Method Validation of Intracellular ATP assay for Potency Test of BCG Vaccine Produced in Thailand

Abstract

An analysis of BCG potency values with the standard method defined by the World Health Organisation is culturable viable count. The potency of vaccine is calculated from the number of live attenuated bacteria in a colony forming unit per dose. The limitations of this method include a lengthy test period (due to slow growth of mycobacteria and 4-week incubation period) and the high variability of CFU assay results. In this study, we used an alternative method, intracellular ATP assay that could be performed more rapidly, in only 2 days to quantify the BCG potency. To ensure that the ATP assay was appropriate to estimate the potency of BCG vaccines, it is essential to conduct the bioassay validation by studying main parameters such as accuracy, precision (both repeatability and intermediate precision) as well as the robustness of test. The study showed good accuracy (91.73% recovery), precision tests (geometric coefficient of variation (GCV) less than 20%) for both repeatability and intermediate precision. For robustness test, there was no significant difference of the test results by two analysts (p>0.05); and the GCV was less than 10%, indicating that this assay showed good robustness as well. In addition, a high correlation was observed between intracellular ATP concentrations and the number of viable bacilli in vaccine samples. Overall, these data indicate that the intracellular ATP assay can be done rapidly; it is sensitive, less time-consuming, and reliable. It is should be a suitable alternative method for potency test of BCG vaccines; and could be routinely used as a standard method for the lot release of BCG vaccines in Thailand.