PK15 cell acclimation suspension method and two-stage virus production process

Abstract

The invention relates to a PK15 cell acclimation suspension method and a two-stage virus production process. According to the PK15 cell acclimation suspension method, PK15 cells are acclimated to adapt to serum-free suspension culture and used as host cells for producing viruses which can have cytopathic effect or sensitivity on suspension PK15 cells by two-stage culture virus inoculation; when the cells grow to 2.0-20.0*10<6> cells / mL, diluting with a production culture medium by 1-5 times is performed to reach a density range of 1.0-10.0*10<6> cells / mL, and then virus inoculation is carriedout or virus inoculation is performed before dilution. The suspension PK15 cells are adaptive to suspension growth in the serum-free chemical-defined culture medium, and the two-stage virus productionprocess is simple and easy in amplification; liquid changing is avoided, and high culture medium utilization efficiency is achieved; production and growth culture media can be identical or not and can be mixed proportionally to realize cell secondary growth and viral expression promotion.

Description

technical field [0001] The invention relates to a PK15 cell acclimation suspension method and the field of production of suspension PK15 cell-related vaccines, in particular to a technique for cultivating suspension PK15 cells in a bioreactor of 30 L or above and cultivating the suspension PK15 cells by a second-stage culture method to produce vaccines. Background technique [0002] Mammalian cell large-scale culture technology is one of the downstream common technologies of biopharmaceutical companies, and it plays an extremely important role in this industry. The key to this technology is to achieve large-scale culture of mammalian cells in suspension serum-free, thereby increasing production capacity, reducing costs, and facilitating scale-up. The usual practice in culture of adherent cells is to add a certain amount of serum to the culture medium to allow the cells to grow on the wall. However, the chemical composition of the serum is uncertain, and the quality is unstab...

AI Technical Summary

Problems solved by technology

[0004] The defects of the existing PK15 cell acclimatization and suspension are: 1. Some cannot achieve complete serum-free culture; 2. Generally, a two-step method is adopted, that is, first reduce to serum-free and then suspend culture, because the medium used for adherent culture Serum is usually added to the basal medium. The nutrient content of the basal medium is weak, and the surface area / volume ratio of the adherent culture during the domestication process is usually small, which limits the intake of nutrients. The entire domestication process takes a long time; 3. After genetic modification, domestication, That is, through genetic modification, insert gene fragments that are not conducive to adherence into the adherent cell line, change the original adherent culture method, and make the cells grow in suspension, and then through continuous optimization and change of the medium, the cells can be serum-free and high-density. Growth, this method is easy to implement, but the insertion of foreign gene fragments affects the quality of the product

Up to now, there is no relevant report on the second-stage culture method of suspension PK15 cells

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