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).