Method for producing classical swine fever live vaccine by using microcarrier high-density cell culture technology

A swine fever live vaccine and cell culture technology, applied in the field of veterinary biological products, can solve the problems of poor product uniformity, fluctuating vaccine product quality level, low production efficiency, etc., to achieve improved uniformity and stability, and good immune protection. , the effect of reducing production costs

Active Publication Date: 2015-07-22
成大生物(本溪)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the use of porcine testis passage cells to produce live swine fever vaccines has been widely used in China. The cells used for production are mainly cultured in roller bottles. Large-scale production requires a lot of manpower and material resources, and the production efficiency is low; When growing and producing toxin in the spinner bottle, the nutrient supply, environmental pH, and oxygen content in the medium cannot be adjusted in time, and the cell density in the spinner bottle is only 0.5-1×10 6 Each of these will affect the quality and yield of the vaccine; as the cell state in each bottle is not the same, resulting in poor product uniformity, resulting in greater fluctuations in the quality of the vaccine product
These are not conducive to high-quality and efficient production of swine fever vaccine

Method used

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  • Method for producing classical swine fever live vaccine by using microcarrier high-density cell culture technology
  • Method for producing classical swine fever live vaccine by using microcarrier high-density cell culture technology
  • Method for producing classical swine fever live vaccine by using microcarrier high-density cell culture technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] (1) Select microcarrier: Fibra Disk from NBS Company.

[0024] (2) Choose a reactor: a 5-liter cell culture tank with BASKET from NBS Company.

[0025] (3) In this example, miniature pig kidney cells (MPK) were selected, and the MPK cells were subcultured in cell culture flasks or spinner bottles at a ratio of 1:2 to 8. Usually, the growth time of each generation of cells was 48 to 72 hours. . When the cell confluency exceeds 80%, inoculate the cells into the bioreactor.

[0026] (4) Inoculate the cells in the bioreactor, with the optimized microcarrier load of 100 grams, after complete attachment, the cell density reaches 1.5×10 6 cells / ml, start to continuously perfuse the medium, and adjust the control conditions of the culture process: growth temperature 36.5-37.5°C, stirring speed 60-120rpm, pH value 7.0-7.4, dissolved oxygen concentration 30-80% and glucose content 1500 ~4500mg / L, the density of cells in the 5L bioreactor stabilized at 14×10 6 ...

Embodiment 2

[0032] (1) Choose a bioreactor: NBS cell lift impeller (CELL LIFT IMPELLER) bioreactor.

[0033] (2) Select microcarrier: Cytodex 1 type microcarrier from GE Company.

[0034] (3) In this example, miniature pig kidney cells (MPK) were selected, and the MPK cells were subcultured in cell culture flasks or spinner bottles at a ratio of 1:2 to 8. Usually, the growth time of each generation of cells was 48 to 72 hours. . When the cell confluency exceeds 80%, inoculate the cells into the bioreactor.

[0035] (4) The suitable range of microcarrier Cytodex 1 in the reactor is 10-25 grams per liter. This time, 20 grams per liter was used, and the density after inoculation of cells was 1.2×10 6 cells / ml, and add an appropriate amount of medium, start continuous perfusion of medium, adjust the control conditions of the culture process: growth temperature 36.5 ~ 37.5 ℃, stirring speed 30 ~ 70rpm, pH value 7.0 ~ 7.4, dissolved oxygen concentration 20 ~ 50% and glucose conte...

Embodiment 3

[0041] (1) Choose a bioreactor: NBS cell lift impeller (CELL LIFT IMPELLER) bioreactor.

[0042] (2) Select microcarrier: Cytodex 3 type microcarrier from GE Company.

[0043] (3) In this example, miniature pig kidney cells (MPK) were selected, and the MPK cells were subcultured in cell culture flasks or spinner bottles at a ratio of 1:2 to 8. Usually, the growth time of each generation of cells was 48 to 72 hours. . When the cell confluency exceeds 80%, inoculate the cells into the bioreactor.

[0044] (4) The suitable range of microcarrier Cytodex 3 in the reactor is 10-20 grams per liter, and 15 grams per liter is used this time, and the density after inoculation of cells is 0.9×10 6 cells / ml, and add an appropriate amount of medium, start continuous perfusion of medium, adjust the control conditions of the culture process: growth temperature 36.5~37.5℃, stirring speed 50~80rpm, pH value 7.0~7.4, dissolved oxygen concentration 25~ 50% and glucose content of 1...

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Abstract

The invention relates to a method for producing a classical swine fever live vaccine by using a microcarrier high-density cell culture technology. The method is characterized in that passage adherent cells MPK cells are inoculated into a bioreactor filled with a microcarrier, a culture medium required by cell growth is poured into the bioreactor, the MPK cells rapidly proliferate in the reactor, classical swine fever viruses are inoculated when the cell density is stable, a classical swine fever virus culture liquid is continuously harvested, the obtained virus culture liquid is clarified and concentrated, a stabilizer and a buffer are added to prepare the vaccine, and vacuum freeze-drying is performed to prepared the classical swine fever live vaccine. According to the invention, the microcarrier high-density cell culture technology is adopted, such that the cell density in the bioreactor can reach 8-16*10<6> / ml, the yield of the virus culture liquid by using the method of the present invention is increased by 20-40 times compared to the yield of spinner flask culture, production efficiency and yield of the classical swine fever live vaccine are significantly increased, production cost is reduced, side reactions of the vaccine are reduced, vaccine production batch stability is enhanced, and the classical swine fever live vaccine with characteristics of safety and effectiveness is obtained.

Description

Technical field [0001] The present invention involves a kind of beast biological products, especially the method of using microcariac cell culture technology to produce swine fever vaccines with micro -carrier high -density cell culture technology. Background technique [0002] At present, the methods of producing swine fever vaccines in China are mainly spleen vaccine (animal tissue method) and cell vaccine.Cell vaccines are divided into two cell production methods: primary (beef testicular cells) and biography (pig testicular cells).The immune protection of the spleen vaccine is better than the cell vaccine, but a rabbit can only produce 500 parts of the effect of not less than 100 PD 50 Swine fever vaccine.In China alone, the market capacity of swine fever vaccines per year is not less than 1.4 billion, which is equivalent to 2.8 million rabbits to prepare these vaccines. Such a huge number is impossible to achieve.Therefore, cell vaccines are the development direction of th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/187C12N7/00A61P31/14
Inventor 陈晓锋高军侯剑英
Owner 成大生物(本溪)有限公司
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