Cells were transfected using PEIpro. On the day of transfection, cells with viability greater than 98% were centrifuged and resuspended at a density of 2× 10^6 cells/mL supplemented with 4 mM glutamine and 0,1% Kolliphor P-188. The cell suspension was distributed in 6-well plates (1.8 mL/well). The DNA:PEIpro complexes were prepared at a ratio of 1:5 (w:w), with a total of 2 μg DNA per well to transfect in 100 μL of complete culture medium. Equal volume of diluted PEIpro (10 μg per well to transfect) was added to the diluted DNA and the mixture was vortexed 4 s and incubated for 3 min at room temperature. Polyplexes were then added to the cells (200 μL per well) and the plates were returned to the incubator at 37°C and 5% CO2 under constant agitation (120 rpm).

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHOBRI/rcTA) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHOBRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1alpha-HTLV constitutive promoters. These results suggest that the cumate-inducible CHOBRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins.