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 in vivo 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.

A robust and efficient alluvial filtration method for the clarification of adeno-associated viruses from crude cell lysates.

Adeno-associated virus (AAV) based vectors have recently been gaining importance as DNA delivery systems. Efficient downstream processing of AAV remains a major challenge as serotypes differ in physicochemical properties, making it difficult to design uniform purification processes. Clarification of AAV is an especially critical step. Harvesting of AAV, like other viruses, often requires cell lysis, resulting in a difficult-to-filter cell lysate. In this study, we evaluated the applicability of diatomaceous earth (DE) as a filter aid for clarification of AAV crude cell lysates. DE filtration proved to be a viable clarification method for AAV2, AAV5 and AAV8. Based on a design of experiment approach, the DE concentration was identified as the main factor influencing AAV particle loss. The loss of AAV during DE filtration was limited to < 2% by maintaining the DE quantity below 0.181 mg DE/1010 AAV. Use of DE reduced manual handling time 3-fold and increased the filter capacity 3.5-fold compared to filtration combined with a prior centrifugation step. Moreover, we showed that the DE type had only a minor influence on the filtration performance. This study demonstrated that filtration with DE as a filter aid is an efficient clarification method for different AAV serotypes.