Effect of refeed strategies and non-ammoniagenic medium on adenovirus production at high cell densities

This work describes the characterisation and optimisation of the infection process in order to improve recombinant adenovirus type 5 yields at high cell densities. For that purpose, 293 cells adapted to suspension were grown in 2 l bioreactors and infected at different cell concentrations, using different refeed strategies, while evaluating cell metabolism. The consumption of amino acids is enhanced during infection, although no amino acid limitation was detected for cells infected at concentrations in the range of 2 ×106 cell/ml, for which the highest volumetric productivity was obtained in batch mode. Conversely, infecting at cell concentrations in the range of 3 × 106 cell/ml led to complete depletion of glucose, glutamine and threonine before the optimal harvesting time, a significant decrease in volumetric productivity being observed; the effect of amino acids and glucose addition at infection time on cell specific and volumetric productivity of adenovirus was assessed, no improvement on adenovirus production being achieved. The effect of ammonia, present in high concentrations at 3 × 106 cell/ml, was evaluated and seem to be detrimental; an 1.8-fold increase on adenovirus volumetric productivity was obtained for infections performed at 3 × 106 cell/ml when non-ammoniagenic medium was used.