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Method for safe continuous enclosed cell culture, virus production/ inactivation

A cell culture, closed technology, applied in the field of bioengineering, can solve problems such as unfavorable large-scale, continuous, safe production of virus vaccines, non-enclosed, etc., to achieve the effect of facilitating automatic control, improving operation flexibility, and reducing pollution opportunities.

Active Publication Date: 2008-05-21
上海丽坤生物科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, in the above two methods, the two processes of cell culture and virus multiplication can be seen as continuous production, but the whole process is intermittent and not closed, and the next process unit starts after the previous process unit ends. , which is not conducive to large-scale, continuous and safe production of virus vaccines

Method used

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  • Method for safe continuous enclosed cell culture, virus production/ inactivation

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Embodiment

[0022] The above description generally illustrates the contents of the present invention, and the following examples will more directly reflect the situation of the present invention, but it should be pointed out that the examples listed here are only for the purpose of illustration, rather than limitation.

example 1

[0023] Example 1 Vero cell culture production / inactivation rabies virus

[0024] Medium: Medium 199 (Gibco), added with 5% fetal bovine serum, adjusted to pH7.0-pH7.2 with sodium bicarbonate.

[0025] Microcarriers and their treatment: Cytodex-1 (Pharmacia) is used as microcarriers, and the matrix is ​​cross-linked dextran. When in use, dry microcarriers are added to a container siliconized with dimethyldichlorosilane, and the microcarriers are heated at room temperature with PBS solution. Fully soak, discard the supernatant, wash twice with PBS solution, replace with new PBS, sterilize at 121°C for 30 minutes, aspirate the supernatant, wash once with Medium 199 containing 5% fetal bovine serum, replace with new culture Base, equilibrate overnight in the incubator.

[0026] 1) Primary culture of cells

[0027] In the self-made siliconized 500mL stirred bioreactor (Spinner Flasks), the treated microcarriers were added at 3g / L, and Vero cells were inserted, and the inoculation...

example 2

[0036] Example 2 MDCK cell culture production / inactivation H3N2 influenza virus

[0037] This example was carried out with reference to the same steps as Example 1, and MDCK cells were used to culture H3N2 influenza virus.

[0038] Medium: Axcevir-MDCK serum-free medium (Excele Biotechnologies, France) was adjusted to pH7.0-pH7.2 with sodium bicarbonate

[0039] Microcarrier: Cytodex-3, dosage 5g / L.

[0040] Cell culture process: seeding density 1.5×10 5 cells / mL, microcarrier Cytodex-3, dosage 5g / L, culture bioreactor is self-made 500mL stirred bioreactor (Spinner Flasks) with siliconization treatment, cultured at 37℃, dissolved oxygen 40%, stirring speed 50rpm, primary The rate was 600mL / day, and after 2 days of inoculation, the cell density was 4.67×10 6 About cells / mL, continuous culture was started, the sampling rate was 300mL / day, samples were taken and counted regularly within 5 days, and the cell density was maintained at 4.21×10 6 cells / mL, it was found that the c...

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Abstract

A method of cells culture, virus production / deactivation in safe, continuum and blocked style, belonging to bioengineering technical field, compring steps of: 1) Inoculate cells and suspension culture, therebycarry out the primary culture of cells; 2)Flowing add fresh medium, simultly pump out cells suspension to maintain cell culture biological reactor stable and guarantee cell amplificatation during residence time in reactor, accordingly carry out cell's continuous cultivation; 3) Pump out cells suspension into virus-containing virus breeding reactor to make virus infect and amplificate in cells preliminary; 4)As the inflow of cells suspension and outflow of virus-containing upper clean liquid, system dynamic balancing is maintained and virus continuous cultivation is achieved; 5) Collect the upper clean liquid pumped from virus breeding reactor and deactivate the virus, accordingly achieve continuously and obturately cells cultivation and virus deactivation. This method is convenient for automatic control and standardized production, also depresses the pollution chances and heightens production assurance coefficient.

Description

technical field [0001] The invention relates to a method in the technical field of bioengineering, in particular to a method for safe continuous closed cell culture, virus production and inactivation. Background technique [0002] There are two main ways to use cells to produce viruses: (1) Cells and virus seeds are cultivated together. During the process of cell proliferation, viruses are replicated to achieve the purpose of mass production of viruses. This is a way to simulate the natural living state of organisms. Method; (2) Separate the amplification process of cells and viruses, that is, a large number of cells are first amplified, and then virus seeds are added after reaching a certain level to maintain the activity of cells, and through the continuous infection and replication of progeny viruses between cells, to To achieve the purpose of mass production of viruses. With the continuous deepening of virus research, it is found that there are certain differences betwe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N7/04A61K39/12C12N5/02C12N5/071
Inventor 罗凤山齐瀚实陈彦田孙海英耿涛
Owner 上海丽坤生物科技股份有限公司
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