Process optimization for the rapid production of Enterovirus 71.

Abstract

Enterovirus 71 (EV71) infection can cause hand-foot-and-mouth disease (HFMD). Inactivated EV71 vaccine was effective to prevent EV71 derived HFMD. A highly efficient and economical process for producing EV71 is needed. In our study, the epidemic strain of EV71 (EV71-2013ZJHFMD) was obtained and purified. The Vero cells were cultured for production of EV71. The mini-bioreactor vessel (Amprotein Inc., China) packed with a 0.6g polymer fiber carrier was used to determine the best seeding cell density, multiplicity of infection (MOI) and temperature. Then the optimized procedure was further applied in a 10L disposable perfusion bioreactor ACPB (AmProtein Current Perfusion Bioreactor). The Vero cell culture and viral titer were monitored. The seeding density of 1.5 × 107cells per 0.6g disk was considered to be the most appropriate for the culture. The best MOI was 0.1 and the temperature was 32°C. The total cell number increased from 1.5 × 109 to 3.0 × 1010. The maximum viral titers reached 1.0 × 108/mL 3days post-infection in our optimized special culture procedure (serum-free during the harvest period, supplemented with 0.25% Lactalbumin Hydrolysate). The total volume of the harvested supernatant was 25L and the total virus yield was 1.93 × 1012. The procedure using Vero cells grown on polymer fiber paper carriers was effective for the large-scale production of EV71.