Development of a High-Yield AAV Production Platform Using DOE and Automated Bioreactors

Recombinant adeno-associated virus vectors (AAV) are increasingly adopted for clinical gene therapy applications due to their relative safety, broad tropism, and durable transgene expression. As regulatory approvals and clinical trials continue to expand, scalable and cost-effective manufacturing methods are imperative to meet growing demand. We report here the development of a robust, high-throughput production platform that integrates Design-of-experiments (DOE) methodology with automated, single-use micro bioreactors to optimize AAV2 production yield in Expi293F HEK 293 suspension cells. Using the triple-plasmid transfection system, we conducted five multifactorial experimental runs in the Sartorius Ambr^® 15 micro bioreactor system, systematically varying key process parameters including pH, culture media, feeding strategy, agitation rate, transfection cell density, plasmid DNA load, FectoVIR^®-AAV to DNA ratio, and plasmid stoichiometry. Concurrently, we implemented pPLUS^® AAV-Helper, a novel helper plasmid designed to enhance the efficiency of viral particle packaging.

Process performance was evaluated by measuring vector genome titer via quantitative and digital PCR methods, total capsid particle concentration by ELISA, and % full capsids by mass photometry, alongside cell viability and growth kinetics. DOE analysis with Sartorius MODDE^® Pro software revealed critical interactions between transfection reagent ratio and cell density and identified an optimal operating window that delivered up to 8.8x1011 vg/mL, a 70-fold improvement over baseline conditions, while achieving a favorable % full capsids of ~25%. To demonstrate scalability, the optimized conditions were translated to the UniVessel^® 2L SU bioreactor system. Preliminary scale-up results from three runs indicate comparable productivity and product quality, confirming the robustness of the Ambr^® 15-derived scale-down model. This approach not only accelerates process development by enabling simultaneous, data-rich screening of multiple variables, but also reduces reliance on traditional shake flask workflows, offering significant time and resource savings. Our findings underscore the utility of combining DOE strategies with automated, single-use bioreactors to establish a scalable, high-yield AAV manufacturing platform suitable for preclinical and clinical supply.