CHO cell culture method

A cell culture and cell technology, applied in the field of CHO cell culture, can solve problems such as affecting the expression of target proteins, and achieve the effects of inhibiting the formation of aggregates, increasing cell density and viability, and reducing the formation of metabolites and aggregates.

Active Publication Date: 2020-07-28
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] In view of the many defects in the prior art, the present invention provides a simple and feasible method for culturing CHO cells to improve the expression of fusion protein and protein content, and then proceed step by step Scale up production to solve the problems in the current technology that affect the expression of target proteins due to factors such as abnormal cell growth, cell clumping, and protein aggregation

Method used

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Embodiment 1

[0054] CHO cells containing the gene of interest encoding the FGF21-Fc fusion protein are provided; in the N38 basal medium (the medium described in Example 1 in the patent CN104513805A), the cells are gradually enlarged and cultured, and the cells are subcultured in roller bottles every three days. After cell expansion in 90mL, 300mL, and 900mL spinner bottles, when the cell density reaches 2×10 6 The cells / ml was transferred to a 5L fermenter; the high-density culture stage was carried out in a 5L fermenter, the stirring speed was 180-250rpm, the dissolved oxygen content was 35-60%, the temperature was 37±0.5°C, the pH was 7.2±0.1, and the cultivation period was 3 ~4 days later when the cell density reaches 7×10 6 When cell / ml, begin to flow and add feeding medium (feeding medium is N38Feed, is the 10 times concentrate of N38 basal medium, and wherein N38 basal medium is the substratum described in embodiment 1 among the patent CN104513805A; Control sulfuric acid Copper 150...

Embodiment 2

[0059] Provide CHO cells comprising the gene of interest encoding the TNFR-Fc fusion protein; in the N38 basal medium (the medium described in Example 2 in the patent CN104513805A), the cells are gradually amplified and cultured, and the culture is carried out in spinner bottles every three days. After cell expansion in 90mL, 300mL, and 900mL spinner bottles, when the cell density reaches 2×10 6 The cells / ml was transferred to a 5L fermenter; the high-density culture stage was carried out in a 5L fermenter, the stirring speed was 180-250rpm, the dissolved oxygen content was 35-60%, the temperature was 37±0.5°C, the pH was 7.2±0.1, and the cultivation period was 3 ~4 days later when the cell density reaches 5×10 6 When cell / ml, begin to flow and add feed medium (feed medium is N38Feed, is the 5 times concentrate of N38 basal medium, and wherein N38 basal medium is the substratum described in embodiment 2 among the patent CN104513805A; Control sulfuric acid Copper 200umol / L, ma...

Embodiment 3

[0064] Provide CHO cells comprising the gene of interest encoding the FGF21-Fc fusion protein; in the N38 basal medium (the medium described in Example 4 in the patent CN104513805A), the cells are gradually scaled up and cultured, and the culture is subcultured in roller bottles every three days. After cell expansion in 90mL, 300mL, and 900mL spinner bottles, when the cell density reaches 2×10 6 The cells / ml was transferred to a 5L fermenter; the high-density culture stage was carried out in a 5L fermenter, the stirring speed was 180-250rpm, the dissolved oxygen content was 35-60%, the temperature was 37±0.5°C, the pH was 7.2±0.1, and the cultivation period was 3 ~4 days later when the cell density reaches 6×10 6 When cell / ml, start to flow and add feeding medium (feeding medium is N38Feed, is 8 times concentrated solution of N38 basal medium, and wherein N38 basal medium is the substratum described in embodiment 4 among the patent CN104513805A; Control sulfuric acid Copper 1...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a CHO cell culture method. According to the CHO cell culture method, a culture medium is fed in a flow feeding waywhen cells are in a logarithmic phase, and the fed culture medium contains manganese ions, copper ions, cysteine and choline chloride. By adding the manganese ions, the copper ions, the cysteine and the choline chloride, the phenomena of abnormal cell growth, cell agglomeration and easy generation of aggregates in the cell culture process are effectively solved. The invention further provides application of the CHO cell culture method in production of fusion protein, and the expression quantity and the protein quality of the fusion protein are effectively improved. By adjusting the dosage of the manganese ions and the dosage of the choline chloride, the sialic acid content in glycosylation modification is high, the mannan content is low, and the stability of the expressed fusion protein isgood.

Description

technical field [0001] The invention belongs to the technical field of animal cell culture, and in particular relates to a CHO cell culture method. Background technique [0002] Cell culture technology is to achieve cell culture in vitro by simulating the growth environment of cells in vivo. Culture medium provides nutrients for in vitro culture of cells and promotes cell growth and proliferation. The development of cell culture medium has gone through chicken embryo juice, natural medium, synthetic medium, and currently commonly used low serum medium, serum-free medium, protein-free medium and chemically defined medium. [0003] Culture medium refers to a liquid or solid medium that can maintain animal cells, microbial cells, plant cells, etc. growing in vitro. The medium currently used for animal cells can be divided according to the development history of animal culture medium: serum-containing medium, low-serum medium, serum-free medium (medium containing some plant hy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0682C12N2500/32C12N2500/30C12N2500/10
Inventor 赵丽丽马动刘忠
Owner LUNAN PHARMA GROUP CORPORATION
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