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Method for production of live smallpox vaccine

A vaccine and solution technology, applied in biochemical equipment and methods, medical preparations containing active ingredients, pharmaceutical formulations, etc., can solve problems such as adverse reactions, and achieve the effects of preventing skin reactions, reducing content, and eliminating adverse side effects

Active Publication Date: 2009-10-14
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

[0066] Embodiment 1: the preparation of the 1st generation working seed solution

[0067] (1) Prepared with 6-well culture plate

[0068] 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

[0079] Embodiment 2: the preparation of the 2nd generation working seed solution

[0080] (1) Prepared with T150 culture flask

[0081] 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).

[0082] Harvest the virus-infected cells by shaking the container quickly to dislodge the virus-infected cells from ...

Embodiment 3

[0090] Embodiment 3: the preparation of vaccine stock solution

[0091] 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).

[0092] 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 solution obtained 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 E6 cells; 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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IPC IPC(8): A61K39/285
CPCC12N2710/24134A61K39/275A61K2039/5254A61K39/12A61P31/12A61P31/20A61K39/285C12N7/02C12N7/04
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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