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Method for production of live smallpox vaccine

A technology for vaccines and attenuating pox, applied in biochemical equipment and methods, medical preparations containing active ingredients, pharmaceutical formulations, etc. Effect

Active Publication Date: 2012-01-25
KM生物医药股份公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These vaccines have caused serious adverse reactions such as post-vaccination encephalitis in infants who received the first vaccination, with an incidence of several in one million cases (see e.g. Non-Patent Document 2)

Method used

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  • Method for production of live smallpox vaccine
  • Method for production of live smallpox vaccine
  • Method for production of live smallpox vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1: the preparation of the 1st generation working seed solution

[0066] (1) Prepared with 6-well culture plate

[0067] The kidneys of 7-day-old SPF rabbits were aseptically removed, washed with sterile PBS / 2ES, chopped, and placed in the dispersion solution. After digestion with overnight stirring at 4°C, the cells were washed again with PBS / 2ES. The cells were collected by centrifugation, and then the collected cells were suspended in a proliferation medium to obtain a rabbit kidney primary cell suspension. The suspension was dispensed into 24-well culture plates and cultured at 37°C until the cells formed a monolayer sheet. The main virus seed solution was diluted with maintenance medium, and viruses were inoculated into each well at MOI=0.3, 0.1, 0.03, 0.01, 0.003, 0.001. Incubation was continued at 30°C until a substantial cytopathic effect was observed. Thereafter, the cells were frozen in a -80°C refrigerator, and after complete freezing was conf...

Embodiment 2

[0077] Embodiment 2: the preparation of the 2nd generation working seed solution

[0078] (1) Prepared with T150 culture flask

[0079] The rabbit kidney primary cell suspension was prepared by the same method as in Example 1 (1), and the suspension was dispensed into T150 culture flasks and cultured at 37° C. for 4 days. After confirming that the cells form a monolayer sheet, the proliferation medium is removed by aspiration. After washing the cells with PBS / ES, the first generation working seed solution obtained in Example 1(1) was diluted with maintenance medium to MOI=0.1, 0.3, 1.0, and then added to the culture flask. Continue culturing at 30°C for 1-2 hours until the virus is fully adsorbed on the cells. Thereafter, a maintenance medium was added, and the culture was continued at 30° C. until a large amount of cytopathic effect was observed (3 days).

[0080] Harvest the virus-infected cells by shaking the container quickly to dislodge the virus-infected cells from ...

Embodiment 3

[0088] Embodiment 3: the preparation of vaccine stock solution

[0089] The rabbit kidney primary cell suspension was prepared by the same method as in Example 1 (1), and the suspension was added into a culture spinner bottle, and cultured at 37° C. for 5 days. After confirming that the cells form a monolayer sheet, the proliferation medium is removed by aspiration. After washing the cells with PBS / ES, dilute the second-generation working seed solution obtained in Example 2(1) or Example 2(2) with maintenance medium to MOI=0.1, 0.3, 1.0, and then add it to the culture spinner bottle Inside. Continue culturing at 30°C for 1-2 hours until the virus is fully adsorbed to the cells. Thereafter, a maintenance medium was added, and the culture was continued at 30° C. until a large amount of cytopathic effect was observed (3 days).

[0090] Harvest the virus-infected cells by shaking the container quickly to dislodge the virus-infected cells from the container. The cells were ce...

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PUM

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Abstract

A safer live smallpox vaccine, which contains a lowered content of revertants, is provided. A process for manufacturing a live smallpox vaccine which comprises steps of: inoculating a master seed solution of an attenuated vaccinia virus to an appropriate number of containers (1 to n wherein n is an integer) of rabbit kidney cells and incubating them; inoculating a portion of the cultured solutionobtained from each container to RK-13 cells and to Vero E6 cells and incubating them to thereby select containers which contain a cultured solution that forms plaques in RK-13 cells but not in Vero E6cells; and preparing a drug substance of vaccine using the aforementioned cultured solution (working seed solution), and a live smallpox vaccine prepared in the aforementioned process.

Description

technical field [0001] The present invention relates to a method for preparing a live vaccinia vaccine containing attenuated vaccinia virus, which produces a revertant immediately while proliferating, as an active ingredient. More specifically, the present invention relates to a method for preparing a live vaccinia vaccine, the method comprising the steps of: inoculating a master seed solution containing attenuated vaccinia virus to an appropriate number of groups (1-n, wherein n is an integer) of rabbit kidney cells Inoculate a part of the culture solution in each container into RK-13 ​​cells and Vero E6 cells, and culture them, so as to select cells containing cells that form plaques in RK-13 ​​cells but not in Vero E6 cells. The container of the culture solution that does not form plaques in the cells, collect various culture solutions obtained in the selection container; use the culture solution collected above (working seed solution) to prepare the vaccine stock solution....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/285
CPCC12N2710/24134A61K2039/5254A61K39/275A61K39/12A61P31/12A61P31/20A61K39/285C12N7/02C12N7/04
Inventor 金原知美横手公幸大隈邦夫仓永雅彦森川茂
Owner KM生物医药股份公司
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