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A method for large-scale production of rotavirus vaccine

A rotavirus and vaccine technology, which can be used in biochemical equipment and methods, antiviral agents, viruses/phages, etc. The effect of industrialized mass production, improving yield and quality

Active Publication Date: 2019-09-13
LIVZON GROUP VACCINE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

An object of the present invention is to solve the defect that rotavirus is difficult to release from cells in large-scale rotavirus vaccine production, resulting in low virus virulence titer, thereby obtaining high-titer rotavirus virus, greatly improving the rotavirus yield of virus
Another object of the present invention is to solve the defect of low adsorption and infection of rotavirus in the large-scale production process, thereby greatly improving the quality of rotavirus, and then improving the antigenicity and immunogenicity of rotavirus inactivated vaccine

Method used

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  • A method for large-scale production of rotavirus vaccine
  • A method for large-scale production of rotavirus vaccine
  • A method for large-scale production of rotavirus vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the preparation of rotavirus liquid

[0043] 1) Recovery and passage of Vero cells: After the cryopreservation tube containing the working seeds of Vero cells (the cells are from ATCC, the working seeds of the cells are self-made) is taken out from the liquid nitrogen, it is quickly put into a 40°C water bath to thaw, and then placed in the Take out the cell suspension in the ultra-clean bench, add it to the cell culture bottle, add the cell growth solution, and put it into the CO2 incubator for cultivation; after the cells grow into a single layer, digest the adherent cells and make a uniform cell suspension solution, according to the seeding ratio of 1:3, inoculated in the cell bottle, after adding the cell growth solution, put it into the CO2 incubator to continue culturing. 10 layers of cell factory culture;

[0044] 2) Cell collection and inoculation bioreactor: when the cells of the third passage are overgrown to a monolayer, digest the adherent cel...

Embodiment 2

[0051] 14L bioreactor cultivates the method for rotavirus vaccine, and preparation method is shown in embodiment 1, and wherein step 4) described bioreactor enters liquid, and the perfusion velocity of changing liquid also has effect to bioreactor production rotavirus Certain effects are shown in Table 1.

[0052] Table 1:

[0053] serial number Bioreactor feed Perfusion velocity during fluid change (WV / D) Titer (lgCCID50 / ml) Antigen content (ug / ml) 1 M199 culture medium with 0.5ug / ml trypsin 6 7.3 2.5 2 M199 culture medium with 0.5ug / ml trypsin 5 7.5 2.2 3 M199 culture medium with 0.5ug / ml trypsin 7 7.0 2.3 4 M199 culture medium with 0.5ug / ml trypsin 8 6.0 1.5 5 M199 medium with 6% bovine serum 5 3.0 0.2

[0054] Conclusion: As shown in Table 1, compared with Group 1 and Group 5, Group 5 only did not change the medium, that is, the cell growth medium containing 6% bovine serum was always used as the cell maint...

Embodiment 3

[0056] 14L bioreactor cultivates the method for rotavirus vaccine, and preparation method is the same as shown in embodiment 1, wherein step 5) described virus seed is activated and bioreactor controls the condition of bioreactor during cell infection 24 hours after liquid change As shown in table 2.

[0057] Table 2:

[0058]

[0059] Conclusion: As shown in Table 2, the combined liquid of rotavirus harvested by groups 1-3 can reach more than 65L, and the virus titer of the collected liquid reaches 7.0 lgCCID 50 / ml or more, the antigen content reaches more than 2.2ug / ml, which meets the requirements of large-scale production, wherein the titer and antigen content of the rotavirus pooled liquid of group 1 (i.e. Example 1) are optimal. And group 4, the harvest liquid of group 5 harvest, the virus titer of harvest liquid is lower, and antigen content is also lower, therefore, the control condition of the bioreactor described in step 5) is: temperature 34.0~36.5 ℃, pH The v...

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Abstract

The invention relates to a large-scale production method of a rotavirus vaccine, particularly a large-scale culture method of a rotavirus inactivated vaccine by using a 14L bioreactor. The method comprises the following steps: 1) revival and subculture of Vero cells; 2) cell collection and bioreactor inoculation; 3) bioreactor culture of cells; 4) solution exchange and cell washing; 5) rotavirus activation and infection; and 6) sustaining culture and collection of rotavirus. The invention aims to overcome the defect of low infectivity titer in the obtained virus since the rotavirus can not easily release cells in rotavirus vaccine large-scale production, thereby obtaining the high-titer rotavirus, and greatly enhancing the rotavirus yield. The invention also aims to overcome the defect of low adsorption infectivity of rotavirus in the mass production process, thereby greatly enhancing the rotavirus quality and further enhancing the antigenicity and immunogenicity of the rotavirus inactivated vaccine.

Description

[0001] Technical field: [0002] The invention belongs to a method for preparing a virus inactivated vaccine, in particular to a method for cultivating rotavirus on a large scale with a 14L bioreactor. [0003] Background technique: [0004] Rotavirus is the main pathogen causing severe dehydrating diarrhea (severe dehydratingdiarrhea) in infants and young children worldwide. Almost all children are infected with rotavirus before the age of 2 to 3 years. Even in developed countries with high levels of hygiene, rotavirus is the most common pathogen causing severe diarrhea. According to reports, diarrhea caused by rotavirus accounts for about 50% of autumn diarrhea. Around the world, rotavirus infection can lead to about 24 million visits to the doctor every year, of which about 2.3 million are hospitalized, and about 600,000 people die from rotavirus infection. The introduction of oral rehydration therapy in developing countries has reduced mortality from diarrhea through a m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K39/15A61P31/14
Inventor 李云富李刚黄勇付臻鹏刘春庭罗翀胡雪芹肖俊光
Owner LIVZON GROUP VACCINE ENG
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