Basic Protocol: Prior to transfection of HEK293T, cell culture media was replaced by fresh serum free media consisting of DMEM supplemented with 2mM of L-Glutamine. Triple plasmid system was performed using a helper plasmid (E2A, E4, and VA), a GFP reporter gene plasmid, and a rep and cap plasmid encoding the AAV2 particle, in a 1:1:1 M ratio, respectively for a total DNA amount of 0.143 μg per cm^2 complexed with PEIPro with 1:3 (DNA:Reagent) ratio. PEIpro and the DNA were diluted into OptiMEM serum-free medium (5% of final volume each) before being complexed and incubated for 20 min prior to transfection. 60h post-transfection, cell media was replaced by fresh DMEM media supplemented with 5% FBS and 2mM of L-Glutamine. Hollow-Fiber Bioreactor Protocol: Piror to transfection, a slow media exchange was performed by opening the IC outlet for 4 h to reduce serum concentration to 5.0%. Triple plasmid system was performed using a helper plasmid (E2A, E4, and VA), a GFP reporter gene plasmid, and a rep and cap plasmid encoding the AAV2 particle, in a 1:2:2 M ratio, respectively respectively for a total DNA amount of 1.5 μg per million predicted cells complexed with PEIPro with 1:2 (DNA:Reagent) ratio. PEIpro and the DNA were diluted into OptiMEM serum-free medium (60 mL final volume each) before being combined and incubated for 20 min. The final complexe solution of 120 mL was then added directly to the bioreactor, which was then left to incubate for 6 h. After incubation, media feeding is automatically restarted. During the 3 days of production, cells were fed with media containing 5.0% serum, 2mM of GlutaMAX, and 10.0% v/v CDM4HEK293 media.

Adeno-associated viral (AAV) vectors have been established as a safe and effective delivery vehicle for gene therapy. However, current methods for AAV production using adherent approaches are suboptimal due to their reliance on a substantial number of plastic-based flasks, manual labor, and a significant manufacturing footprint. Consequently, a protocol for generating AAV2 was developed on the Quantum, a semi-automated closed hollow-fiber bioreactor platform. In this system, Human Embryonic Kidney 293T cells were successfully expanded and transfected to produce an average crude AAV2 titer of 4.92 × 10¹⁴ viral particles and 6.81 × 10¹³ viral genomes from 1.2 L of harvested cell lysate. The application of a standard AAV downstream process confirmed normal processability of the material. A cost of goods model comparing the Quantum bioreactor with the current standard HYPERStack36 and Corning CellSTACK 10-layer systems demonstrated that the Quantum bioreactor reduced the number of open steps by more than 40-fold, production time by up to 3.6-fold (HYPERStack36) and 7.5-fold (CellSTACK 10-layer), and costs by up to 2-fold (HYPERStack36) and 20.7-fold (CellSTACK 10-layer). Therefore, the Quantum bioreactor is an effective alternative to plastic flasks for the manufacturing of AAVs at both R&D and early translational scale, as it reduces production time, operating costs, and process risk.