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Prepn process of inactivated rotavirus vaccine

A technology of rotavirus and inactivated vaccine, which is applied in the field of preparation of rotavirus inactivated vaccine and can solve problems such as hypersensitivity reactions

Active Publication Date: 2007-08-22
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The amplified rotavirus is physically mixed, concentrated and purified, inactivated with formalin or βpropiolactone, and used as a vaccine stock solution after sterility testing, but the cultured cell line used is still not human diploid cells , possibly with high hypersensitivity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] ——The preparation method of monovalent to pentavalent inactivated rotavirus adopts the following steps:

[0022] (1) Cultivation of cells: human diploid cells 2BS, SL-7, WI38, KMB17, preferably K / MB17 are cultivated with MEM medium, and the composition of medium is: 5%-10% fetal bovine serum (volume ratio), 300-592mg / L of L-glutamine, 100-200U / mL of penicillin and streptomycin, 0.66g-1.32g / L of sodium bicarbonate; cultured at 37°C, 5% carbon dioxide until grown into single layer, used to inoculate rotavirus.

[0023] (2) Virus inoculation: the titer of the virus seed is greater than or equal to 10 7.5 TCID 50 / mL, use trypsin with a final concentration of 15-50 μg / mL, preferably 25 μg / mL trypsin, activate at 37°C for 20-45 minutes (preferably 45 minutes), inoculate cells at a multiplicity of infection of m.o.i=0.1~3, and Adsorb at 37°C for 40-90 minutes, discard the virus liquid, add serum-free MEM medium containing trypsin at a final concentration of 0.5-1 μg / mL, an...

Embodiment 2

[0030] The preparation method of quadrivalent rotavirus inactivated vaccine adopts the following processing steps:

[0031] (1) Cell preparation: Digest and disperse KMB17 cells with trypsin, add complete MEM medium, culture at 37°C until they grow into a confluent monolayer, and replace the medium with serum-free MEM 12 hours before inoculation maintenance fluid.

[0032](2) Virus inoculation and culture: activate rotavirus strains G1, G2, G3, and G4 with trypsin at a final concentration of 20 μg / mL at 37°C for 45 minutes, discard the maintenance solution, and use the same The KMB17 cells were washed once more with the maintenance solution, and the cells were inoculated at a multiplicity of infection of m.o.i=3, adsorbed at 37°C for 90 minutes, discarded the virus liquid, and added serum-free MEM medium containing trypsin at a final concentration of 0.5 μg / mL, in Culture at 37°C until obvious cytopathic changes occur (generally 3-5 days, m.o.i=3).

[0033] (3) Virus collect...

Embodiment 3

[0039] ——Refer to the Chinese regulations on biological products for the verification of the original solution,

[0040] (1) Determination of protein content

[0041] As determined by the Lowry method, the protein content should not be higher than 20 μg / mL. The determination results were all less than 20 μg / mL.

[0042] (2) Antigen amount

[0043] Coat the microtiter plate with purchased or self-made commercialized monoclonal antibodies specific to the two types of rotavirus for detection. Double-antibody sandwich enzyme-linked immunosorbent assay is used, and the amount of antigen should not be less than 3200EU / mL.

[0044] (3) Sterility inspection

[0045] According to the inspection according to law, the sample is sterile and complies with the regulations.

[0046] (4) Residual amount of bovine serum albumin

[0047] When detected by enzyme-linked immunoassay, the residual bovine serum albumin should not be higher than 50ng / mL. The average value of the test results w...

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Abstract

The present invention discloses preparation process of inactivated rotavirus vaccine. Rotavirus strains G1, G2, G3 and G4 are made to adapt human diploid cell line and propagate on human diploid cell line to obtain the producing strain of inactivated rotavirus vaccine, with the human diploid cell line being KMB17. The human diploid cell line used as the cell matrix for producing inactivated rotavirus vaccine is safer compared with available cell matrixes. Therefore, by means of the established technological platform, it is possible to develop inactivated rotavirus vaccine with other serotypes of rotavirus as the target.

Description

technical field [0001] The invention relates to a method for preparing an inactivated rotavirus vaccine, more specifically, the invention relates to a method for cultivating rotavirus by using a human diploid cell line, and then preparing a monovalent or multivalent inactivated vaccine. Background technique [0002] Human rotavirus (human rotavirus, HRV) infection is the main cause of acute dehydrating diarrhea in infants, and HRV is spreading worldwide. At present, rotavirus is still the main cause of infant disease and death in both developed and developing countries. Rotavirus is highly contagious and can spread and become popular in a relatively short period of time. The development of vaccines is an effective way to control and prevent rotavirus infection. [0003] In the early stage of rotavirus vaccine development, oral attenuated live vaccines were mainly used. In order to have a good protective effect on multiple HRV serotypes, researchers developed multivalent re...

Claims

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Application Information

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IPC IPC(8): A61K39/15A61P31/14C12N7/00C12N5/08
Inventor 刘馨孙茂盛
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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