4.4 Effect of Operation Mode
The low cell density culture process operating under a batch mode of
cultivation generally results in a lower volumetric yield of AAV
necessitating large scale bioreactor production to meet exceptionally
high demand of the late clinical phase studies. When coupled with a
sustained cell-specific yield, the high cell density culture process
under fedbatch or perfusion mode of operation provide a mean to enhance
the volumetric yield. Both modes have been successfully implemented to
achieve a high cell density culture of Sf9 cells[58], [59]. The
nutrient feeding strategy in a fedbatch process aims at maintaining the
cells in the mid-exponential phase for a sustained time to achieve a
high cell density before infection. When coupled with nutrient feeding
in the post-infection phase, metabolic limitations are alleviated.
Bedard et al. described an exemplary work where the nutrient feed
formulation was designed to meet metabolic need of Sf9 cells to support
the sustained expression of β-galactosidase following BV
infection[60]. Building on this, Elias et al. reported
β-galactosidase production at a cell density of 14 million cells/mL
without reduction of the cell-specific yield[61]. Meghrous et al.
reported that when applied at the time of infection for the triple BV
co-infection system, a complete medium replacement strategy that
practically mimics the perfusion at shake flask scale, resulted in a
linear increase in volumetric yield at various cell densities at the
time of infection without any loss of cell-specific yield of
AAV[48]. When further scaled up in a 3L bioreactor, the infection of
Sf9 cells at 2.5 million cells/mL in a fresh medium offered similar
cell-specific yield and a proportionate increase in volumetric yield
(Table 1). In a further study by Mena et al. the medium replacement
strategy coupled with infection at 5 million cells/mL with high MOI of
triple BVs (3 MOI of each) resulted in around three fold increase in
genomic AAV particles in comparison to the control, low cell density
batch process[59]. This increase in volumetric yield was
proportionate to the increase in cell density, which demonstrated
sustained cell-specific yield. Furthermore, when carefully tailored, the
fedbatch strategy in combination with low MOI applied to AAV production
in Sf9, resulted in almost 1 log increase in volumetric yield of
functional AAV particles (Table 1)[59]. In this case, Sf9 cells were
infected at 5 million cells/mL at an MOI of 0.1 (of each BVs), which
resulted in cell density increase up to 9.6 million cells/mL at 48 hpi.
The nutrient supplementation was provided at the time of infection, and
24 h before and after infection. A more recent study by Joshi et al.
involving AAV5 production employing One-Bac under fedbatch mode of
operation also suggested improved yield of genomic and functional
particles of AAV5 (Table 1)[58]. The high cell density at the time
of infection (~10 million cells/mL) was achieved via
bolus nutrient supplementation followed by high MOI infection, which
resulted in an immediate transition of cell culture from the growth
phase to the productive phase within 24hpi under synchronous infection
of baculovirus. Coupled with an additional bolus feeding, the process
exhibited sustained AAV production in post-infection phase[58].