An Optimized AAV Sample Preparation Method for Robust Quantification of Viral Titers by PCR

Discover a novel approach to processing adeno-associated virus (AAV) samples prior to digital polymerase chain reaction (dPCR) to increase titer accuracy and reproducibility. For AAV applications ranging from research tools to clinical-stage gene therapeutic strategies, the ability to quantify AAV vector concentration precisely and reproducibly is crucial. While several analytical methods are available, digital polymerase chain reaction is one of the most robust and reliable ways to determine absolute quantification of viral DNA at various stages of production.

To ensure accurate titer measurements by dPCR from transgene DNA, unpurified AAV samples require DNase-I treatment to remove exogenous DNA prior to capsid disruption. Multiple factors, such as ethylenediaminetetraacetic acid (EDTA) concentration and the DNase activation and inactivation method, can significantly impair the amplification efficiency of the protected viral vector genome.  This highlights the potential for large discrepancies between the results of different laboratories using analogous protocols.

In this study, using a design of experiments approach, we have extrapolated an optimal DNase-I application that can be used as a standard dPCR treatment for AAVs.

Join this webinar to gain insights into an optimal DNase-I application that can be used as a standard dPCR treatment for AAVs and the best concentration of EDTA to allow the inactivation of DNase-I, while not inhibiting the dPCR reaction following the treatment.

Speaker

Zoe Bowman, Research Associate, Teknova

Zoe Bowman is a Research Associate at Teknova, where she focuses on advanced analytical development and tissue culture work for their novel AAV product portfolio. She is a key team member responsible for the launch of the company’s first proprietary product — the AAV-Tek™ AEX Buffer Screening Kit — and is currently working on solutions to help common bioprocessing bottlenecks such as AAV sample treatment prior to analytical testing.

Zoe graduated with honors from the University of Missouri (MU) with a Bachelor of Science degree in Biomedical Engineering, where she was awarded the 2020 Biomedical Engineering Outstanding Senior Student Award from the College of Engineering. During her time at MU, she worked in a biomedical microfluidics laboratory developing an automated antibiotic resistance testing system. After graduating, she joined Viracor Eurofins where she worked on projects involving PCR assay development for vaccines.

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