Investigating critical thermal parameters for pre-analytical preparation of adeno-associated virus vector genome titration by droplet digital polymerase chain reaction

抄録

Several recombinant adeno-associated virus-based gene therapy products have been developed recently. The vector genome (VG) titer is a critical quality attribute associated with the clinical dosing of these products and, thus, requires accurate quality control measures. Typically, VG titers are measured by quantitative polymerase chain reaction (qPCR). Droplet digital PCR is more reliable than qPCR and a powerful analytical tool for quantifying genome copies with high accuracy and precision; however, VG titers cannot be correctly quantified without the appropriate preparation of analytical test samples. In this study, we systematically assessed the role of each component and treatment comprising DNase treatment for free and residual DNA, DNase inactivation, and single-stranded DNA extraction from capsids in VG titration results during pre-analytical sample preparations. Incubation near the single-stranded DNA-release temperature decreased the number of recombinant adeno-associated virus genome copies. Moreover, we developed a simplified three-step pre-analytical procedure with concurrent DNase inactivation and single-stranded DNA extraction at a much higher temperature than the release temperature. Developing an analytical procedure for recombinant adeno-associated virus genome titration by droplet digital PCR based on release temperature is a science- and risk-based approach that would improve quality control testing of recombinant adeno-associated virus-based gene therapy products.