Insights into adenoviral vector production kinetics in acoustic filter‐based perfusion cultures

Abstract

One of the major limitations in the production of adenoviral vectors is the reduction in cell-specific productivity observed for increasing cell density at infection in batch cultures. This observation strongly suggests some nutrient depletion and/or metabolite inhibition in the media. These limitations have been partially overcome through other feeding strategies, such as fed-batch and sequential batch operations. To improve these results, we evaluated perfusion as a strategy to increase the volumetric productivity of HEK-293 cell cultures, by allowing productive infection at higher cell densities. An acoustic cell separator was employed in consideration of the increased shear sensitivity of the cells during the infection phase. The effects of perfusion rate and cell density at infection on the production of a recombinant adenovirus expressing the GFP were investigated. The perfusion mode allowed successful infection at cell densities in the range of 2.4-3 x 10(6) cell/mL, while maintaining a similar cell specific productivity (17,900 +/- 2400 VP/cell) to that of a batch infected at a low cell density (5 x 10(5) cell/mL). The highest virus concentrations (4.1 +/- 0.6 x 10(10) VP/mL) were attained for a feed rate of 2 vol/d and constituted a fivefold increase compared to a batch with medium replacement. Rapid assessment of the infection status was achieved through the use of on-line monitoring of respiration, fluorescence, and biovolume. Analysis of the kinetics of nutrient consumption and metabolite production revealed that a reduction in specific productivity is correlated with reduced metabolic activity.

Multifrequency permittivity measurements enable on‐line monitoring of changes in intracellular conductivity due to nutrient limitations during batch cultivations of CHO cells

“…Further applications should be processes in which changes in the physiological state are of primary importance for productivity, for example, viral vector production schemes and/or perfusion cultures. 26,69 To our knowledge, this article is the first report that interprets multifrequency permittivity measurements and the resulting b-dispersion parameters and relates them to the physiological state of mammalian cells. These measurements appeared to us as being a powerful on-line monitoring tool.…”

Production of lentiviral vectors by large‐scale transient transfection of suspension cultures and affinity chromatography purification

“…Typically, transfection at 1 Â 10 6 cells/mL gives optimum levels of recombinant protein expression in our laboratory. However, it has been observed that at these high cellular densities adenovirus vector production is limited by reduced cell specific productivity (Nadeau and Kamen, 2003;Kamen and Henry, 2004;Henry et al, 2004). This so called ''cell density effect'' is believed to be either due to key nutrient depletion or inhibitor byproduct accumulation.…”

Monitoring lentiviral vector production kinetics using online permittivity measurements

“…The bioreactor setup was identical to the one used in a previous study [29]; similar conditions have been described earlier in detail [33]. In brief, a Chemap 3.5 L type SG bioreactor vessel (working volume 2.7 L) was equipped with probes to measure and control the temperature and dissolved oxygen (DO) at 37 • C and 40% air saturation.…”