BACKGROUND: Transient gene expression (TGE) provides a rapid way to generate recombinant protein biologics for pre-clinical assessment. Human embryonic kidney (HEK293) cells have traditionally been used for TGE; however, there is demand from industry for efficient, high-producing TGE systems that utilize Chinese hamster ovary (CHO) cells. A polyethyleneimine (PEI)-based TGE process has been developed for CHO cells using an episomal expression system to generate enhanced recombinant protein titers. RESULTS: A five-fold improvement in monoclonal antibody (mAb) volumetric productivity was achieved by examining key parameters including transfection medium, cell density, transfection reagent, DNA: reagent ratio, the time of transfer to mild hypothermia and feeding strategy post-transfection. The Epi-CHO system allowed for a six-fold expansion in culture volume post-transfection without significantly affecting specific productivity. This system generates 400% more mAb per mu g of plasmid DNA when compared with a non-episomal system. In addition, the use of X-box binding protein 1 to enhance secretion capacity and provide further improvements in mAb production with TGE was investigated. CONCLUSION: Through optimization of key parameters, our results demonstrate the development of a low-cost, high-yielding, episomal TGE system that may be adopted during pre-clinical biologic drug development. (C) 2011 Society of Chemical Industry