Process Development Of Newcastle Disease Virus Based On BHK-21 Suspension Cells With Serum-Free Medium

Newcastle disease(ND)causes a worldwide threat to poultry farming resulting from its high morbidity and mortality.Vaccination is the most effective measure to prevent ND.It still remains some unsolved problems such as shortages of SPF embryonated eggs,barriers in quality control and limitations in scale up during the production of Newcastle disease virus(NDV)vaccine with traditional embryonated eggs process.And because of the high cost,complicated operation and difficulty in process control utilizing cell adherent culture process,we aim to overcome those problems with the application of serum-free suspension culture process in production of NDV vaccine.While there are still remaining other obstacles,such as lacking of NDV-sensitive cell lines,lacking of serum-free maintenance medium and culture process which is suitable for NDV proliferation.Accordingly,it is urgent to develop cell-based suspension culture process for NDV vaccine production and the serum-free medium suitable for NDV proliferation.Firstly,the serum-free medium SF201 developed by our laboratory was used as the growth and maintenance medium,and serum-free suspension culture process for NDV production was established by screening NDV-sensitive cell lines,virus adaptation,virus seed preparation and optimizing the conditions of virus inoculation.The process was determined as follows:BHK-v002 cells were diluted into 6.0×106 cells/mL with fresh medium when the viable cell density reached to 9.0×106 cells/mL.Under the conditions of 0.005 MOI and 5 ?g/mL TPCK-trypsin,the hemagglutination titer(HA titer)could reach up to 8.5 log2HAU/25?L and the cell-specific virus yield(Svy)was 1685.9 virions/cell in 96 hours post infection.Thereafter,six basic serum-free medias were implemented the mixture design experiments by using Design Expert software to optimize the culture process and increase the virus yield,then a serum-free maintenance medium BMM11 was obtained.In this culture system,the HA titer and Svy were 9.5 log2HAU/25?L and 3185.1 virions/cell,respectively.Furthermore,F hydrolysate was added to BMM11 medium(BMM11-F)at a concentration of 8 g/L to improve the BMM11 medium.The HA titer and Svy reached 10.4 log2HAU/25?L and 6981.5 virions/cell,respectively.To guide the development of serum-free and chemically defined maintenance medium,the effects of maintenance medium BMM11 and its key components glucose and Gln on cell growth,metabolism and virus proliferation were investigated.The depletion of glucose and Gln during the anaphase of virus proliferation in maintenance medium BMM11 was not conducive to the proliferation of virus and the maintenance of cell viability.What's more,high concentration of glucose led to acidification of the culture environment which led to reduction in virus titer,so the level of glucose should be controlled.When Gln in BMM11 medium was added to 4.088 g/L,the virus HA titer and Svy were 10.5 log2HAU/25?L and 6704.4 virions/cell,respectively.And this titer was similar to the level in BMM11-F medium.In this paper,the production process of NDV based on serum-free suspension culture system of BHK-21 cells was established,and a maintenance medium which is suitable for efficient proliferation of NDV was successfully developed.It provides a method for establishing NDV production process based on animal cell serum-free suspension culture technology and replacing current embryonated eggs process.