Unveiling the secrets of adeno-associated virus: novel high-throughput approaches for the quantification of multiple serotypes.

Adeno-associated virus (AAV) vectors are among the most prominent viral vectors for gene therapy, and their investigation and development using high-throughput techniques have gained increasing interest. However, sample throughput remains a bottleneck in most analytical assays. In this study, we compared commonly used analytical methods for AAV genome titer, capsid titer, and transducing titer determination with advanced methods using AAV2, AAV5, and AAV8 as representative examples. For the determination of genomic titers, we evaluated the suitability of qPCR and four different digital PCR methods and assessed the respective advantages and limitations of each method. We found that both ELISA and bio-layer interferometry provide comparable capsid titers, with bio-layer interferometry reducing the workload and having a 2.8-fold higher linear measurement range. Determination of the transducing titer demonstrated that live-cell analysis required less manual effort compared with flow cytometry. Both techniques had a similar linear range of detection, and no statistically significant differences in transducing titers were observed. This study demonstrated that the use of advanced analytical methods provides faster and more robust results while simultaneously increasing sample throughput and reducing active bench work time.