METHOD VALIDATION OF A MOUSE POTENCY BIOASSAY FOR THE RECOMBINANT BOTULINUM NEUROTOXIIN TYPE A, C-FRAGMENT rBoNTA(Hc) VACCINE (GLP STUDY)

Abstract

The method validation of the mouse potency assay examined the critical parameters of the assay and determined the suitability of the assay to produce credible data. The basic potency assay was examined to determine what impact different analysts, dose-challenge schedules and mouse shipments had on the method and its reproducibility. Groups of ICR mice were inoculated with varying doses of rBoNTA(Hc) vaccine, and 21 or 23 days later the animals were challenged with botulinum type a toxin, Hall A strain. The mice were observed for 5 days after challenge, and survival rates and the 50% effective doses (ED50) were calculated. The analysis of the potency assay was based on Repeatability, Intermediate Precision, Accuracy, Linearity, Specificity, Ruggedness and Suitability. The assay was highly precise for an in vivo assay, with an overall coefficient of variation (CV) of 9.02 % for the Log10 ED50. The potency exceeded expected values and thereby failed the acceptance criteria. The results of the assay were determined to be linear and followed the probit model in all but one assay, with an average p-value of 0.664. The specificity experiments demonstrated that the method could precisely measure (CV=11.8%) the potency of the vaccine despite changes in buffers and the addition of non-specific proteins, as long as the adjuvant concentration remained constant. The method was not rugged enough to allow for a change in the immunization and toxin challenge interval. Based on the results and the acceptance criteria set forth in the validation protocol, the mouse bioassay is a highly reproducible, specific method that can be used to evaluate the rBoNTA(Hc) potency of pilot and production lots.