Multi-parameter process optimization for high yield AAV2 vector production using scale-down multi-parallel bioreactor systems and high-throughput analytical tools

Adeno-associated virus (AAV) vectors are recognized as highly efficient carriers for genetic material. Among the various manufacturing platforms, transient production is the most established one for AAV production. Despite this, the transient production is influenced by numerous variables that can significantly impact the outcome. In this study, we employed 15 ml multi-parallel scale-down bioreactor system to evaluate critical process parameters like cell densities and plasmid DNA concentration. Additionally, a 250 ml bioreactor system was used to explore factors, such as bioprocess mode, comparing batch versus perfusion processes under optimized conditions. Results showed that in low cell density AAV production, where cell specific productivity is highest, the perfusion process yielded over three-fold lower functional titers compared to the batch process at 37°C. Furthermore, the impact of temperature-shifts, a yield improvement strategy that is rarely explored in the context of AAV production, was examined, resulting in increased functional AAV yields - almost two-fold in batch process and over three-fold increase in perfusion process. The temperature-shift not only increased total number of functional AAV particles but also improved the ratio of functional to capsid titers, suggesting enhanced encapsidation or reduced genomic loss from filled particles. After identifying the optimal setup, the process was successfully scaled up to a benchtop bioreactor, demonstrating the scalability and reproducibility of the optimized process. Production results were rapidly assessed using high-throughput analytical techniques, evaluating capsid titers via the biolayer interferometry (BLI)-based Octet® platform and functional titers through an in vitro assay using the high-throughput imaging-based Incucyte® system.