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

Nguyen, Tam N. T., Park, Damdae, Canova, Christopher T., Sangerman, Jose, Srinivasan, Prasanna, Ou, Rui Wen, Barone, Paul W., Neufeld, Caleb +4 more·2025·Biotechnology and Bioengineering
DOI 10.1002/bit.28967PMC PMC12067042
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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 suboptimal 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 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.

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  • Most existing viral vector models are vector specific and typically require re-derivation or extensive re-parameterization when applied to other viral systems.

    “Most existing models are vector specific and typically require re-derivation or extensive re-parameterization when applied to other viral systems.”

    From Mechanistic model for HEK293 viral vector processes and its application in a digital shadow framework (2026) · cites this paper as [16]

    The Discussion (page unavailable) says this rAAV model can be adapted to different serotypes and production methods through mathematical modifications and reparameterization. It does not establish that most existing models are vector-specific or typically require extensive reparameterization or re-derivation for other viral systems. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • For AAV, several mechanistic frameworks describe triple transfection dynamics, cell growth, metabolism, genome replication, and capsid formation.

    “For AAV, several mechanistic frameworks describe triple transfection dynamics, cell growth, metabolism, genome replication, and capsid formation [5, 16, 17].”

    From Mechanistic model for HEK293 viral vector processes and its application in a digital shadow framework (2026) · cites this paper as [16]

    The Materials and Methods section describes a mechanistic triple-transfection model that includes cell growth, viral DNA replication, and capsid formation. The Results report metabolite measurements, but the modeling section uses dead-cell concentration as a surrogate for harmful species rather than describing a mechanistic model of metabolism. No page numbers are available in our copy. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Several mechanistic modeling efforts have characterized HEK293 based viral vector production, although most of them remain vector specific.

    “Several mechanistic modeling efforts have characterized HEK293 based viral vector production, although most of them remain vector specific.”

    From Mechanistic model for HEK293 viral vector processes and its application in a digital shadow framework (2026) · cites this paper as [16]

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Cited by

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Authors
Nguyen, T. N. T., Park, D., Canova, C. T., Sangerman, J., Srinivasan, P., Ou, R. W., Barone, P. W., Neufeld, C., Wolfrum, J. M., Springs, S. L., Sinskey, A. J., & Braatz, R. D.
Affiliations
  • Department of Chemical Engineering Massachusetts Institute of Technology Cambridge Massachusetts USA
  • Center for Biomedical Innovation Massachusetts Institute of Technology Cambridge Massachusetts USA
  • Department of Biology Massachusetts Institute of Technology Cambridge Massachusetts USA
ORCID
Venue
Biotechnology and Bioengineering
Publisher
Wiley
Published
2025-03-18
Volume
122
Issue
6
Pages
1424-1440
ISSN
  • 0006-3592
  • 1097-0290
DOI
10.1002/bit.28967
PMC
PMC12067042
Cited by
9
References
3
Language
en
Licence
creativecommons.org/licenses/by-nc-nd/4.0/ (vor)
Funders
Food and Drug Administration

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