2.1. Cell line development, culture conditions and stability evaluation
In order to generate a cell line capable of stably expressing the structural premembrane (prM) and envelope (E) proteins, HEK293SF-3F6 cells (NRC, Canada) were transfected by lipofection in a shaken vented tube (spin tube, TPP AG, Switzerland) using a chemically-defined medium (HEK TF, Xell AG, Germany) and Lipofectamine 3000 reagent (Invitrogen, USA), as described previously (Alvim et al., 2019). Transfection volume was 4 mL.
The gene construct used (named LECC15) was based on an in-house designed signal peptide followed by the sequence encoding prM-E (683 amino acids) of the yellow fever virus (South-America I genotype, strain BeH655417, GenBank # JF912190). The gene construct was codon-optimized, synthetized, subcloned into pCIneo vector (Promega, USA) and supplied ready to transfect by Genscript (USA).
Approximately 48h post-transfection (hpt), cells were transferred to a vented 125 mL-erlenmeyer flask (Corning, USA) and diluted with fresh HEK TF medium in the presence of 100 μg/mL G418 sulfate (Gibco, USA) for selection of stable transfectants. Every 3-4 days, viable cell concentration was adjusted to 1 × 106 cells/mL by diluting cell suspension with fresh HEK TF medium containing 100 μg/mL G418 sulfate. Documented research cell banks of the stable cell pool were cryopreserved at 30 and 60 days post-transfection.
In order to evaluate production profile over time, the cell pool was kept in culture in the presence of the selection antibiotic for up to approximately 120 days post-transfection. During the whole period, cells were kept in a humidified incubator at 37 °C and 5 % CO2 and shaken at 180 rpm (50 mm orbit). Cell count was performed using an automated cell counter (Vi-Cell®XR, Beckman Coulter, USA) and, when necessary, glucose and lactate concentrations were monitored using a YSI 2700 Select Biochemistry Analyzer (YSI Inc., USA).