Antigen Molecular Characterization Stability Analysis Of Recombinant Protein Vaccine

In recent years,virus-like particles(VLP)based recombinant protein vaccines have been developed.Three kinds of recombinant protein vaccines.At present,recombinant protein vaccines against HBV,HPV and HEV have been successfully launched.Key epitopes can be fully displayed on the VLP surface,antigen presentation and immune responses are stimulated,which is favored in the research and development of vaccines.The stability of the recombinant protein is important to ensure the immunogenicity and antigenicity of the vaccine.In this paper,the stability of two recombinant proteins was studied.On the one hand,the real-time stability and thermal stability assessment of commercial hepatitis E vaccine were carried out.On the other hand,the thermal stability of potential candidate vaccine hepatitis B therapeutic antigen was explored.As for hepatitis E vaccine stability assessment,five lots of hepatitis E vaccines stored at 2-8 ? for 36 months were selected as tested samples for real-time stability analysis.The results of in vivo potency assay showed that the hepatitis E vaccine still had good immunogenicity after long-term storage.As for in vitro immunochemical detection,eight specific monoclonal antibodies with different characteristics were selected to characterize molecular stability of different epitopes on the recombinant protein p239,and consistent conclusions were obtained by different in vitro methods.After long-term storage,hepatitis E vaccine still had at least 50%antigenicity compared with Mock formulation.Results from physicochemical assessment(LC-MS,MALDI-TOF MS,TEM,E-SDS,HPSEC)showed that the protein integrity and particle size of p239 antigen in the hepatitis E vaccine had no significant change after long-term storage.The stability of thermal stressed hepatitis E vaccine samples was detected.The in vivo potency test showed that the ED50 of hepatitis E vaccine was still less than the release standard(1.0 ?g)within 2-week thermal stress at 42 ?.Stressed hepatitis E vaccine still had at least 50%antigenicity compared with time zero.Thermal stressed results provided feasible data for hepatitis E vaccine application for CTC.In the study of the thermal stability of hepatitis B therapeutic antigen,firstly,the thermal stability of hepatitis B therapeutic antigen in aqueous solution was studied.In order to explore the best buffer conditions,HPSEC,DSC and sandwich ELISA were used.The optimal buffer formulation(5 mM PB 7.0+100 mM NaCl+0.3%Tween 80)was finalized.Under the optimum buffering conditions,the antigenicity of hepatitis B therapeutic antigen in solution could remain nearly 100%after thermal acceleration at 50 ? for 7 days.Subsequently,the thermal stability of hepatitis B therapeutic antigen adsorbed on adjuvant was detected by SDS-PAGE,DSC and sandwich ELISA.It was found that the existence of adj uvant would not weaken the thermal stability of hepatitis B therapeutic antigen.Two new adjuvants Zol and Ris can promote the maintenance of protein integrity.In conclusion,this study completed the real-time stability of hepatitis E vaccine,which proved that hepatitis E vaccine still has good immunogenicity,antigenicity,particle size and protein integrity after 36-month storage.Hepatitis E vaccine has good thermal stability.This result prompts that hepatitis E vaccine has potential to be used outside cold chain in the future.Hepatitis B therapeutic antigen has good thermal stability both in the aqueous solution and after adsorption to adjuvant.These results provide data for hepatitis B therapeutic antigen to be a potential candidate for therapeutic hepatitis B vaccine.