Large-Scale Transient Transfection of Serum-Free Suspension-Growing HEK293 EBNA1 Cells: Peptone Additives Improve Cell Growth and Transfection Efficiency Phuong Lan Pham, Sylvie Perret, Huyen Chau Doan, Brian Cass, Gilles St-Laurent, Amine Kamen, Yves Durocher Animal Cell Technology Group, Bioprocess Platform, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Canada H4P 2R2; Telephone: 514-496-6192; fax: 514-496-6785; e-mail: yves.durocher@cnrc-nrc.gc.ca Received 28 January 2003; accepted 5 June 2003 Published online 21 August 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10774 Abstract: Large-scale transient transfection of mamma- lian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant pro- teins (r-proteins). As many r-proteins used for therapeu- tic and structural studies are naturally secreted or engi- neered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purifi- cation is required. In an attempt to design such a serum- free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent pro- tein (GFP) and human placental secreted alkaline phos- phate (SEAP) as reporter genes. The suspension grow- ing, serum-free adapted HEK293SF-3F6 cell line was sta- bly transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bear- ing plasmids. Compared to our standard serum-free me- dium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection effi- ciency and significantly increased volumetric productiv- ity fourfold. Using the optimized medium formulation, transfection efficiencies between 40–60% were routinely obtained and SEAP production reached 18 mg/L −1 . To date, we have successfully produced and purified over fifteen r-proteins from 1–14-L bioreactors using this se- rum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropi- lin-1 extracellular domains (ED) in bioreactors. Each pro- tein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuro- pilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free me- dium significantly improves protein expression com- pared to peptone-less medium, and significantly in- creases their purification efficiency compared to serum- containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milli- gram amounts of r-proteins within 5 days posttransfec- tion. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 332– 342, 2003. Keywords: polyethyleneimine; secreted proteins; biore- actor; human embryonic kidney cells; immobilized metal affinity chromatography INTRODUCTION The great advances made in genomics and proteomics dur- ing the past decade have positioned recombinant proteins (r-proteins) as major therapeutic biomolecules and drug tar- gets (Chu and Robinson, 2001; Kelley, 2001). The increas- ing demand for milligrams of r-proteins to be used in pre- clinical, biochemical, and biophysical studies justifies the need of a rapid and scaleable expression system. While bacterial expression systems often satisfy these needs, ex- pressed proteins frequently suffer from poor solubility or lack of proper processing. In this regard, mammalian ex- pression systems are more appropriate for providing soluble and fully processed r-proteins. Although the usual means of producing r-proteins in mammalian cells is to establish clones stably expressing the gene of interest, this technology is not readily amenable to a high-throughput mode. The recent large-scale transient transfection technology is now generating great interest because of its demonstrated ability to produce large amounts of r-proteins within a few days (Durocher et al., 2002; Girard et al., 2002; Jordan et al., 1998; Meissner et al., 2001; Schlaeger and Christensen, 1999). The use of nonviral gene-transfer systems such as cationic liposomes and polymers, or calcium-phosphate pre- cipitates has recently been shown to be highly effective in transfecting cells grown in suspension, a prerequisite for scale-up. The cationic polymers polyethyleneimines (PEIs) further offer the advantages of being cost-effective, sim- ple to use, non-cytotoxic, and stable (Boussif et al., 1995). Polyethyleneimines polymers are available in linear and branched forms with various molecular weights and poly- dispersities. The most important characteristic of PEIs con- sists of their cationic charge density due to the presence of a potentially protonable amino nitrogen at every third atom which may participate in DNA condensation. These pro- Correspondence to: Yves Durocher © 2003 Wiley Periodicals, Inc.