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].