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A kind of combination culture medium expressing adalimumab and application thereof

A technology of adalimumab and culture medium, which is applied in the field of biotechnology and fermentation, and can solve problems such as large errors, affecting the expression of cell growth products, and cytotoxicity

Active Publication Date: 2018-10-16
通化东宝生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are few studies on adalimumab cell culture. The production of adalimumab is mainly carried out by batch fed culture, using CHO cells and CD FortiCHO medium. The expression level is about 2-3g / L. The amount is low, and the by-product lactic acid will be generated and accumulated during the cell culture process, which has a certain toxic effect on the cells and affects the growth of the cells and the expression of the product
[0005] Patent document CN104212769 A provides a laboratory culture method with a yield of 5.33g / L (also the highest in the prior art), but this is based on the general medium, adding up to 16 kinds of additives , if this method is used for large-scale cell culture, there will be large errors in the operation process, and the stability between batches cannot be guaranteed

Method used

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  • A kind of combination culture medium expressing adalimumab and application thereof
  • A kind of combination culture medium expressing adalimumab and application thereof
  • A kind of combination culture medium expressing adalimumab and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] shake flask culture

[0093] Preparation of basal medium:

[0094] Take 0.86g CD FortiCHO, 0.175g glucose, and 0.035g poloxamer 188 to prepare solution A, in which the concentration of CD FortiCHO is 24.56g / L, the concentration of glucose is 5g / L, and the concentration of poloxamer 188 is 1g / L.

[0095] Take 0.96g M20A and prepare solution B whose concentration is 27.62g / L.

[0096] Mix 35ml of solution A with 35ml of solution B to obtain a basal medium.

[0097] Preparation of feed medium:

[0098] Take 4.45g of F001S and prepare solution C (27ml) with a concentration of 164.9g / L.

[0099] Take 0.2g of F001B to make solution D (2.7ml) with a concentration of 76g / L.

[0100] During the cell culture process, solution C and solution D were used as feed medium, and were poured into shake flasks at a volume ratio of 10:1.

[0101] Resuscitate cells, after expansion, dilute and inoculate the cells into CD FortiCHO:M20A=1:1 basal medium, the initial volume is 70ml, at 36...

Embodiment 2

[0111] shake flask culture

[0112] Preparation of basal medium:

[0113] Take 0.69g CD FortiCHO, 0.14g glucose, and 0.028g poloxamer 188 to prepare solution A, in which the concentration of CD FortiCHO is 24.56g / L, the concentration of glucose is 5g / L, and the concentration of poloxamer 188 is 1g / L.

[0114] Take 1.16g M20A and prepare solution B whose concentration is 27.62g / L.

[0115] Mix 28ml of solution A with 42ml of solution B to obtain a basal medium.

[0116] Preparation of feed medium:

[0117] Take 4.45g of F001S and prepare solution C (27ml) with a concentration of 164.9g / L.

[0118] Take 0.2g of F001B to make solution D (2.7ml) with a concentration of 76g / L.

[0119] During the cell culture process, solution C and solution D were used as feed medium, and were poured into shake flasks at a volume ratio of 10:1.

[0120] Resuscitate cells, after expansion, dilute and inoculate the cells into CD FortiCHO:M20A=1:1.5 basal medium, the initial volume is 70ml, at 3...

Embodiment 3

[0123] shake flask culture

[0124] Preparation of basal medium:

[0125] Take 1.075g CD FortiCHO, 0.22g glucose, and 0.044g poloxamer 188 to prepare solution A, in which the concentration of CD FortiCHO is 24.56g / L, the concentration of glucose is 5g / L, and the concentration of poloxamer 188 is 1g / L.

[0126] Take 0.725g M20A and prepare solution B whose concentration is 27.62g / L.

[0127] Mix 43.75ml of solution A with 26.25ml of solution B to obtain a basal medium.

[0128] Preparation of feed medium:

[0129] Take 4.12g of F001S and prepare solution C (25ml) with a concentration of 164.9g / L.

[0130] Take 0.38g of F001B and prepare solution D (5ml) with a concentration of 76g / L.

[0131] During the cell culture process, solution C and solution D were used as feed medium, and were poured into shake flasks at a volume ratio of 5:1.

[0132] Resuscitate cells, after expansion, dilute and inoculate the cells into CD FortiCHO:M20A=1:0.6 basal medium, the initial volume is ...

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Abstract

The invention discloses a combined medium for expressing adalimumab. The combined medium is composed of a basic medium and a fed medium, wherein the basic medium is composed of CD FortiCHO and M20A ina ratio of 1: 0.5-1.5; the fed medium consists of F001S and F001B in a ratio of 5-15: 1; and the above mediums are both commercial mediums. Individual usage of any of the mediums cannot produce high-yield stable adalimumab; but the combined medium of the basic medium and the fed medium can significantly increase the expression quantity of adalimumab and improve cell density and cell viability, stably overcomes the problem of accumulation of a large amount of lactic acid and low yield of target proteins in conventional adalimumab cell culture processes, and is beneficial for further improvement of the output and quality of adalimumab.

Description

technical field [0001] The invention relates to a combination culture medium for expressing adalimumab and application thereof, belonging to the fields of biotechnology and fermentation. Background technique [0002] Chinese hamster ovary (Chinese hamster ovary, CHO) cells are derived from the ovary of Chinese hamster. Commonly used in biological and medical research, as well as in the production of recombinant proteins for therapeutic use. Compared with other cell expression systems, the CHO cell expression system can stably integrate with foreign genes and express proteins that are almost identical to natural protein structures, immunogenicity, glycosylation types and methods through complex post-translational modifications, thereby It can guarantee the medicine and efficacy of the recombinant protein. In addition, CHO cells rarely secrete their own endogenous proteins, which is also conducive to the later isolation and purification of exogenous proteins. Therefore, CHO...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C07K16/24
CPCC07K16/241C12N5/0682
Inventor 冷春生周芳陈子君杨旭
Owner 通化东宝生物科技有限公司
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