Recombinant adeno-associated virus (rAAV) has been utilized successfully for in vivo gene delivery for the treatment of a variety of human diseases. There is a critical need for the development of robust analytical methods to sustain the growth of rAAV gene therapy products and ensure safe and appropriate therapeutic dosages selected for treatment. TCID50 assay (50% Tissue Culture Infectious Dose) is an in vitro cell-based method widely used to determine AAV infectivity and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative real-time PCR (qPCR) serves as the end-point method to detect the amount of replicated viral genome after infection. Here, we evaluate how well the droplet digital PCR (ddPCR) can be adapted into TCID50 assay as an end-point detection method. By direct comparison between qPCR and ddPCR read-out, we observed a trend of improved inter-assay precision when the ddPCR method is utilized. Particularly, we offer improvements of TCID50 infectious titer assay with: (1) higher precision and sensitivity by adapting ddPCR as an end-point method without standard curve preparation; (2) identification of an important second “set threshold” value in infectivity scoring; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID50 method with improved accuracy and robustness that is important for rAAV infectious titer testing during process development and manufacturing.
Author: Tam Duong, PhD, Scientist II, R&D