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• An imaging-based high content analysis was used to analyze the binding activity of microparticle adsorbed antigens. • The degree of epitope overlapping was determined on antigens in adjuvanted vaccine formulation. • The activity of proteins adsorbed or coupled onto microparticles could be visualized and quantitatively analyzed. • Subtle conformational changes could be assessed using epitope-specific monoclonal antibodies. Nano/microparticle-based formulations are attracting more and more attention in the drug and vaccine formulation field. Analytical methodologies are needed for analyzing the function of the protein molecules before and after their adsorption onto particles. Here, we report a fluorescence imaging-based high content analysis (HCA) for analyzing bimolecular interactions on the particle surface. Subtle conformational alteration of antigen epitope overlapping were shown to be feasible using this microplate-based HCA method. This HCA is highly amenable for automation, making it a method of choice for early formulation development as well as for stability test of products in late stage development. [ABSTRACT FROM AUTHOR]

• Immunoassays without dissolution process was used in antigenicity assessment. • Multiple assays were used for functional characterization of the HEV vaccine. • Feasibility of labelling the vaccine for the WHO CTC use was demonstrated. • No changes in quality attributes of an HEV vaccine at the end of expiry dating. Hepatitis E, which is caused by infection with hepatitis E virus (HEV), is a global health problem in both developed and developing countries. An efficacious hepatitis E vaccine was licensed (by China) in 2011 with a trade name of Hecolin®. The antigen contained in this vaccine is a truncated version of the sole capsid protein encoded by open reading frame 2, which is designated p239. In this study, the real-time and real-condition stability and accelerated stability of five lots of hepatitis E vaccine products at the end of the designated shelf life, were assessed by a well-established quality analysis platform. The protein integrity of p239 that was recovered from the vaccine lots was demonstrated using CE-SDS, LC-MS and MALDI-TOF MS. The particle characteristics of the recovered vaccine antigen were assessed by TEM and HPSEC. The immunogenicity of hepatitis E vaccines was assessed by a mouse potency assay, which is part of product release and stability testing. Several methods were employed to assess the antigenicity of vaccines with or without adjuvant dissolution. Specifically, the well-established methods of sandwich ELISA and surface plasma resonance (SPR)-based BIAcore were used with unique murine monoclonal antibodies. Most interesting, two 'dissolution-free' immunoassays were also used for in situ antigenicity assessment of the vaccines. In addition to the confirmation of vaccine stability at the end of expiry dating, i.e., after storage in recommended conditions (2–8 °C) for 36 months, the mouse potency assay and sandwich ELISA were used to assess the accelerated stability of prefilled syringes to demonstrate the feasibility of out-of-cold-chain storage. In summary, molecular and functional characterization confirmed the shelf life stability of the vaccine at the end of expiry dating and the feasibility of transporting the hepatitis E vaccine for a given period of time out of cold chains. [ABSTRACT FROM AUTHOR]