4.3 Effect of Cell Density at Infection
The cell density over which the cell-specific yield of recombinant protein expression drops, is an important indicator for selecting the infection time and the cell density at infection for a productive batch. This has been referred to in many studies as the cell density effect or the time of infection. In the case of insect cell-baculovirus system, this critical indicator is a result of the overall nutrient availability, the cellular physiological state, and the ratio of cell population infected versus total viable cells in the culture determined by the MOI (i.e. synchronous Vs. asynchronous) during the production phase. Commercially available serum-free media such as Sf 900-II support a maximum cell density growth in batch mode of around 10 million cells/mL[56]. However, the same medium does not sustain protein expression in the post-infection phase at such a cell density. When analyzed for AAV production using a triple BV co-infection strategy at an overall MOI 5, the optimal cell density at the time of infection was reported to be 1 million cells/mL in the EX-CELL® 420 medium[48]. Below this cell density, the nutrient consumption was suboptimal, and above this cell density, the cell-specific yield dramatically dropped. In another study, Cecchini et al. reported that cell-specific yield of AAV production in Sf9 cells was maintained up to the cell density of 3.5-4 million cells/mL in SFX serum-free medium[57]. When analyzed for AAV5 production in One-Bac Sf9 cells using Sf900-II or Sf900-III medium by Joshi et al., the cell density breakpoint was found to be 1.7-2.0 million cells/mL under synchronous infection [58]. Any combination of cell density at the time of infection and MOI that results in an overall cell density above 2 million cells/mL resulted in a drop in the cell-specific yield.