The demand for recombinant adeno-associated virus (rAAV) is poised to exceed current production capacities, driven by the increasing number of approved and advanced-phase rAAV-based gene therapies [1]. Current rAAV manufacturing processes yield less-than-optimal titers, consisting of a significant proportion of non-functional empty capsids [2]. FDA recently approved the first rAAV-based gene therapies manufactured in the Sf9 insect cell line with the baculovirus expression vector system (BEVS). Within the Sf9/BEVS platform, Sf9 cells produce rAAV as a result of infection with recombinant baculoviruses that carry the genetic blueprint for rAAV vector production. Transitioning from the traditional batch manufacturing to continuous processing would allow to significantly increase the BEVS productivity. However, the formation of baculovirus defective interfering particles severely compromises the process efficiency and leads to shutdown of continuous operation in the BEVS within, at best, 5–7 days from viral inoculation [3]. In this work, a mathematical model is developed and used for in silico design of a process for continuous rAAV manufacturing in the BEVS [4,5]. The mathematical model is used to assess the optimal operating conditions of cell growth and production bioreactors that can counteract the detrimental effect of baculovirus defective interfering particles. The process designed through the mathematical model is implemented and validated on laboratory-scale bioreactors, achieving for the first time continuous production of high titers of rAAV for over 20 days. This study paves the way for efficient and scalable continuous rAAV manufacturing, positioning the industry to meet the rising demand for gene therapies.
References
[1] Au, H.K.E., Isalan, M. and Mielcarek, M., 2022. Gene therapy advances: a meta-analysis of AAV usage in clinical settings. Front. Med. 8, 809118.
[2] Su, W., PatrÃcio, M.I., Duffy, M.R., Krakowiak, J.M., Seymour, L.W. and Cawood, R. (2022). Self-attenuating adenovirus enables production of recombinant adeno-associated virus for high manufacturing yield without contamination. Nat. Commun. 13(1), 1182.
[3] Vignuzzi M. and López, C. B (2019). Defective viral genomes are key drivers of the virus-host interaction. Nat. Microbiol. 4, 1075–1087.
[4] Destro, F. and Braatz, R.D. (2024). Efficient simulation of viral transduction and propagation for biomanufacturing. bioRxiv, DOI: 10.1101/2024.03.30.587435.
[5] Destro, F., Joseph, J., Srinivasan, P., Kanter, J.M., Neufeld, C., Wolfrum, J.M., Barone, P.W., Springs, S.L., Sinskey, A.J., Cecchini, S., Kotin, R.M., and Braatz, R.D. (2023). Mechanistic modeling explains the production dynamics of recombinant adeno-associated virus with the baculovirus expression vector system. Mol. Ther. Methods Clin. Dev. 30, 122-146.
