Non-serum culture medium for multiple animal cell large-scale culture

A serum-free medium and large-scale culture technology, applied to animal cells, etc., can solve problems such as increased costs, differences between product batches, and reduced recovery rates

Inactive Publication Date: 2006-05-31
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] (1) Serum is susceptible to contamination by viruses, mycoplasma or other pathogens;
[0005] (2) Differences between different batches of serum cause differences between product batches;
[0006] (3) The existence of a large amount of serum protein increases the difficulty of downstream separation and purification, which increases the cost and reduces the recovery rate;
[0007] (4) It is difficult to completely remove some serum proteins by means of separation and purification, which seriously affects the final quality of the product
[0012] (1) Due to the hig

Method used

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  • Non-serum culture medium for multiple animal cell large-scale culture
  • Non-serum culture medium for multiple animal cell large-scale culture
  • Non-serum culture medium for multiple animal cell large-scale culture

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Experimental program
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Effect test

Embodiment 1

[0033] The above components are mixed and dissolved in pyrogen-free ultrapure water to obtain a culture medium.

[0034] After the HB58 hybridoma cells (obtained from ATCC) were subcultured and adapted in the serum-free medium of the present invention, they were inoculated in 2 liters of bioreactors of BF-2 (Germany B.BRAUN company), and the inoculation density was 2.0×10 5 cells / ml, start perfusion after 56 hours of cultivation, the perfusion rate is 0.5 (1 / day), the perfusion medium is the serum-free medium of the present invention, and the cell density is maintained at 1.2×10 after 200 hours of cultivation. 7 cells / ml, the monoclonal antibody concentration is maintained at about 500mg / L (see figure 1 ), compared with the results of ordinary culture medium batch culture, the cell density and monoclonal antibody concentration were increased by more than 8 times. In the figure, curve 1 is the viable cell density, curve 2 is the total cell density, and curve 3 is the ex...

Embodiment 2

[0036] Element

[0037] rCHO cells (rCHO SS3 A2, expressing human anticoagulant factor III) were subcultured and adapted in the serum-free medium according to the present invention, and then inoculated in a B.BRAUN 2-liter bioreactor with an inoculation density of 2.0×10 5 cells / ml, start perfusion after 40 hours of cultivation, the perfusion rate is 0.58 (1 / day), the perfusion medium is the serum-free medium of the present invention, and the cell density is maintained at 0.9-1.0×10 after 255 hours of cultivation 7 cells / ml, the product concentration is maintained at about 350-380U / L (see figure 2 ), compared with the results of ordinary culture medium batch culture, the cell density and monoclonal antibody concentration were increased by 6 times and 5 times, respectively. In the figure, curve 4 is the living cell density, the curve is the total cell density, and curve 6 is the expression level of the product.

Embodiment 3

[0039] Element

[0040] After the 293 cells were subcultured and adapted in the serum-free medium of the present invention, they were inoculated in a 2-liter bioreactor of B.BRAUN for batch culture, and the inoculation density was 2.45×10 5 cells / ml, by image 3 It can be seen that there is almost no lag phase in the growth of the cells, and they enter the exponential growth phase after inoculation, with an average specific growth rate of 0.46day -1 , with a maximum viable cell density of 11.0×10 5 cells / ml. In the figure, curve 7 is the living cell density, and curve 8 is the ratio of living cells.

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Abstract

The invention opened non-serum mediums which can proper to culture many kinds of the animal cells. The character of it is to use the DMEM/F12 as the base medium, then add the growth factor, hormone, the essential amino acid and the microelement. It has the functions: (1) it can support many cells and clones; (2) it is same as the serum medium in the growth of the cell and the expression of the product; (3) it support the long heritable culture; (4) it is benefit for the isolation of the product because it contains less protein; (5) it is cheap. We can get the high density of the cells and the product concentration using the medium.

Description

technical field [0001] The invention relates to a serum-free medium used for producing biological products such as antibodies, vaccines, and gene recombinant proteins during large-scale high-density culture of animal cells. technical background [0002] Animal cell culture has been widely used in the production of various biologically active substances such as monoclonal antibodies, virus vaccines, virus vectors, immune regulators, growth factors, specific tumor antigens, and various gene recombinant protein drugs. Add a certain amount (5%-10%) of bovine serum, which contains growth factors, hormones, carrier proteins, adhesion factors, trace elements and other nutrients required for cell growth, which can greatly promote cell growth and product expression. [0003] However, the application of serum also brings many disadvantages: [0004] (1) Serum is susceptible to contamination by viruses, mycoplasma or other pathogens; [0005] (2) Differences between different batche...

Claims

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Application Information

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IPC IPC(8): C12N5/07
Inventor 谭文松朱明龙周燕华平牛红星
Owner EAST CHINA UNIV OF SCI & TECH
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