Process standardization for optimal virus recovery and removal of substrate DNA and bovine serum proteins in Vero cell-derived rabies vaccine.

Purification of a rabies vaccine by a single zonal centrifugation run was replaced by two runs with optimal standardization of the sucrose density gradient. As a result, significant reductions in the levels of substrate DNA and bovine serum protein in the Vero cell-derived human rabies vaccine were achieved. Following many trials, for the first run, loading of the 3.2-l capacity K-3 rotor with 1800 ml of 60% sucrose solution and 1400 ml of vaccine PBS buffer solution gave a satisfactory linear gradient. However, after the first run, the substrate DNA and bovine serum contents exceeded the required levels. After protamine sulphate and Tween-80 treatment of the concentrated inactivated material, a second run using the same procedure as in the first run was tried. However, these purification procedures resulted in low virus recovery. To achieve optimal virus recovery, and removal of substrate DNA and bovine serum protein, the peak fractions from the first run as indicated by the haemagglutination, sucrose concentration, and optical density values were pooled and the sucrose concentration of the pooled fractions was increased to 60%. A second (flotation) run was then carried out. Using this method, the virus recovery rate was more than 95% that of the first run, and the levels of cellular DNA and bovine serum protein were well within the acceptable limits of less than 100 pg/dose and one part per million, respectively. The substrate DNA was quantified by both radioactive labeling and non-radioactive biotin labeling methods. For the quantification of calf serum protein, a counter-immunoelectrophoresis method was developed and effectively applied. A potency assay was performed using the National Institutes of Health (NIH) and well-standardized in vitro single radial immuno diffusion (SRD) methods. Finally, an immunogenicity study was conducted with human volunteers and the results were confirmed by a rapid fluorescent focus inhibition test (RFFIT).