Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti‐mitotic agents

Abstract

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.

Iron (III) citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium

“…PEI condenses DNA into particulate complexes, which are then believed to be taken up by the cell through endocytosis (Kopatz et al 2004). Currently, most reports of transient transfection using PEI have used either HEK 293 or Chinese hamster ovary (CHO) cells (Derouazi et al 2004;Schlaeger and Christensen 1999;Tait et al 2004). It appears that higher titers may be achieved using 293 cells, due to the ability of these cells to utilize the EBNA protein for replicating plasmids (Shen et al 1995).…”

Development of a generic transient transfection process at 100 L scale

“…Although scalable transient expression using CHO cells is frequently reported (Derouazi et al 2004;Tait et al 2004;Kunaparaju et al 2005;Galbraith et al 2006) the human embryonic kidney 293 cell line (HEK293) is the most widely used cell line for transient protein expression. HEK293 cells readily take up DNA using various gene transfer vehicles such as calcium phosphate (Jordan et al 1998;Meissner et al 1999;Girard et al 2001;Meissner et al 2001;Durocher et al 2002;Baldi et al 2005), XtremeGENE Ro1539 (Schlaeger et al 1998;Schlaeger et al 2003) and PEI (Schlaeger and Christensen 1999;Durocher et al 2002;Pham et al 2003;Schlaeger et al 2003;Baldi et al 2005).…”

Effect of addition of ‘carrier’ DNA during transient protein expression in suspension CHO culture

“…PEI/DNA complexes were formed at a N/P molar ratio of 10 for all transfections as it has been shown to be the optimal ratio for transfection of CHO-S cells adapted to grow in CHO-S-SFM II medium (Tait et al 2004). We observed decreased cell viability at higher N/P ratios.…”

“…DNA:PEI complex was allowed to form in 150 mM NaCl at a N:P ratio of 10:1, which has previously been reported to be optimum for transfection of CHO cells (Tait et al 2004;Tait et al 2005). Figure 1a shows that DNA:PEI complex size increased significantly with time in CD-CHO medium indicating aggregation behavior.…”

“…PEI effectively transfers DNA to the nucleus because of its natural buffering capacity which helps in endosomal escape (Boussif et al 1995;Godbey et al 2000). Both linear (Derouazi et al 2004) and branched PEI (Tait et al 2004) have been reported for transient expression. In this work we have restricted our analysis to the use of branched PEI as a delivery agent.…”