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Perfusion culture method of mammal cell

A cell culture and mammalian technology, applied in the direction of animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of insufficient cell density and vigor, expanded culture scale, low expression level, etc., to achieve good operability and Practicality, the effect of expanding the scale of cultivation

Active Publication Date: 2012-03-21
LUNAN PHARMA GROUP CORPORATION
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Problems solved by technology

[0017] At present, the methods for large-scale culture of mammalian cells to produce target proteins generally have defects such as low expression, short fermentation cycle, and insufficient cell density and viability. Some new technologies in perfusion culture have problems such as complicated operation process and unfavorable expansion of culture scale.
The rotation speed is 75 rpm during spinner bottle culture

Method used

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  • Perfusion culture method of mammal cell
  • Perfusion culture method of mammal cell

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Experimental program
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Effect test

Embodiment 1

[0028] The step-by-step expansion of embodiment 1 cell

[0029] The recovery and expansion of seed cells and the high-density fermentation stage all use serum-free medium.

[0030] Take a cryopreserved seed cell from the liquid nitrogen of the cell bank, which is a recombinant CHO cell highly expressing TNFR: Fc fusion protein, quickly put it into warm water at 37°C for recovery, add 37°C preheated culture medium and blow gently, Centrifuge at 800rpm for 5min, discard the supernatant, add 40mL of medium preheated at 37°C to the pellet, and place in 5% CO 2 Constant temperature incubator culture. Subculture after 72 hours, centrifuge at 800rpm for 5min, discard the supernatant, and add fresh 37°C preheated medium to the pellet to make the cell density reach 2.0×10 5 cells / mL above.

[0031] In the process of cell subculture and expansion, the culture container is enlarged step by step, and after cell expansion in 30mL, 90mL, 300mL, 900mL, 1500mL spinner bottles, when the tot...

Embodiment 25L

[0032] Fermentation culture in embodiment 25L tank

[0033] Serum-free medium is still used in the high-density culture stage of fermentation production to maintain the stability of parameters in each stage of fermentation. During the whole process, open perfusion culture and fermentation are adopted to monitor cell density and glucose consumption. According to the consumption of glucose, the glucose concentration is controlled by continuously adding a certain concentration of glucose to 0.5-3g / L; dissolved oxygen is controlled as oxygen dissolved in the air 50% of the amount.

[0034] In the initial stage of 5L fermenter culture, the culture temperature of engineered cell strains was maintained at 36-37°C; the pH was controlled at pH 7.1-7.2, and CO was added during fermentation 2 and NaHCO 3 To maintain its stability; stirring speed is controlled at 150rpm;

[0035] When the cell density reaches 8×10 6 When cells / mL, the cell growth trend slows down and reaches a plateau...

Embodiment 3

[0037] The comparative test of two kinds of fermentation process control of embodiment 3

[0038] 1. Fermentation control 1

[0039] The fermentation process is controlled to pH 7.1-7.2 in the early stage of fermentation, the stirring speed is 150rpm, the dissolved oxygen is 50%, the temperature is 36-37°C, and the cell density reaches 8×10 6 When the cells / mL reached the stable stage, the pH was lowered to 6.9, the temperature was lowered to 33°C, the stirring speed was reduced to 82rpm, the dissolved oxygen was 50%, and the concentration of glucose was controlled to be 0.5-3g / L.

[0040] 2. Fermentation control 2

[0041] During the entire fermentation process, the pH is controlled to be 7.1-7.2, the stirring speed is 150 rpm, the dissolved oxygen is 50%, the temperature is 36-37° C., and the concentration of glucose is controlled to be 0.5-3 g / L.

[0042] The test results are shown in Table 1. figure 1 and figure 2 . The results show that the present invention has ach...

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Abstract

The invention relates to a perfusion operation method for large-scale culture of mammal cells, which is suitable for culturing the mammal cells with recombinant protein, and more suitable for culturing the mammal cells capable of producing TNFR-Fc fusing protein. The method has the following characteristics: firstly, engineering cells are amplified gradually in the generation and amplification process; secondly, the engineering cells are cultured in a of fermentation tank of 5L; and thirdly, during the later stage of the large-scale culture, the expression amount of the protein is improved byreducing the culturing temperature, lowering the pH value, reducing the rotation speed and adjusting the filling speed rate. According to the process, the production period is prolonged, the cell density is increased, the cell viability is improved, and the expression amount of the protein is improved greatly.

Description

technical field [0001] The invention relates to a method for perfusion culture of mammalian cells. Background technique [0002] Animal cell culture began at the beginning of this century, and its scale began to expand in 1962. It has become a widely used technical method in the fields of biological and medical research and application. The use of animal cell culture to produce enzymes, growth factors, vaccines, monoclonal antibodies and other biological products with important medical value has accounted for 50% of the world's bio-high-tech product market share, and has become an important part of the bio-medical high-tech industry. [0003] Large-scale animal cell culture technology is a very important link in biotechnology pharmaceuticals. According to the growth characteristics of cells, it can be divided into adherent cells and suspension cells; in terms of its culture methods, it can be summarized as suspension culture and immobilized culture; in terms of operation met...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 赵志全赵丽丽夏燕
Owner LUNAN PHARMA GROUP CORPORATION
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