Development Of An Efficient Process For Pseudorabies Virus Production Using Mammalian Suspension Cells

Pseudorabies(PR)is a viral disease that may cause huge economic losses to the pig industry worldwide.Currently,vaccination is one of the most effective ways to prevent and control pseudorabies in the most of developing countries.Due to the sensitivity of the pseudorabies virus(PRV),a wide range of cell lines such as BHK-21 cells,PK-15 cells,and Vero cells are considered as good candidates for the production of inactivated PRV vaccines.With the emergence of new PRV mutant strains,which has worsened PRV infection in Chinese pigs and the rapid development of animal husbandry,the demand for PR virus vaccines is increasing dramatically.At present,the production process of PR inactivated viral vaccines in China mainly has issues such as process instability and low production efficiency.The use of roll bottles and microcarriers in the bioreactors to produce PR inactivated viral vaccines is one of the main reasons for the low production efficiency.Therefore,it is of great importance to use suspension culture of mammalian cells in serum-free medium to improve the propagation efficiency.In this work,firstly,the cell growth and virus production was evaluated using the BHK-21 cells,PK-15 cells and Vero cells in an in-house serum-free mdium.Different multiplicity of virus infection(MOI)and target viable cell density at time of infection(TOI)were tested for respective cell lines.Considering the cell growth performance,the virus titers,as well as with the aim of the easy operation for the subsequent scale-up,the BHK-21 cells were used for further process optimization including virus adaptation,infection strategies and feeding strategies.It was found that with a MOI of 0.0005,the viable cell density of 3×106 cells/ml,the P1 seed virus,a 1:2 dilution inoculation strategy,and a 3%feeding strategy,the PRV titer was 9.25 1gTCID50/ml,with an increase of 0.92 1gTCID50/ml compared to unoptimized cultivation.And the establishment of PRV amplification process under the same conditions for 10 batch verification experiments,the maximum PRV titer is stable at(9.30±0.12)1gTCID50/ml.The standard deviation of the maximum PRV titer is 0.12.The relative standard deviation is 1.27%,indicating a stable PRV production process.On this basis,the effect of pH on the PRV titer was investigated with cold model experiments,and the optimal pH of PRV was determined to be 6.90-7.10.Finally,the established PRV efficient production process was evaluated in a 3 L bioreactor,achieving a high PRV titer of 9.33 1gTCID50/ml.The result are promising for further process scale-up for the efficient production of PR inactivated virus vaccines.