Abstract
Increasing demand for recombinant adeno-associated virus (rAAV)-based
gene therapies necessitates increased manufacturing production.
Transient transfection of mammalian cells remains the most commonly used
method to produce clinical-grade rAAVs due to its ease of
implementation. However, transient transfection processes are often
characterized by sub-optimal yields and 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 a temporal misalignment between viral DNA replication and capsid
synthesis within the cells and the repression of later phase capsid
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 mechanistic model to develop an intensified process for
rAAV production that combines continuous perfusion with high cell
density re-transfection. We demonstrate that performing multiple,
time-separated doses at high cell density boosts both cell-specific and
volumetric productivity and improves plasmid utilization when compared
to a single bolus at standard operating conditions. Our results
establish a new paradigm for continuously manufacturing rAAV via
transient transfection that improves productivity and reduces
manufacturing costs.