Process Study On Microcarrier Suspension Culture For Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus Vaccine(Strain TJM-F92)

Porcine reproductive and respiratory syndrome(PRRS),commonly known as blue-ear disease,is a kind of highly contagious disease of pigs caused by porcine reproductive and respiratory syndrome virus(PRRSV).The virus mainly infects porcine alveolar macrophages and other immune cells,causing the suppression of immune system and reproductive disorders in sows,respiratory system diseases in piglets and growing pigs.The PRRSV can also cause serious secondary infection and eventually lead to death in the pig populations.Since 2006,the outbreak of highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRS)caused by highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRS V),results in huge economic losses to the swine industry.It is still one of most important disease,which harms to the pig industry.For prevention and control of the disease,vaccination with the high-quality vaccines is the one of the most important method.Generally,there are two types of vaccines are available on the market,i.e.live vaccine and inactivated vaccine(killed vaccine).The live vaccines are widely used in clinical application and the inactivated vaccines are not commonly used,because of its uncertainty efficacy.For the vaccine manufacturing,the yield and quality of the antigen are very important.However,the productionof the PRRSV vaccine as well as most other animal vaccines is still made by using the roller bottle culture method in our country.In this study,the scale up process technology in microcarier suspension for production of PRRSV strain TJM-F92 vaccine has been developed.The process procedures have been optimized in 3 L and 14 L bioreactors.In brief,the work has been focused on the followings:1.Process study on microcarrier suspension culture of Marc-145 cellIn this study,the scale-up conditions of the Marc-145 cells in 3L and 14 L bioreactors were optimized including stirring speed,percentage of dissolved oxygen(DO),initial cell seeding density and amount of microcarrier(g/L)used.The results showed that the best conditions for the process in microcarrier Marc-145 cell suspension were as following:the temperature was set at 37℃;microcarrier at 3-5g/L;DO at 50%;speed at 40-60 rpm(40-50 rpm for 3L bioreactor and 45-60 rpm for 14L bioreactor);initial cell seeding density at 1.5 x 105 cells/ml.Under this process conditions,the cell densities could reach up to 2.0x106 cells/ml after 72 h culture.2.Process study on microcarrier suspension culture of PRRSV strain TJM-F92In this study,the conditions of the process in microcarrier Marc-145 cell suspension infected with highest passage of the PRRSV strain TJM-F92 production seeds were optimized.The result showed that the optimizations of multiplicity of infection(MOI)for the virus inoculation was 0.1,and the virus titers could reach up to greater than 108.0 TCID50/ml when cells appeared as 70%CPE at 36-48 h post-inoculation.3.The preparation of PRRSV strain TJM-F92 live vaccine using the process in microcarrier suspension culture and its safety/efficacy testIn this study,three batches of laboratory vaccines,lot 2015001,lot 2015002,and lot 2015003 were prepared by the process in microcarrier suspension culture.The vaccine titer was lost lower than 100.5 TCID50/dose during the freeze-drying process.These vaccines were tested in compliance with the regulatory classification required by the PRRSV strain TJM-F92 vaccine.The results of immunogenicity study showed that the 80%of animals in the immunized group were protected and no protection in the control group after the challenge.These results showed that there was no significant difference between the vaccine prepared by the microcarrier suspension culture and the vaccine prepared by traditional roller bottle culture.Therefore,the vaccine prepared by the process in microcarrier suspension culture can fully replace that by the conventional roller bottle culture.In summary,the complete reliable bioprocess procedures and sufficient reliability parameter for large-scale production of PRRSV vaccine were optimized.