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Enhanced recombinant adeno-associated virus (rAAV) biomanufacturing: Design of experiment (DOE) enabled transfection optimization for maximum full capsid yield and robust scale-up
Date
2026-05-28
Abstract
Recombinant adeno-associated virus (rAAV) gene therapies are a promising class of therapeutics. While their production via triple transfection in a single cell line offers flexibility regarding modification of the transgene, it faces significant challenges, particularly regarding predominance of empty capsids, reproducibility of process performance (titre and product quality), and successful scale-up. This study addresses these limitations by employing a two-stage Design of Experiment (DOE) approach to optimize triple transfection for rAAV5 viral vector production. An initial screening design systematically evaluated a comprehensive set of factors for their impact on transfection efficiency, genome titre, capsid titre, and the ratio of full to empty capsids. Four highly influential factors identified during screening were further investigated in a second-stage response surface design. Statistical analysis confirmed that DNA amount, complexation time, FectoVir-AAV volume, and the ratio of pTransgene were the most critical determinants of performance. The optimal conditions were successfully scaled up from 6 well plates to a 50 L Wave reactor. Coupled with the implementation of an alternative harvest strategy, transfection efficiencies, capsid, and genome titre were maintained while there was an increase in % full capsids to 73% at scale demonstrating a viable, robust, and scalable manufacturing strategy for high-quality rAAV5 vectors.
Supervisor
Description
Publisher
Wiley-VCH GmbH
Citation
Biotechnology Journal 21, e70249
Files
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Whelan_2026_Enhanced.pdf
Adobe PDF, 1.3 MB
ULRR Identifiers
Funding code
Funding Information
Sustainable Development Goals
External Link
License
Attribution-NonCommercial-ShareAlike 4.0 International