Abstract Current viral vector manufacturing processes face a number of significant challenges. One of these challenges is the availability of rapid and robust analytics to quantitate viral titer. Typical adeno-associated virus (AAV) vector processes have relied on the traditional quantitative polymerase chain reaction assay (qPCR) to quantitate the genome copy number and cell based assays for determining potency. These assays can be time consuming and have relatively high assay to assay variation. Droplet digital PCR (ddPCR) is emerging as an analytical tool for quantitating genome copies which can solve some of these issues. ddPCR is a new PCR based technique which directly quantifies DNA copies with an un-paralleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties which appear to lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. With the help of Integrated DNA Technologies (IDT), we optimized and qualified a ddPCR assay for the quantification of an AAV2 viral vector. We tested multiple primer/probe sequences targeting different portions of the viral vector genome. We also evaluated and optimized the PCR cycling parameters including cycle number and annealing temperature. The resulting procedure was then characterized for range, precision, repeatability, and robustness. This presentation reports on the optimization and qualification of this assay.

Optimization and qualification of a droplet digital polymerase chain reaction assay for quantitation of an aav2 viral vector

2019 Spring National Meeting

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