This study was undertaken to modify and develop procedures for tissue culture-inactivated Japanese B encephalitis (JBE) virus vaccine production in large quantities. Various types of glass bottles were tried and, considering many advantages, long cylindrical roller (CR) bottles were selected. Several variables were investigated including number and volume of trypsinized cells to be seeded, volume of growth medium required for optimum cell growth, amount of calf serum, and volume of harvest medium for a high-titer virus yield. A good confluent cell sheet in CR bottles was obtained within a week by increasing the calf serum from 4 to 10% and when such tissue in a CR bottle was inoculated with 45,000 viral mean tissue culture infective doses directly into the medium, the cytopathological effects (CPE) appeared on day 5. High-titer virus yields were obtained when the harvests were made at 4+ CPE using medium 199 with 2% human albumin at pH 8.3 to 8.5. No appreciable gain in titer was found from such harvests by blending to release intracellular virions. The production methods finally adopted gave consistently good results, and several inactivated JBE virus vaccine lots with minimum immunizing doses, ranging from 0.005 to 0.017 ml, were prepared using a large number of CR bottles in a simulated commercial-scale production system.
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