Transient gene expression with CHO cells in conditioned medium: a study using TubeSpin® bioreactors

Background Transient gene expression (TGE) allows rapid protein production in mammalian cells and has become an important tool in the pharmaceutical product development pipeline [1]. Polyethylenimine (PEI)-mediated, high-density transfection allowed to express recombinant proteins at yields exceeding 1 g/L in only a few weeks [2]. Although highly efficient protocols are available, volumetric scale-up of TGE is still a challenge. A major issue is the need to perform the transfection in fresh medium rather than in conditioned (spent) medium. This implies a medium exchange step just before transfection. In CHO-DG44 [3] cells we observed up to a 100-fold decrease in volumetric protein production if transfections were performed in conditioned medium, compared to fresh medium. The reasons for such a negative effect of conditioned medium on transfectability and/or protein production expression are not yet known. To study this problem we transfected CHO cells at small-scale in TubeSpin bioreactor 50 tubes using 41 different commercially available serum-free media formulations in combination with different transfection parameters and culture conditions. By comparing the transient production of a recombinant IgG antibody among the different media, we observed variation of up to 400-fold when transfecting in fresh media and up to 20-fold when using conditioned medium. The optimization of the PEI:DNA ratio allowed a significant improvement in yields of transfection in conditioned medium. Methods Suspension-adapted CHO-DG44 cells [3] were routinely cultivated in TubeSpin bioreactor 50 tubes (TPP, Trasadingen, Switzerland) in ProCHO5 medium (Lonza, Vervier, Belgium) supplemented with 13.6 mg/L hypoxanthine, 3.9 mg/L thymidine, and 4 mM glutamine. The 38 media samples for transfection were provided by Excellgene SA. Conditioned medium is defined as a cell culture medium where cells have been growing for more than two days up to a density between 4-5 million cells/mL. Cell growth was accessed with the Packed Cell Volume method. The dual expression vector pXLGCHO-A3, containing the cDNAs coding for human anti-Rhesus D IgG1 heavy and light chain cloned in separate expression cassettes in a head-to-head orientation, was kindly provided by Excellgene SA [4]. Transfections are performed at a cell density of 5.5 million cells/mL at a volume of 5 mL by the direct addition of 15 μg of pDNA and 76 μg of linear 25 kDa Polyethylenimine (PEI, Polysciences, Eppenheim, Germany)