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Automation and comparability of qPCR and ddPCR for AAV genome titration for in-process samples.

C Harrison(1) S Perez(1) A Sergijenko(1) D Marginean(1) R Tarnowska(1) M Bagnati(1) N Sweeney(1)

1:Cell Therapy Catapult

qPCR is one of the assays that is deployed routinely to determine genomic titre of produced AAV samples within the manufacturing process. Titre is used for estimating the effective therapeutic dose for this vector when testing it in preclinical or clinical trials. The method is complex, time consuming, and prone to human error due to the number of manual sample manipulation steps. As such, ddPCR is principally preferred as a technique for genomic titre analysis due to its high reproducibility, low sensitivity to impurities and eschewing calibration curve. However, due the availability of equipment and costs of procedures, this can deter laboratories from transferring genomic titre analysis to ddPCR. Automation has been shown to significantly reduce laboratory hands-on time, costs and errors. In this study we scripted manual steps of those two methods using an advanced liquid handler Tecan Fluent® and compared them by analyzing in process samples during AAV manufacturing. The enhanced precision resulting from reduced human error and elimination of operator-to-operator variability improved the comparability between qPCR and ddPCR methods. Our findings indicated that both techniques yielded comparable titers across sample types with the exception of TFF1. Notably, qPCR demonstrated advantages of lower consumable costs, increased throughput, and a wider dynamic range while both methods exhibited high precision and repeatability. In conclusion, the cost-effective, high-throughput nature, and widespread availability of qPCR make it a suitable choice for AAV genomic titration comparably to ddPCR when both methods are automated, achieving reliable genomic titre results for in-process AAV2 samples.

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