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Process optimization method for culturing oncolytic adenovirus by using bioreactor tank flow

A bioreactor, oncolytic adenovirus technology, applied in the field of bioengineering, can solve problems such as virus amplification

Inactive Publication Date: 2020-10-20
ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because once glucose is depleted, virus amplification stops

Method used

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  • Process optimization method for culturing oncolytic adenovirus by using bioreactor tank flow
  • Process optimization method for culturing oncolytic adenovirus by using bioreactor tank flow

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Embodiment 1

[0024] In order to make the present invention easier to understand, the present invention is explained below with specific embodiments. It should be understood that: this example is only used to illustrate the present invention rather than limit the present invention, and the specific experimental methods not mentioned in the following examples are carried out according to conventional experimental methods.

Embodiment example 1

[0026] Bioreactor: Bioflo 310 reactor (USA NBS company);

[0027] Microcarrier: Fibra-Cel flake carrier (NBS Company, USA);

[0028] Cells: HEK293, purchased from ATCC, USA;

[0029] Virus: Recombinant type 5 adenovirus vector carrying oncolytic adenovirus carrying anti-oncogene IL-24, self-constructed; inoculation MOI between 30-60;

[0030] Cell growth medium: DMEM medium (Gibco, USA) containing 10% serum (Zhejiang Tianhang Company) by volume;

[0031] Virus infection solution: DMEM medium (Gibco, USA) containing 2% serum (Zhejiang Tianhang Company) by volume;

[0032] Determination method of glucose content: direct determination with biological process biochemical analyzer (Shenzhen Silman Company);

[0033] Sugar consumption calculation formula: sugar consumption = (concentration of influent liquid × volume of influent liquid + concentration in the original tank × volume in the original tank) - (concentration of effluent liquid × volume of effluent liquid + concentratio...

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Abstract

The invention relates to the technical field of bioengineering, in particular to a set of technological process for culturing and amplifying recombinant adenovirus by fixing a Fibra-Cel flaky carrierin a bioreactor and amplifying HEK293 cells. Under the condition of comprehensively adapting to a 5-type adenovirus replication and amplification system, a DMEM culture medium containing 10 percent ofserum is used in a cell culture stage, a tank flow culture mode is adopted, the glucose concentration is controlled to be 1 to 2g / L, and the DMEM culture medium containing 2 percent of serum is usedwhen virus inoculation is carried out for 48 hours; the glucose concentration is controlled to be 0.5-1.5 g / L by adopting the tank flow culture mode, a serum-free culture medium is adopted in a virusharvesting stage, the glucose concentration is controlled to be 0.5-1.5 g / L by adopting the tank flow culture mode, and continuous harvesting is performed for 5-6 days. According to the method, the later purification difficulty can be reduced, sub-batches cannot be generated to meet the requirements of biological products, and relatively high virus titer can be achieved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and is particularly suitable for a set of process optimization methods used in cultivating oncolytic adenoviruses with bioreactor tank flow. Background technique [0002] HEK293 cells are packaging cells for adenovirus vectors. Adenovirus is a popular vector for gene therapy research. Oncolytic adenovirus integrates virus therapy, gene therapy and immunotherapy, and has made gratifying progress in the treatment of tumors. In 2004, the first gene therapy product "Jinyousheng" and in 2005, the first oncolytic virus product "Ankerui" were successively launched in China, making adenoviral vectors realize the real clinical application. Therefore, improving the large-scale culture of HEK293 cells and the large-scale culture of oncolytic adenovirus has more and more important market significance. [0003] At present, there have been a lot of research and development on the use of bioreactors for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N5/073C12N5/071C12R1/93
CPCC07K14/54C12N5/0603C12N5/0686C12N7/00C12N2500/34C12N2710/10321C12N2710/10322
Inventor 赵婷张晓珍王如意李成彬朱哲范清林孔圆圆吴婷婷王文静宋礼华
Owner ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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