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Method for fully suspended cell culture of avian influenza (H9) inactivated vaccine

A cell culture, inactivated vaccine technology, applied in the field of biomedicine, can solve the problems of low virus amplification efficiency, waste of culture medium, high virus titer, etc., to achieve the standard of vaccine production, reduce operation and cost, virus immunogen Sexual stability and long-lasting effects

Active Publication Date: 2019-09-17
BEIJING VBIOSCI INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, if the virus amplification efficiency is very low using the usual batch culture method, a sufficiently high virus titer can only be obtained through complex perfusion and fed-batch culture methods
However, because influenza virus is a lytic virus and expands rapidly, it will kill the host cells quickly, so the complex and time-consuming perfusion and fed-batch culture methods can obtain high virus titers, but waste a lot of medium

Method used

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  • Method for fully suspended cell culture of avian influenza (H9) inactivated vaccine
  • Method for fully suspended cell culture of avian influenza (H9) inactivated vaccine
  • Method for fully suspended cell culture of avian influenza (H9) inactivated vaccine

Examples

Experimental program
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Effect test

Embodiment 1

[0030] 1. Acclimatized and cultured MDCK cells adapted to culture in serum-free medium

[0031] The MDCK cryopreserved adherent cells were revived with DMEM / D32 medium containing 10% imported fetal bovine serum, and passaged after the cells grew densely.

[0032] After subculture, select cells with good shape and good growth, and gradually carry out domestication and culture. The specific steps are as follows:

[0033] The DMEM / D32 medium with imported fetal bovine serum content of 6% was continuously subcultured for 4 times;

[0034] Continuously subculture twice with DMEM / D32 medium containing 2% imported fetal bovine serum;

[0035] DMEM / D32 medium and serum-free medium are used for culture in a mixed culture medium with a volume ratio of 2:1, wherein the content of imported fetal bovine serum is 2%, and the culture is continuously subcultured for 6 times;

[0036] Use DMEM / D32 medium and serum-free medium for culture in a mixed culture medium with a volume ratio of 1:4, ...

Embodiment 2

[0061] 1. Acclimatized and cultured MDCK cells adapted to culture in serum-free medium

[0062] The MDCK cryopreserved adherent cells were revived with DMEM / D32 medium containing 7% imported fetal bovine serum, and passaged after the cells grew densely.

[0063] After subculture, select cells with good shape and good growth, and gradually carry out domestication and culture. The specific steps are as follows:

[0064] The DMEM / D32 medium with imported fetal bovine serum content of 4% was continuously subcultured for 4 times;

[0065] Continuously subculture twice with DMEM / D32 medium containing 1% imported fetal bovine serum;

[0066] Use DMEM / D32 medium and serum-free medium to culture in a mixed culture solution at a volume ratio of 2:1, in which the content of imported fetal bovine serum is 1%, and continuously subculture for 6 times;

[0067] Use DMEM / D32 medium and serum-free medium to culture in a mixed culture medium with a volume ratio of 1:4, in which the imported f...

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Abstract

The invention relates to a method for fully suspended cell culture of an avian influenza (H9) inactivated vaccine. The method comprises the following steps that MDCK monolayer cells are subjected to habituated culture, an avian influenza (H9) inactivated vaccine chick embryo virus is directly inoculated into MDCK suspension cells for culture, enlargement culture is carried out in a bioreactor, an harvested culture is cultured until the proliferation speed of the avian influenza (H9) inactivated vaccine is stable, and the virus content is greater than or equal to 108.5 EID50; when culture is carried out until the cytopathy reaches 80% or above, a virus solution is harvested, and the avian influenza (H9) inactivated vaccine is obtained. The chick embryo virus is directly inoculated into the MDCK suspension cells for habituated culture, which effectively increases the performance stability, continuity and safety of the produced suspended virus, the obtained suspended virus HA is greater than or equal to 1:4096; the virus content per 0.1 ml is greater than or equal to 109.5 EID50, and the virus content per 1 ml is greater than or equal to 109.5TCID50.

Description

technical field [0001] The patent of the present invention relates to the technical field of biomedicine, in particular to a method for culturing an inactivated avian influenza (H9) vaccine with whole suspension cells. Background technique [0002] In recent years, the technology of full-suspension animal cell culture has developed rapidly and is increasingly used in the production of avian influenza vaccines to gradually replace the traditional chicken embryo culture technology. [0003] However, in the existing production technology, the cells selected in the avian influenza vaccine production process are usually single-layer adherent cells, which are cultured and grown in a single-layer attachment mode. Therefore, a culture process with serum plus microcarriers is required. However, serum has disadvantages such as high price, large batch-to-batch variation, and the risk of contamination by exogenous pathogens. Moreover, since the serum contains a large number of unknown ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16C12N7/04
CPCA61K39/12A61P31/16C12N7/00A61K2039/5252C12N2760/16134C12N2760/16161
Inventor 侯野李俊峰王璐马玉鑫邓志平张凌云张乾顺刘少奇
Owner BEIJING VBIOSCI INC
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