SCALABLE DOWNSTREAM PROCESS FOR PURIFICATION AND INACTIVATION OF MICROCARRIER-PROPAGATED JAPANESE ENCEPHALITIS VIRUS ANTIGEN: PROCESS DEVELOPMENT AND VALIDATION

Japanese encephalitis is a mosquito-borne viral disease of major public-health importance in Asia, and vaccination remains the principal strategy for disease prevention because no specific curative therapy is available for this disease. Inactivated whole-virion vaccines require robust and reproducible manufacturing processes that ensure adequate viral yield, efficient impurity clearance, and reliable viral inactivation for safe product release into the market. The current manuscript describes an integrated process for development, purification, and inactivation of Japanese encephalitis viral antigen propagated in microcarrier-based Vero cell culture and its validation. The upstream platform employed Vero cells expanded on Cytodex 1 microcarriers in a controlled bioreactor environment to support reproducible cell growth and virus production. The viral harvests were concentrated by tangential flow filtration, purified by Celufine Sulfate affinity chromatography, further processed through Sartobind Q membrane filtration for reduction of host-cell DNA, and subsequently inactivated with formaldehyde under defined conditions. The downstream process was assessed across the three independent batches of viral antigen using plaque assay, protein estimation, host-cell protein analysis, host-cell DNA testing, and in vitro and in vivo inactivation confirmation. The workflow consistently generated purified viral antigen with acceptable impurity reduction and complete loss of detectable infectivity after inactivation. Bioreactor harvests yielded higher virus titers than static cultures, and the downstream processing remained reproducible across three batches prepared using the described workflow. Collectively, these findings support the suitability of the proposed platform for scalable manufacture of purified and inactivated JEV antigen for vaccine applications.