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A method for large-scale production of rotavirus vaccine using bioreactor

A bioreactor and rotavirus technology, applied to biochemical equipment and methods, viruses, microorganisms, etc., can solve the problems of low virus virulence titer, low rotavirus adsorption and infection, and improve production and quality Effect

Active Publication Date: 2019-07-09
LIVZON GROUP VACCINE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One object of the present invention is to solve the defect that rotavirus is difficult to release from cells in large-scale production, resulting in low viral virulence titer, and obtain a high-titer, high-yield inactivated rotavirus vaccine. Another aspect of the present invention One purpose is to solve the defect of low adsorption and infection of rotavirus in the mass production process, thereby greatly improving the yield and quality of rotavirus

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the preparation of rotavirus liquid

[0042] 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 1:4, inoculated in the cell bottle, added the cell production solution, put it into the CO2 incubator to continue culturing, after two passages, inoculated the cells in the 10-layer cell factory, and After adding cell growth medium, culture;

[0043] 2) Cell collection and inoculation bioreactor: When the c...

Embodiment 2

[0050] 75L 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.

[0051] Table 1:

[0052] 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 8 7.5 2.3 2 M199 culture medium with 0.5ug / ml trypsin 6 7.3 2.2 3 M199 culture medium with 0.5ug / ml trypsin 5 7.0 2.0 4 M199 culture medium with 0.5ug / ml trypsin 9 6.7 1.5 5 M199 medium with 0.2% bovine serum 8 3.5 0.5

[0053] Conclusion: As shown in Table 1, compared with Group 1 and Group 5, Group 5 is only the cell growth solution in which the serum concentration in the culture solution has been reduced to 0.2% as the ...

Embodiment 3

[0055] 75L 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 the bioreactor when cell infection 24 hours after liquid change As shown in table 2

[0056] Table 2:

[0057]

[0058] Conclusion: As shown in Table 2, the virus titer of the rotavirus pooling solution harvested by groups 1-3 reaches 7.0lgCCID 50 / ml or more, the antigen content reaches more than 2.0ug / 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. embodiment 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 35.0~36.0 ℃, pH The value is 7.2-7.5, the dissolved oxygen is 15%-35%,...

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Abstract

The invention relates to a method for producing rotavirus vaccines in large scale by utilizing a bioreactor, and in particular relates to a method for culturing rotavirus inactivated vaccines in large scale by utilizing a 75L bioreactor. The method comprises the following steps: (1) carrying out reviving and passage on Vero cells; (2) collecting the cells and inoculating the cells into the bioreactor; (3) culturing the cells in the bioreactor; (4) changing the liquid and washing the cells; (5) activating and infecting rotaviruses; and (6) carrying out maintenance culture and harvesting on the rotaviruses. The method aims at overcoming the defect that in the large-scale production, the rotaviruses can not release cells easily, so that the obtained viruses are low in toxicity titer, and the inactivated rotavirus vaccines with high titer and high yield are obtained; the method also aims at overcoming the defect that in the large-scale production, the adsorption force and infection force of the rotaviruses are poor, and thus the yield and quality of the rotaviruses are greatly improved.

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 75L 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
CPCA61K39/12C12N7/00C12N2720/12334C12N2720/12351
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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