AAV Process Development in Culture Biosciences’ 250mL Bioreactors

Adeno-associated viruses (AAVs) have emerged as an extremely promising and widely-used delivery vehicle for gene therapy. However, a major hurdle in the development of AAV-based gene therapy is the high cost of manufacturing AAV on the clinical and commercial scale due to the difficulty of scale-up and the inherently high variability during AAV production. Using triple transfection in suspension HEK293 has been shown to yield viral genome titers in excess of 1x1014 genomes/L. However, the percentage of filled AAV capsids and genome titers can range widely across batches and adjustments in upstream and downstream processing are required for each unique AAV serotype, making it difficult to standardize AAV production. Additionally, AAV production processes are not particularly robust; even after optimization, simply switching plasmids or transfection reagents can result in order-of-magnitude differences in viral titers, necessitating additional rounds of process optimization. These challenges are difficult to overcome when running just a few experiments at a time and are exacerbated by the high variability of AAV processes, which can mask the effect of the parameters being test-ed. However, they can be addressed through high-throughput, intentional exploration of design space using sufficient replicates to confidently identify critical process parameters that drive efficient and robust AAV production. Here we show production of multiple AAV serotypes in our 250mL bioreactors and demonstrate how running multiple small-scale bioreactors in parallel enabled us to quickly identify transfection reagents, complexation conditions, and bioreactor process parameters to improve overall yields.

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