Production method of pseudorabies virus vaccine

A pseudorabies virus and production method technology, which is applied to the production field of pseudorabies virus vaccine, can solve the problems such as hidden quality risks, high production cost and high labor intensity of the vaccine, and achieve simple and stable production process, balanced and stable quality, and a degree of automation. high effect

Active Publication Date: 2014-02-05
成都史纪生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional process is labor-intensive, time-consuming, low in efficiency, and high in production cost; it is easy to be polluted by the environment; the difference between different batches of spinner bottle culture is large; it requires a lot of space; it may be contaminated by bacteria or other viruses during the spinner bottle culture operation Increased toxicity may lead to quality problems of the vaccines produced; once contamination occurs during spinner bottle cultivation, due to the large number of spinner bottles, it is difficult to harmlessly dispose of them, which involves biosafety and public health issues
In addition, there have been reports on the use of bioreactor microcarriers to cultivate rabies vaccines, but there has been no report on the use of bioreactor microcarriers to produce pseudorabies virus

Method used

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  • Production method of pseudorabies virus vaccine

Examples

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Effect test

Embodiment 1

[0026] Example 1: (1) Passaging and culturing of cells for seedling production: The suckling hamster kidney (BHK-21) cell line was selected as the cells for seedling production. Take well-grown BHK-21 cells in cell culture flasks, digest and passage them with EDTA-trypsin cell dispersion solution (Hank's solution containing 0.25% trypsin (1:250), 0.02% EDTA), and replace with cell growth solution (containing 90 %DMEM solution, 10% bovine serum, 200 units / ml of penicillin sodium and streptomycin sulfate each, pH 7.4) continue to culture in cell culture flasks, culture temperature is 37 ℃, when a dense and good cell monolayer is formed, use Carry out microcarrier suspension culture in continuous subculture or inoculation in bioreactor, wherein the bioreactor is a bioreactor suitable for microcarrier suspension culture that can automatically control temperature, pH, dissolved oxygen and stirring speed parameters;

[0027] (2) Propagation of virus seeds for seedling production: Di...

Embodiment 2

[0034]Example 2: (1) Passage and culture of cells for seedling production: the porcine kidney (PK-15) cell line was selected as the cells for seedling production. Take well-grown PK-15 cells in cell culture flasks, digest and passage them with EDTA-trypsin cell dispersion solution (Hank's solution containing 0.25% trypsin (1:250), 0.02% EDTA), and replace with cell growth solution (containing 98 %DMEM solution, 2% bovine serum, 250 units / ml of penicillin sodium and streptomycin sulfate each, pH 7.0) continue to culture in cell culture flasks, culture temperature is 37 ℃, when a good cell monolayer is formed, use Continue to subculture or inoculate in a bioreactor for microcarrier suspension culture, wherein the bioreactor is a bioreactor suitable for microcarrier suspension culture that can automatically control temperature, pH, dissolved oxygen and stirring speed parameters;

[0035] (2) Propagation of virus seeds for seedling production: Dilute virus seeds 10 times with cell...

Embodiment 3

[0042] Example 3: (1) Passaging and culturing of cells for seedling production: the monkey kidney (Marc-145) cell line was selected as the cells for seedling production. Marc-145 cells growing well in the cell culture flask were digested and passaged with EDTA-trypsin cell dispersion solution (Hank's solution containing 0.25% trypsin (1:250), 0.02% EDTA), and cell growth solution (containing 94 %DMEM solution, 6% bovine serum, 100 units / ml of penicillin sodium and streptomycin sulfate each, pH 6.8) continue to culture in the cell culture flask, the culture temperature is 37 ℃, when a good cell monolayer is formed, use Continue to subculture or inoculate in a bioreactor for microcarrier suspension culture, wherein the bioreactor is a bioreactor suitable for microcarrier suspension culture that can automatically control temperature, pH, dissolved oxygen and stirring speed parameters;

[0043] (2) Propagation of virus seeds for seedling production: Dilute virus seeds 10 times wit...

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Abstract

The invention discloses a production method of a pseudorabies virus vaccine. The production method comprises the following steps: (1) adopting a cell of a continuous cell line, subjecting the cell to digestive passage, and continuously culturing the cell in a cell culture flask by using a cell growth medium; (2) diluting a virus seed into 10 times by using a cell maintenance medium, and inoculating a cell monolayer to obtain an infected cell virus liquid, that is, a virus seed for production; (3) preparing the cell monolayer formed in the step (1) into a cell suspension through digestion, inoculating the cell suspension into a bioreactor, and adding a microcarrier into the bioreactor; (4) performing virus-introduction operation on the cell, wherein the introduced virus seed is the virus seed produced in the step (2); and (5) harvesting liquid in the reactor together with the microcarrier until all of the cells on the microcarrier fall off and a dissolved oxygen value increases obviously, placing the liquid and the microcarrier under conditions of minus 20 DEG C, repeatedly freezing and thawing the liquid and the microcarrier twice, and removing the microcarrier and cell fragments to prepare the pseudorabies virus vaccine. The production method of the pseudorabies virus vaccine is short in production cycle and large in yield.

Description

technical field [0001] The embodiment of the present invention relates to a production method of a pseudorabies virus vaccine, in particular to a method for producing a pseudorabies virus vaccine by using microcarrier cell suspension culture in a bioreactor. Background technique [0002] At present, domestic pseudorabies virus vaccine production only relies on the spinner bottle culture method. The traditional process is labor-intensive, time-consuming, low in efficiency, and high in production cost; it is easy to be polluted by the environment; the difference between different batches of spinner bottle culture is large; it requires a lot of space; it may be contaminated by bacteria or other viruses during the spinner bottle culture operation Increased toxicity may lead to quality problems of the vaccines produced; once contamination occurs during spinner bottle cultivation, due to the large number of spinner bottles, it is difficult to harmlessly dispose of them, which invo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/245A61P31/22C12N7/04C12R1/93
Inventor 徐宏军胡来根任丽张奕强岳丰雄
Owner 成都史纪生物制药有限公司
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