Model-Based Design of Recombinant Adeno-Associated Viral Vector Production

Recombinant adeno-associated virus (rAAV) is one of the most commonly used platforms for in vivo gene therapy treatments.¹ Their reduced toxicity, robust and long-term transgene expression, and ability to transduce both dividing and non-dividing cells as well as target a wide range of tissues have made rAAV the most widely used viral vector.^1,2 However, current state-of-the-art viral vector manufacturing methods still fall short of meeting current and future demands. rAAV production in HEK293 cells has been successfully adapted to suspension cultures,^3–5 for which we have developed a mechanistic model based on the published understanding of the underlying biology and existing data. The objectives of the model development are to confirm understanding of the underlying mechanisms and to have a way to predict responses to various process inputs. This approach mirrors the development of mechanistic models for other biotherapeutics, which have been used to facilitate process development and reduce the high cost of experimentation compared to empirical optimization.⁶

This presentation discusses the adaptation of a mechanistic model of rAAV production via triple transfection into a macroscopic bioreactor model. Various bioreactor modes and designs are evaluated using the adapted model framework. A production platform design is proposed, with viral titer and full capsid ratio as the critical quality attributes,⁷ that optimizes process parameters relating to transient transfection, including transfection time, plasmid ratio, and harvest time.

References:

(1) Wang, D.; Tai, P. W. L.; Gao, G. Adeno-Associated Virus Vector as a Platform for Gene Therapy Delivery. Nat. Rev. Drug Discov. 2019, 18 (5), 358–378. https://doi.org/10.1038/s41573-019-0012-9.

(2) Nonnenmacher, M.; Weber, T. Intracellular Transport of Recombinant Adeno-Associated Virus Vectors. Gene Ther. 2012, 19 (6), 649–658. https://doi.org/10.1038/gt.2012.6.

(3) Chahal, P. S.; Schulze, E.; Tran, R.; Montes, J.; Kamen, A. A. Production of Adeno-Associated Virus (AAV) Serotypes by Transient Transfection of HEK293 Cell Suspension Cultures for Gene Delivery. J. Virol. Methods 2014, 196, 163–173. https://doi.org/10.1016/j.jviromet.2013.10.038.

(4) Grieger, J. C.; Soltys, S. M.; Samulski, R. J. Production of Recombinant Adeno-Associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector. Mol. Ther. 2016, 24 (2), 287–297. https://doi.org/10.1038/mt.2015.187.

(5) Blessing, D.; Vachey, G.; Pythoud, C.; Rey, M.; Padrun, V.; Wurm, F. M.; Schneider, B. L.; Deglon, N. Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells. Mol Ther Methods Clin Dev 2019, 13, 14–26. https://doi.org/10.1016/j.omtm.2018.11.004.

(6) Kyriakopoulos, S.; Ang, K. S.; Lakshmanan, M.; Huang, Z.; Yoon, S.; Gunawan, R.; Lee, D. Y. Kinetic Modeling of Mammalian Cell Culture Bioprocessing: The Quest to Advance Biomanufacturing. Biotechnol. J. 2018, 13 (3), e1700229. https://doi.org/10.1002/biot.201700229.

(7) Gimpel, A. L.; Katsikis, G.; Sha, S.; Maloney, A. J.; Hong, M. S.; Nguyen, T. N. T.; Wolfrum, J.; Springs, S. L.; Sinskey, A. J.; Manalis, S. R.; Barone, P. W.; Braatz, R. D. Analytical Methods for Process and Product Characterization of Recombinant Adeno-Associated Virus-Based Gene Therapies. Mol. Ther. Methods Clin. Dev. 2021, 20, 740–754. https://doi.org/10.1016/j.omtm.2021.02.010.