Validation of medium-cell-density production at bioreactor scale
Since the MCD production resulted in a significant improvement in titer and no loss in functionality, we decided to evaluate the scalability of this process. For that, we conducted the production in a 3-L bioreactor, where the cells were seeded at 4 × 106 cells/mL in fresh medium and were immediately transfected with plasmids delivered on a cell basis. Whole broth culture was sampled at 8-16h intervals. A satellite culture, with a volume of around 25 mL taken from the bioreactor post-transfection, was evaluated in parallel in a shake flask (Figure S1). At 24 hpt, a transfection efficiency of 36.9% was observed, similar to the production in shake flasks (33.8%, Figure 2C). Figure 7 shows cellular and production kinetics in the bioreactor, demonstrating that MCD production is feasible at a bioreactor scale. Low-cell-density 3-L bioreactor productions previously completed in our laboratory were used as control. In these LCD runs, AAV6 vectors packaging a genome without fluorescent marker were produced; thus, it was impractical to assess their functional titer. Cell density in the MCD bioreactor reached its maximum at 59 hpt with 9.5 × 106 viable cells/mL. Vector production peaked around 60 hpt with a titer of 5.9 × 1010 VG/mL, a ~4-fold increase compared to control bioreactors (Figure 7C). At this point, the cell-specific titer was around 15,000 VG/cell, a similar value to the one obtained by LCD production in bioreactors (Figure 7D). Regarding the functional titer, the bioreactor yielded 7.8 × 109 ETU/mL 48 hours post-transfection (Figure 7E). As a result, the VG/ETU ratio was as low as 4.6 at 48 hpt, plateauing around 10 at subsequent time points (Figure 7F).