High-cell-density cultivations to increase MVA virus production

To facilitate optimization of the virus propagation phase at HCD, a larger set of feeding strategies was analyzed in small-scale cultivations using shake flasks. Densities up to 63 × 106 cells/mL were obtained at the end of the cell growth phase applying a discontinuous perfusion mode (semi-perfusion) with the same cell-specific perfusion rate as in the bioreactor (0.060 nL/(cell d)). At this cell concentration, a medium exchange at time of infection was required to obtain expected virus yields during the first 24 h after infection. Applying an additional fed-batch feeding strategy during the whole virus replication phase resulted in a faster virus titer increase during the first 36 h after infection. In contrast, a semi-continuous virus harvest scheme improved virus accumulation and recovery at a rather later stage of infection. Overall, a combination of both fed-batch and medium exchange strategies resulted in similar cell-specific virus yields as those obtained for CCD processes but 10-fold higher MVA-CR19 titers, and four times higher volumetric productivity.