Tech Note: High Throughput AAV Viral Titering Using Nanoplate-Based Digital PCR

Center for Breakthrough Medicines

Viral vectors are effective vehicles for delivering genetic material into cells and widely used in gene therapies, gene-modified cell therapies and certain vaccines. Vectors based on adeno-associated viruses (AAVs) are preferred for gene therapies due to their low immunogenicity, nonpathogenicity and ability to readily enter multiple cell types.

 AAVs used in drug products must be fully characterized during process development and manufacturing to ensure product quality, safety and efficacy. Physical viral titer – the concentration of viral particles containing viral genomes – is an important critical quality attribute (CQA) to enable informed decision-making in trial design and regulatory data submissions.

Physical titer can be calculated using polymerase chain reaction (PCR)-based methods such as quantitative PCR (qPCR) or digital droplet PCR (ddPCR). Historically, ddPCR is the preferred method for determining AAV titers over qPCR because it is more precise and repeatable and does not require the use of a standard reference curve. However, droplet-based methods can be challenged by matrix effects and long analysis times. It can be difficult to implement ddPCR methods across all phases of AAV development and manufacturing, particularly early ones when resources are scarce. With limited data, companies run the risk of developing processes that fail during clinical and commercial scale, resulting in time and resources to correct.

The microfluidic nanoplate technology leveraged in the QIAcuity Digital PCR System from QIAGEN overcomes the challenges with inconsistent droplet generation, complex workflows, and slow droplet readouts.

In this technical application note, nanoplate-based dPCR is compared to ddPCR analyses for different types of AAV samples to demonstrate that the QIAcuity is a successful platform for AAV titering. The results show that the nanoplate-based dPCR system rapidly delivers accurate titer values, providing more data across more sample types, faster, and with higher throughput to drive towards first-time right process development

What you will learn:

– How new advances in digital PCR provide more data, across more sample types, faster, and with higher throughput.

– Critical factors to consider when choosing automated platforms for AAV viral titer determination.

– Strategies to efficiently quantify more targets in the same sample.

– Pushing past common droplet busting matrices to expand testing throughout the AAV process continuum.

Download the full Tech Note here.

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