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Improvements of ATP Assay as a Substitute for the CFU Method in Estimating Viable Cell Count for BCG/rBCG Vaccine Preparations

Tom H. Jin, Tianli Qu, Anant Raina, Peter Alexander and Eric Tsao

Bacillus Calmette–Guérin (BCG) and recombinant BCG (rBCG) vaccines can be genetically traced back to a live attenuated strain of Mycobacterium bovis. As organism viability is essential for the stimulation of a protective immune response, monitoring the count of viable organisms is an integral part of vaccine quality control. The colonyforming unit (CFU) test has been the conventional assay for determining BCG viability, and is a widely accepted surrogate for BCG potency. CFU analysis, however, is problematic and time consuming. The slowness and high variability of CFU test results are the main driving forces for manufacturers and control laboratories to look for a rapid, more reproducible alternative viable count assay. A modified adenosine triphosphate (ATP) luminescence assay was developed by Statens Serum Institut and was promoted by the WHO as an alternative viable count assay. However, certain conditions during the processes of sample preparation and ATP extraction have to be established before the ATP assay can meet the requirements of robustness and reproducibility. This study is focused on identifying the conditions necessary for a reliable process of ATP analysis for BCG/rBCG preparations. Using our improved ATP assay protocol, we demonstrated that the correlation coefficient between CFU count and ATP concentration of BCG/rBCG vaccines was high (R2=0.83 for accelerated stability samples and R2>0.97 for all other preparations). The ATP luminescence assay is a rapid, sensitive, reliable, strain-non-specific method in quantification of the viability of live attenuated mycobacterial vaccine preparations.