Characterization And Stabilization Of Inactivated Virus And Virus Like Particles

Inactivated virus and virus-like particles (VLPs) represent two classes of important vaccine antigens with a common feature of multimeric subunit structure. The complex particulate assembly is susceptible to the environments of production, transportation and storage, resulting in conformational change which may bring about safety issues and decrease the efficiency of the vaccines. It is therefore an important subject to study the stability and stabilization of these particulate antigens.For these purposes, the inactivated foot and mouth disease virus (FMDV) and hepatitis B virus surface antigen VLPs (HBsAg-VLPs) were studied as models. Analytical methods of conformational changes of particulate antigens in solution and at solid-liquid interface, the mechanisms of conformational changes during purification, and factors affecting stabilization were systematically studied. The detailed contents and results are as follows.1) We developed a size-exclusion high-performance liquid chromatography (SE-HPLC) method for rapid characterization of the assembly structure of vaccine antigens. The HPSEC method was successfully used to analyze inactivated FMDV, hepatitis B core antigen (HBcAg) VLPs, and HBsAg-VLPs in different processing steps such as fermentation, purification and storage. Compared with ultracentrifugation, HPSEC is simpler, rapid, and reliable. The method has been recommended to companies by China Institute of Veterinary Drugs Control, and applied in industrial practice.2) FMDV and HBsAg-VLPs were characterized by field flow fraction (FFF) and various spectroscopic techniques including circular dichroism, flurescence spectroscopy and dynamic light scattering. The results further verified the HPSEC method, and provided more detail information of conformational changes.3) Dual polarization interferometry (DPI) was developed for real-time monitoring the adsorption and conformational changes of HBsAg-VLPs at solid-liquid interface. The thickness of HBsAg-VLPs at DEAE ion exchange surface varied with pH. At low pH like pH 5.0, possible bi-layer adsorption was involved as evidenced by an adsorbed layer thickness higher than average diameter of the HBsAg-VLPs. At pH 7.0 or 9.0, the HBsAg-VLPs spread thinly or even adsorbed in disassembled formation on the surface as revealed by significant decrease in thickness. By comparing the DPI results with chromatography experiments, the significant loss of antigen recovery was proven to be highly related to conformational changes at solid-liquid surface.4) Stabilization of vaccine antigen particles during processing was investigated. For FMDV, the most stable pH was found between 7.5-8.0, and the most suitable salts were ammonium salts. Addition of multi-hydroxy compounds significantly increased the stability of FMDV as indicated by HPSEC and DSC. By adding multi-hydroxy compounds as purification companion, the FMDV recovery increased for 10.1% during ultrafiltration, and increased from 17.1% to 84.0% during chromatography. The entire process was completed with 56.7% final FMDV recovery, and the purity above 98%.