Perfusion‐Based Production of rAAV via an Intensified Transient Transfection Process

Abstract

Increasing demand for recombinant adeno‐associated virus (rAAV)‐based gene therapies necessitates increased manufacturingproduction. Transient transfection of mammalian cells remains the most commonly used method to produce clinical‐graderAAVs due to its ease of implementation. However, transient transfection processes are often characterized by suboptimal yieldsand low fractions of full‐to‐total capsids, both of which contribute to the high cost of goods of many rAAV‐based gene therapies.Our previously developed mechanistic model for rAAV2/5 production indicated that the inadequate capsid filling is due to atemporal misalignment between viral DNA replication and capsid synthesis within the cells and the repression of later phasecapsid formation by Rep proteins. We experimentally validated this prediction and showed that performing multiple, time‐separated doses of plasmid increases the production of rAAV. In this study, we use the insights generated by our mechanisticmodel to develop an intensified process for rAAV production that combines perfusion with high cell density re‐transfection. Wedemonstrate that performing multiple, time‐separated doses at high cell density boosts both cell‐specific and volumetricproductivity and improves plasmid utilization when compared to a single bolus at standard operating conditions. Our resultsestablish a new paradigm for continuously manufacturing rAAV via transient transfection that improves productivity andreduces manufacturing costs.