Cell line and culture
HEK293SF cells were maintained in serum-free suspension cultures at 37°C, 5% CO2, and 75% relative humidity in a shaker incubator (Infors, Switzerland) at 135 rpm speed of agitation or specified otherwise. The basal maintenance and production medium was HyCell TransFx-H (Cytiva Life Sciences, USA) supplemented with 0.1% w/v of Kolliphor P188 (Sigma-Aldrich, USA) and 4 mM GlutaMAX (Gibco, USA). GlutaMAX was replaced by 6 mM L-Glutamine (Gibco, USA) for experiments in which nutrients and metabolites were analyzed. The supplemented medium was prepared by adding either glucose for a final concentration of 7.2 g/L or 15% (v/v) Cell Boost 5 (Cytiva Life Sciences, USA) to achieve a similar glucose concentration.
Low- and medium-cell-density cultures (1 and 4 × 106cells/mL, respectively, at the time of transfection - TOT) were done in 125-mL polycarbonate shake flasks (TriForest, USA) with a working volume of 25 mL. High-cell-density (10 × 106 cells/mL at TOT) transfection was performed in 50-mL TubeSpin bioreactors (TPP, Switzerland) with a working volume of 10 mL and 180 rpm. For achieving and maintaining high cell densities, from 48 hours post-seeding, a perfusion-like mode of operation was followed with medium exchange of 1 vessel volume per day (VVD) both before and after transfection, where cells were centrifuged and resuspended in fresh medium.
The bioreactor production was performed in a 3-L vessel (Applikon Biotechnology, The Netherlands) equipped with a double marine impeller, a pH sensor, a temperature sensor, a dissolved oxygen (DO) concentration sensor, a micro sparger with 100-nm pore size, and a capacitance probe for monitoring of cell culture characteristics, such as cell growth. The pH, temperature, and DO parameters were set at 7.15 ± 0.05, 37°C, and 40% oxygen saturation, respectively. The HEK293SF cells from shake flasks were inoculated into the bioreactor at 4 × 106cells/mL in a fresh culture medium. The transfection was conducted immediately after cell inoculation with plasmid delivered on a cell basis (1 µg/106 cells).