On day 3 after inoculation with HEK293T/17 cells, the vessel is transfected with two plasmids at a plasmid mass ratio of 3:1, respectively, using PEIpro in a total of 816 ml IMDM or DMEM supplemented with 10% FBS and 6mM glutamax (I-10 or D-10 medium). Transfection begins by mixing PEIpro with IMDM or DMEM (without serum) to a final concentration of 40 µg/ml. The plasmids are then added to a separate tube of IMDM or DMEM to a final concentration of 20 µg/ml. PEIpro and DNA (2:1 Ratio) solutions are then combined in equal volumes and placed on a shaker at 140 rpm for 2 min at room temperature. After shaking, the transfection solution is allowed to sit for 20 min at room temperature and then added to 750 ml of I-10 or D-10 medium. The iCELLis vessel is removed from its base, the growth medium is aspirated from the vessel, and the I-10 or D-10 medium with transfection solution is added to the bioreactor. Stir speed is decreased for the remainder of the culture to give a 0.5 cm/sec flow rate at the increased volume.

Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication-dependent, less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV hFIX in a disposable fixed bed bioreactor, the iCELLis^(R) Nano (PALL Corporation). A fixed bed in the center of the iCELLis^(R) bioreactor is surrounded by culture media which is pumped through the bed from the bottom of the bioreactor so that a thin film of media overflows the bed and is replenished with oxygen and depleted of CO₂ as it returns to the surrounding media reservoir. We show that this fixed bed bioreactor can support as many as 2.5x10⁸ cells/mL of fixed bed. By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection media, and adding an additional media change to the process, we increased our yield to as high as 9.0x10¹⁴ viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis^(R) a promising scalable technology for the clinical production of AAV gene therapy products.