Key Technologies In Large Scale Production Of Vriuses And Vaccine Development

Emerging infectious diseases have seriously affected social stability and become a major threat to human health.At present,the prevention and treatment of Emerging infectious diseases has become the focus of this area.The development and reserve of vaccines have been the focus and hotspot of Emerging infectious diseases,and have great social significance.In this study,we used two different kinds of bioreactors to product several different viruses(potential vaccine candidates)on a large scale,optimizing the cultivation of conditions,physical and chemical parameters,and evaluation of the quality control and the screening of different adjuvants.Recombinant adenoviral(Ad)vectors are highly efficient gene transfer vectors widely used in vaccine development and immunotherapy.To promote the industrial application of Ad vectors,studies focusing on reducing the cost of manufacturing,shortening the preclinical research period and improving the quality of products are needed.Here,we describe a highly efficient and economical process for producing Ad vector in a novel,single-use bioreactor system suitable for clinical trials.A mini-bioreactor was used for parameter optimization and development of medium replacement protocols for Ad5-GFP production before scale-up.HEK293 cell culture and virus infection were monitored in a disposable AmProtein Current Perfusion Bioreactor and Bioflo310 bioreactor using optimized parameters and medium replacement protocols.The total cell number increased from 2.0 x 109 to 3.2 × 1010 after six days of culture.The total number of viral particles obtained in a single batch was 1.2× 1015.We also evaluated the quality of Ad5 produced by different strategies.These results demonstrate the efficiency and suitability of this system for Ad vector production for research and GMP applications.Coxsackie virus A16(CVA16)and Enterovirus 71(EV71)infections can cause hand-foot-and-mouth disease(HFMD).There is currently no available vaccine for CVA16 caused HFMD.In this study,we isolated epidemic strains of CVA16 and EV71,developed an optimized procedure(serum-free during the harvest period,supplemented with 0.5%(w/v)Lactalbumin Hydrolysate)for CVA16/EV71 production using Vero cells grown on polymer fiber paper carriers in a mini-bioreactor.The Vero cell culture and virus infection were then monitored in a disposable perfusion bioreactor(AmProtein Current Perfusion Bioreactor,ACPB)and a Bioflo310 bioreactor(New Brunswick Scientific,USA),respectively.The total cell number increased from 1.5 x 109 to 3.0 × 1010.The maximum viral titers reached 7.8 ×107(CVA16)and 1.0× 108(EV71)three days post-infection in our optimized special culture procedure.These results provide useful information regarding the use of a novel bioreactor system for the large-scale production of CVA16 and EV71.Enterovirus 71(EV71)and Coxsackievirus A16(CVA16)have caused severe epidemics of hand,foot and mouth disease(HFMD)in the Asia Pacific in recent years,particularly in infants and young children.This disease has become a seriouspublic health problem,as no vaccines or antiviral drugs have been approved for CVA16 infections.In this study,we compared CVA16 vaccines with different adjuvant(AL,MF59 and D-Trehalose).The neutralization antibodies titers(GMT)were evaluated.The result show that MF59 and Trehalose dehydrate are also promising adjuvants for bivalent vaccines of formalin-inactivated CVA16/EV71.A process for human influenza H1N1 virus vaccine production from Madin-Darby canine kidney(MDCK)cells using a novel packed-bed bioreactor is described.The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor(AmProtein Current Perfusion Bioreactor)with proportional-integral-derivative control of pH,dissolved 02(DO),agitation,and temperature.During 6 days of culture,the total cell number increased from 2.3×109 to 3.0×1010 cells.The maximum virus titers of 7.8×107 TCID50/mL.These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells,is a convenient and stable platform for industrial scale production of influenza vaccines.