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Method for culturing suspended cell virus capable of maintaining production stability of virus antigens and improving effective content of viruses

A technology of suspension cells and culture methods, applied in biochemical equipment and methods, viruses, animal cells, etc., can solve the problems of virus quantity culture cell density, unstable state relationship, poor virus quality, and not necessarily large virus quantity, etc. Achieve the effect of ensuring stable growth, improving sensitivity and high stability

Active Publication Date: 2019-03-29
内蒙古必威安泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, with the application of large-scale culture of BHK suspension cells in the industrialization of virus culture, some problems have also been magnified. The most prominent and confusing phenomenon is the relationship between the number and quality of viruses in virus culture in BHK suspension cells. The density-state relationship of cells is unstable
Often the cells are growing well but the quality of the cultured virus is poor.
In this regard, a large number of scientific research institutes and vaccine manufacturers at home and abroad have done a lot of research work. At present, the more common solutions mainly include adjusting the amount of inoculation, adjusting the time of inoculation, and changing the culture medium. However, there is no one at present. Systematic, stable, viable solutions that meet mass production conditions
[0004] The main problem of the existing technology is that cell culture and virus culture are separated and judged. The existing technology mainly focuses on the culture density and cell growth state of the cell culture in the early stage. However, the actual situation is that when the cell density is high, the virus amount after virus culture not necessarily much

Method used

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  • Method for culturing suspended cell virus capable of maintaining production stability of virus antigens and improving effective content of viruses
  • Method for culturing suspended cell virus capable of maintaining production stability of virus antigens and improving effective content of viruses
  • Method for culturing suspended cell virus capable of maintaining production stability of virus antigens and improving effective content of viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 carries out foot-and-mouth disease kind ONXC / 92 strain cultivation in three consecutive batches

[0031] 1.1 Recovery of BHK-21 cell line

[0032] Resuscitate the BHK-21 cells frozen in liquid nitrogen, dilute and count the BHK-21 suspension cells that can be stably passaged, and divide the cell suspension in the logarithmic growth phase before freezing at a density of 0.5×10 6 -0.6×10 6 The cells / ml were inoculated into 125ml shake flasks, and the medium was DMEM / F12 without any additives. Shake flasks were placed at 37°C, 5% CO 2 Cultivate in an incubator; use a miniature magnetic stirrer to provide magnetic drive, and the speed is set to 105r / min. Sampling and counting every 48h, and dilution passage. After passage, the cell density was controlled at 0.5×10 6 -0.6×10 6 cells / ml.

[0033] 1.2 Cultivation of seed cells in bioreactors

[0034] Insert domesticated seed cells into the bioreactor (initial density 0.5×10 6 cells / ml, the viability of th...

Embodiment 2

[0070] Embodiment 2 uses the method of the present invention to change serum-free nutrient solution to prepare foot-and-mouth disease virus antigen and does not change the preparation method difference contrast of nutrient solution

[0071] 2.1 Recovery of BHK-21 cell line

[0072] Dilute and count the BHK-21 suspension cell lines that can be stably passaged, and divide the cell suspension in the logarithmic growth phase at a density of 0.5×10 6 -0.6×10 6 cells / ml were inoculated into 125ml shake flasks. Shake flasks were placed at 37C, 5% CO 2 Cultivate in an incubator; use a miniature magnetic stirrer to provide magnetic drive, and the speed is set to 105r / min. Sampling and counting every 48h, and dilution passage. After passage, the cell density was controlled at 0.5×10 6 -0.6×10 6 cells / ml.

[0073] 2.2 Cultivation of seed cells in bioreactors

[0074]Insert domesticated seed cells into the bioreactor (initial density 0.5×10 6 cells / ml, the viability of the seed c...

Embodiment 3

[0089] Example 3 Changes in virus storage valence

[0090] The virus harvested in Example 1.6 was subjected to repeated freeze-thaw and high-temperature challenge experiments to verify the stability of the virus.

[0091] result

[0092] 3.1 Detection of the effect of different freeze-thaw times on virus stability

[0093] Store the virus antigen in a refrigerator at -70°C, then place it at room temperature until it completely thaws into one freeze-thaw, and test the stability of the antigen by freezing and thawing ten times in a row. TCID 50 and LD 50 , the test results are shown in Table 8:

[0094] Table 8 The results of ten consecutive freeze-thaw virus stability tests

[0095]

[0096] 3.2 Effect of temperature on virus stability

[0097] The virus was cultured in a water bath at 42°C for different times, and the antigen stability was tested. The test results are shown in Table 9:

[0098] Table 9 Test results of virus stability at different times for viruses cu...

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Abstract

The invention provides a method for culturing a suspended cell virus capable of maintaining the production stability of virus antigens and improving the effective content of viruses. The method uses aserum-free culture method in a cell culture stage, and the first-time medium change reduces the influence of cell metabolites on cell growth and reduces the interference on late-stage virus production. After the medium change, the added dextran sulfate effectively plays the effects of improving virus infection sensitivity and cell dispersion. PEG 6000 and a high-calcium solution are added to a later-phase virus inoculation and medium change system so as to improve the virus inoculation efficiency and to greatly increase the stability of foot-and-mouth disease virus. The titer of the preparedvirus solution can reach 107.0-109.01 ogTCID50 / mL, the content of complete antigen 146S can reach 6 [mu]g / ml or above, and the cultured foot-and-mouth disease virus is more stable.

Description

technical field [0001] The invention belongs to the field of cell culture and maintenance, and preparation of virus antigens, in particular to a method for culturing suspension cell viruses for maintaining stability and producing high-titer virus antigens, cultured viruses, and preparation of vaccine compositions containing the virus antigens. Background technique [0002] The process of using animal cell culture technology to produce viruses refers to the process of using liquid medium to culture animal cells on a large scale in specific containers, and then inserting viruses to make them proliferate in large quantities in the cells. In 1928, Maitland created the technology of using tissue culture to propagate virus in vitro. In 1949, Enders used Hela cells to culture poliovirus in vitro and produce vaccines, which marked the official application of animal cell culture technology in the field of vaccine production. In 1962, Capsti et al. domesticated BHK adherent cells int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/02C12N7/00C12N7/02
CPCC12N5/0686C12N7/00C12N2500/25C12N2500/34C12N2770/32151
Inventor 张涛刘月何召庆李如珩徐婉英武俊兰王永伟孙聪张春辉
Owner 内蒙古必威安泰生物科技有限公司
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