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Continuous Production of rAAV via an Intensified Transient Transfection Process
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  • Tam N. T. Nguyen,
  • Damdae Park,
  • Christopher T. Canova,
  • Jose Sangerman,
  • Prasanna Srinivasan,
  • Rui Wen Ou,
  • Paul Barone,
  • Caleb Neufeld,
  • Jacqueline Wolfrum,
  • Stacy Springs,
  • Anthony Sinskey,
  • Richard Braatz
Tam N. T. Nguyen
Massachusetts Institute of Technology Department of Chemical Engineering
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Damdae Park
Massachusetts Institute of Technology Department of Chemical Engineering
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Christopher T. Canova
Massachusetts Institute of Technology Department of Chemical Engineering
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Jose Sangerman
Massachusetts Institute of Technology
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Prasanna Srinivasan
Massachusetts Institute of Technology
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Rui Wen Ou
Massachusetts Institute of Technology Department of Biology
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Paul Barone
Massachusetts Institute of Technology
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Caleb Neufeld
Massachusetts Institute of Technology
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Jacqueline Wolfrum
Massachusetts Institute of Technology
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Stacy Springs
Massachusetts Institute of Technology
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Anthony Sinskey
Massachusetts Institute of Technology
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Richard Braatz
Massachusetts Institute of Technology Department of Chemical Engineering

Corresponding Author:[email protected]

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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.
15 Sep 2024Submitted to Biotechnology and Bioengineering
16 Sep 2024Submission Checks Completed
16 Sep 2024Assigned to Editor
16 Sep 2024Review(s) Completed, Editorial Evaluation Pending
11 Oct 2024Reviewer(s) Assigned