The current manufacturing process for AAV therapies is expensive, time consuming and unlike the production of monoclonal antibodies, their manufacture has not been standardized across the industry. Many different production systems and downstream purification processes are used across the industry, resulting in high production costs and processes that lack flexibility, scalability and quality. [2]
AAV therapies are produced through triple transient transfection into a single cell line where three plasmids are combined with a transfection reagent that undergoes interactions with DNA. While transfection of multiple plasmids gives flexibility and allows modification of transgene regulatory elements, there are complexities associated with the triple transfection resulting in challenges around reproducibility of process performance in terms of titre and product quality. Production of full AAV capsids, which correspond to the desired product, is typically between 10-20% due to complexities associated with triple transfection. This low yield negatively impacts the downstream purification of the therapies with overall process yields remaining below 50%. A high-yield production process would enable a more efficient and cost-effective downstream process which would reduce the costs of the medicines for patients.
This study describes a 2 stage DoE approach to optimize triple transfection using FectoVir-AAV and a HEK293F cell line. The three plasmids allowed for production of an AAV5 capsid containing the transgene encoding Green Fluorescent Protein (GFP). An initial screening design was implemented due to the large number of factors with potential to have significant effects on transfection efficiency, genome titre, capsid titre, and ratio of full to empty capsids. The factors screened included DNA amount, plasmid ratios, volume of FectoVir-AAV, complexation volume, complexation time and cell culture density. Four factors were taken forward to the second stage which implemented a central composite design. This design was chosen to accurately estimate quadratic terms in responses and expand the design space beyond levels included in the screening. Once optimum conditions for transfection were identified, the conditions were further verified at 125 mL shake flask, 3L shake flask and 10L Wave reactor scales.
The optimum conditions resulted in ~76% transfection efficiency, genome titre of ~1.3 x 1011 vg/mL, capsid titre of ~2.2 x 1011 capsids/mL and a percentage of full capsids of ~60%. The conditions identified showed reproducible performance across each scale assessed with % full capsids ranging from 60-73% - over a 3-fold increase in full capsids in comparison to industry standard. This 2-stage DoE workflow is a powerful tool to develop an understanding of the triple transfection process and ensure a high yield production process is developed enabling an efficient and cost-effective downstream process.
Acknowledgements:
This research is supported by the Taighde Éireann through EBPPG/2022/38s in conjunction with APC Ltd and the School of Chemical and Bioprocess Engineering, University College Dublin (UCD). The authors acknowledge the support of Enterprise Ireland under its Research, Development and Innovation Fund, Grant Award 180753/RR for N. Donohue.
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