YVES DUROCHER, SYLVIE PERRET AND AMINE KAMEN RECOMBINANT PROTEIN PRODUCTION BY TRANSIENT TRANSFECTION OF SUSPENSION- GROWING CELLS Keywords: Polyethyleneimine, mammalian cells, EBNAI, green fluorescent protein, SEAP. Abstract. Motivated by an increasing demand for the rapid production of milligrams of r-proteins, we have optimized a transfeetion procedure using polyethyleneimine (PEI) for the human embryonie kidney 293 cells (293) grown in suspension that is scalable. The human placental secreted alkaline phosphatase (SEAP) and the green fluorescent protein (GFP) were used as reporter genes to respectively monitor productivity and transfeetion efficiency. The 293 ceilline and two genetic variants expressing the SV40 large T-antigen (293T) or the EBNAI protein (293E) were tested for transfeetion and protein expression using the pcONA3.1 (Invitrogen) and pTT vectors. Up to ten-fold higher expression level was obtained when transfecting 293E cells with the oriP-containing pTT vector compared to transfeetion of 293T cells with the SV40 ori-containing pcONA3.1 vector. Various PEI polymers were tested and both linear and branched 25 kOa PEI proved to be the most efficient. Transfeetion efficiencies, as determined by FACS- analyses of GFP-positives cells 48 hours post-transfection (hpt), varied from 60 to 98%. The presence of serum in the culture medium significantJy increased gene transfer and expression. High levels of expression were also reached when transfeetion mixture was added to cells in their conditioned medium (24h post-seeding) containing 1% serum. Transfeetion of a 500 ml bioreactor culture with pTT/SEAP (0.5 mg DNA) led to the accumulation of over 10 mg r' of active SEAP at 120 hours post-transfection. 1. INTRODUCTION Transient transfection of suspension-growing cells provides a mean to generate mg amounts of recombinant proteins (r-proteins) within few days (see Wurm and Bernard, 1999 for review). The human embryonic kidney 293 cell line (293) is a host of choice for this application as it can be transfected with high efficiency using most common transfection vehicIes. This cell line can also be easily adapted to suspension culture in the presence or absence of serum (Cöte et al., 1998). Two genetic variants expressing the large T antigen (293T) or EBNAI (293E) are available. These allow for episomal maintenance and amplification of plasmid DNA bearing specific viral origins of replication, namely the SV40 ori and oriP respectively. The cationic polymer polyethyleimine (PEI) was recently described as a good and cost-effective transfection reagent for many cell lines (Boussif et al., 1995). It has also been shown to efficiently mediate gene transfer in 293 cells grown in suspension culture (Schlaeger and Christensen, 1999). This polymer is available under both branched and linear isomers with various molecular weight and polydispersity, these physicochemical parameters being of importance for gene transfer activity (Godbey et al., 1999). In this study, we first looked at transfection efficiency using various PEI polymers. We then evaluated the best vector/cell line 331 o-w. Merlen et al. (eds.), Recombinant Protein Production with Prokaryotic and Eukaryotic Cells, 331-337. © 2001 Kluwer Academic Publishers.