16/00
CPCC12N5/0682C07K16/00C12N2500/90
Inventor 孙明王红叶李冰香朱小永
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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