Abstract: In this study, we screened the BHK-21 cells which were sensitive to pseudorabies (PRV) and analyzed their growth and virus production characteristics. The conditions of cell culture and virus production in the reactor were optimized, so we finally established the PRV propagation process of BHK-21 suspension cells in bioreactor. The optimal culture and virus propagation conditions of BHK-21 suspension cells in 1.2 L bioreactor based on cell growth dynamics and TCID₅₀ virus titer, were optimized by response surface methodology and single factor optimization method, and further batch culture was carried out in 5 L bioreactor. The results showed that one of adherent cells named BHK-21-02 and one of suspension cells named BHK-21-XF02 with high PRV sensitivity were screened. BHK-21-XF02 suspension cells could achieve good growth and virus propagation in serum-low medium containing 3% serum and serum-free medium. Using response surface methodology, the optimum cell culture conditions of 1.2 L reactor were as follows:inoculation density was 1.20×10⁶ cells·mL^-1, stirring speed was 120 r·min^-1 and DO was 40%, the cell density could reach (7.61±0.18)×10⁶ cells·mL^-1 and the cell viability was (96.93±1.18)% after batch culture in 5 L reactor for 72 hours. Using single factor method, the optimal conditions of virus propagation in 1.2 L reactor were as follows:MOI was 0.001, temperature was 37℃, cell density for virus inoculation was 2.0×10⁶cells·mL^-1 and stirring speed was 80 r·min^-1. Under conditions, the virus titer reached (7.13±0.11) lgTCID₅₀·mL^-1 at 48 hours in 5 L reactor batch culture. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of PRV vaccines.

Key words: BHK-21 suspension cell, bioreactor, pseudorabies virus, susceptibility