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Pharmaceutical Technology Regulatory Sourcebook October 2020 57 fers key advantages. One important benefit is that ddPCR provides absolute quantification of vector genomes, whereas qPCR uses indirect quantita- tion relative to a reference standard, which may or may not be available or properly represent the rAAV product. Additional ddPCR advantages result from the droplet compartmentalization of individual DNA molecules that reduces the potential for inhibi- tory effects of sample matrix or impurities, and droplets concentrate the target sequence. When compared, ddPCR has shown increased accuracy and precision (16). It should be noted that VG titers derived from these methods have been observed to differ sig- nificantly when applied to individual vector lots, and that "viral structure, aggregates, and impu- rities" and results obtained can " be altered by sample preparation" (17). While determination of vector particle quantity can be derived by the physicochemical and molecu- lar methods already described, another important parameter for estimation of potency is the ability of the particle preparation to deliver the transgene to cells; referred to as the product's "infectious titer." The combination of VG and infectious titer are ap- plied to derive the virus particles:infectious units ratio referenced in the CMC guidance. Determination of the infectious titer applied to QC testing of AAV drug substance and drug product generally follows the TCID50 method de- scribed by Zhen et al. (18). Although this method was developed using qPCR, ddPCR is also poten- tially an option. Impor ta nt ly, t he accu rac y of t he TCID50 method can be inf luenced by the presence of ag- gregates and impurities, increasing assay vari- ability (18,19). For this reason, other methods have been developed and evaluated with varying success at discriminating between infectious par- ticles and non-infectious controls. These include infectious center assays that add complexity by use of membrane transferred nucleic acid and probe hybridization, and a method that applies permissive cell lines to eliminate the necessity of using helper virus (20). More recently, a relative infectivity method has been proposed as an alternative to the TCID50 method for use in screening early AAV vector candidates that also eliminates the need for a helper virus (19). The variability associated with infectivity methods may be further exacerbated when measures of potency progress to expression or functional activity assays, as these measure transgene activity downstream from the trans- duction events quantitated by PCR methods. As a result, it is unlikely that infectious titer mea- Digital droplet polymerase chain reaction (ddPCR) provides absolute quantification of vector genomes, whereas quantitative polymerase chain reaction (qPCR) uses indirect quantitation relative to a reference standard, which may or may not be available or properly represent the recombinant adeno associated virus (rAAV) product.