Method for producing rabies viruses by suspension culture of BHK21 cells

A rabies virus and suspension culture technology, which is applied in the field of suspension culture BHK21 cells to produce rabies virus, can solve the problems of inability to realize automation, large product batch differences, and high risk of contamination, and achieve large-scale automated and continuous production, The effect of reducing batch variance and increasing virus titer

Inactive Publication Date: 2010-10-06
BEIJING SKYWING TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. When large-scale spinner bottles are used to culture BHK21 cells, hundreds of spinner bottles will be used, but each spinner bottle is an independent culture unit, and the state of cells and virus yields cultured in each spinner bottle are different. Therefore, the quantity and quality of the rabies virus produced cannot be effectively controlled, and the quality of the vaccine produced by the rabies virus cannot be effectively controlled.
[0007] 2. BHK21 cells are cultured in spinner bottles. Due to the adherent growth characteristics of BHK21 cells, BHK21 cells need to be digested for passage; in addition, after inoculating virus, adding and replacing virus maintenance solution, adding NaHCO 3 Adjusting the pH value and finally harvesting the culture requires multiple o

Method used

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  • Method for producing rabies viruses by suspension culture of BHK21 cells
  • Method for producing rabies viruses by suspension culture of BHK21 cells
  • Method for producing rabies viruses by suspension culture of BHK21 cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] This example is used to illustrate the method for producing rabies virus by suspension culture BHK21 cells of the present invention.

[0060] Rabies virus: Flury strain.

[0061] Cell culture medium: the dispersant is ultrapure water, and the components contained in the medium are shown in Table 3 (wherein the molecular weight range of soybean protein is 0.1-5Kb).

[0062] Virus maintenance solution: BHK21 cell culture medium including 0.2% by volume of serum.

[0063] Bioreactor: 100 liter stirred bioreactor.

[0064] Add 55 liters of cell culture medium to the bioreactor in advance, and complete the sterilization and cooling of the cell culture medium. Under the conditions of rotation speed of 60 rpm, temperature of 36°C, pH value of 7.3 and dissolved oxygen concentration of 60%, the cell density of inoculation and culture was 3×10 6 cells / ml of 5 liters of BHK21 cell seed solution. Cultured for 6 days, the BHK21 cells were cultured to a cell density of 6×10 6 ce...

Embodiment 2

[0067] This example is used to illustrate the method for producing rabies virus by suspension culture BHK21 cells of the present invention.

[0068] Rabies virus: ERA strain.

[0069] Cell culture medium: the dispersant is ultrapure water, and the components contained in the culture medium are shown in Table 4 (wherein the molecular weight range of soybean protein is 0.1-5Kb).

[0070] Virus maintenance solution: BHK21 cell culture medium with 0.5 volume % serum.

[0071] Bioreactor: 100 liter stirred bioreactor.

[0072] Table 4

[0073] components

mg / L dispersant

components

mg / L dispersant

L-alanine

105

Calcium Nitrate Tetrahydrate

65

L-arginine hydrochloride

191

potassium chloride

420

L-Aspartic Acid

11

Anhydrous Magnesium Sulfate

75

L-Asparagine

48

Sodium chloride

7800

[0074] components

mg / L dispersant

components

mg / L dispersant

L...

Embodiment 3

[0078] This example is used to illustrate the method for producing rabies virus by suspension culture BHK21 cells of the present invention.

[0079] Rabies virus: aG strain.

[0080] Cell culture medium: the dispersant is ultrapure water, and the components contained in the culture medium are shown in Table 5 (wherein the molecular weight range of soybean protein is 0.1-5Kb).

[0081] Virus maintenance solution: BHK21 cell culture medium including 1% by volume of serum.

[0082] Bioreactor: 100 liter stirred bioreactor.

[0083] table 5

[0084] components

mg / L dispersant

components

mg / L dispersant

L-alanine

116

Calcium Nitrate Tetrahydrate

60

L-arginine hydrochloride

198

potassium chloride

400

L-Aspartic Acid

19

Anhydrous Magnesium Sulfate

72

L-Asparagine

43

Sodium chloride

7600

L-cysteine ​​hydrochloride

22

Anhydrous Disodium Hydrogen Phosphate

260...

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Abstract

The invention provides a method for producing rabies viruses by the suspension culture of BHK21 cells, which comprises a step of performing suspension culture of BHK21 cells in bioreactor containing a BHK21 cell culture medium, wherein the conditions for the suspension culture in the step include a temperature of 32 to 36 DEG C, a pH value of 7.0 to 8.0 and a dissolved oxygen concentration of 30 to 50 percent; and the BHK21 cell culture medium comprises the components shown in a table 1. The method overcomes the biases of the prior art and realizes the production of the rabies viruses by the suspension culture of BHK21 cells in the bioreactor. The obtained rabies viruses can be used for producing rabies vaccine. Due to the automatic culture environment parameter control of the bioreactor, the cells grow and the viruses propagate in more favorable environments, the virus titer is improved and the large-scale automatic continuous production can be realized.

Description

technical field [0001] The invention relates to a method for culturing cells to produce virus, in particular to a method for suspending culture BHK21 cells to produce rabies virus. Background technique [0002] Rabies is a zoonotic infectious disease caused by Rabies Virus, which has the highest fatality rate (almost 100%) in the world. Mammals, especially dogs and cats, are susceptible. And cats and dogs as hosts of rabies virus can also transmit rabies virus to humans. Therefore, the epidemiological control of rabies mainly relies on expanding the coverage of animal vaccination and immunization; especially with the increase in the number of domestic pets in recent years, it is even more necessary to strengthen the strict management of pet immunization such as dogs and cats. High-quality veterinary rabies vaccines are the basis for successful immunization; and to prepare rabies vaccines, especially veterinary rabies vaccines, large-scale production of rabies virus species...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12R1/93
Inventor 陈文庆王建超刘俊生
Owner BEIJING SKYWING TECH CO LTD
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