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ABSTRACT Recombinant adeno associated virus (rAAV) vectors have become popular delivery vehicles for in vivo gene therapies, but demand for rAAVs continues to outpace supply. Platform processes for rAAV production are being developed by many manufacturers, and transient chemical transfection of human embryonic kidney 293 (HEK293) cells is currently the most popular approach. However, the cutting edge nature of rAAV process development encourages manufacturers to keep cell culture media formulations, plasmid sequences, and other details proprietary, which creates hurdles for small companies and academic labs seeking to innovate in this space. To address this problem, we leveraged the resources of an academic‐industry consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) to develop an rAAV production system based on transient transfection of suspension HEK293 cells adapted to an in‐house, chemically defined medium. We found that balancing iron and calcium levels in the medium were crucial for maintaining transfection efficiency and minimizing cell aggregation, respectively. A design of experiments approach was used to optimize the transient transfection process for batch rAAV production, and PEI:DNA ratio and cell density at transfection were the parameters with the strongest effects on vector genome (VG) titer. When the optimized transient process was transferred between two university sites, VG titers were within a twofold range. Analytical characterization showed that purified rAAV from the AMBIC process had comparable viral protein molecular weights versus vector derived from commercial processes, but differences in transducing unit (TU) titer were observed between vector preps. The developed media formulation, transient transfection process, and analytics for VG titer, capsid identity, and TU titer constitute a set of workflows that can be adopted by others to study fundamental problems that could improve product yield and quality in the nascent field of rAAV manufacturing.
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Toward the quantification of adeno-associated virus titer by electrochemical impedance spectroscopy
Gene therapies have shown great promise for the potential treatment of a broad range of diseases. Adeno-associated viruses (AAVs) are popular gene vectors because of their ability to target specific tissues, and they have demonstrated high transduction efficiencies in multiple neurological targets. While these therapeutics hold great promise, their biomanufacturing has limited potential cost-reduction and more widespread adoption. Herein, we report the preliminary development of an immunosensor for measuring the titer of adeno-associated virus 2 (AAV2), which may be deployed for rapid quantification of product yield during AAV biomanufacturing. We functionalized an interdigitated electrode array with anti-AAV2 antibodies, and electrochemical impedance spectroscopy was employed to investigate the response to AAV2 titer. A Faradaic sensing principle was utilized, in which the charge transfer resistance (Rct) of an electrochemical reporter was monitored after capture of AAV2 on the surface of the sensor. A linear response was measured over titers 1012 - 1013 capsids/mL.
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- Award ID(s):
- 1846911
- PAR ID:
- 10493598
- Publisher / Repository:
- IEEE
- Date Published:
- Journal Name:
- IEEE BioSensors
- ISBN:
- 979-8-3503-4604-6
- Page Range / eLocation ID:
- 1 to 4
- Subject(s) / Keyword(s):
- biosensor virus AAV impedance spectroscopy
- Format(s):
- Medium: X
- Location:
- London, United Kingdom
- Sponsoring Org:
- National Science Foundation
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