Scalable transient protein expression.

Transient transfection is a well-established method to rapidly express recombinant proteins from mammalian cells. Accelerating activity in biotherapeutic drug development, demand for protein-based reagents, vaccine research, and large initiatives in structural and functional studies of proteins have propelled the need to generate moderate to high amounts of recombinant proteins and other macromolecules in a flexible and rapid manner. Progress over the last 10-15 years has demonstrated that transient transfections can be reliably and readily scaled up to handle milliliters to tens of liters of cells in suspension culture and obtain milligrams to grams of recombinant protein in a process that requires only days to weeks. This review will summarize developments in this field, properties of the components of a transient expression system that enable maximal protein production, and detailed protocols for this application.

Transient transfection factors for high-level recombinant protein production in suspension cultured mammalian cells.

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.

Non-viral human IL-10 gene expression reduces acute rejection in heterotopic auxiliary liver transplantation in rats.

We studied nonviral delivery, expression, and the effect of the human interleukin-10 (Hu IL-10) gene on the rat model of heterotopic auxiliary liver transplantation (HALT). Two previous pilot studies showed remarkable expression of the Hu IL-10 gene in donor and recipient rats, and a decreasing effect of acute rejection in certain cases. In this study, we focused on the efficacy of Hu IL-10 gene expression to decrease acute rejection compared with cyclosporine A (CyA) in a HALT model. Three study groups and one control group were designed. Each group consisted of 6 DA donor and 6 Lewis recipient rats, which underwent HALT. In the control group, donors and recipients were not treated at all. In group II, recipients were treated with one dose of CyA. In group III, donors were treated with Hu IL-10 plasmid. In group IV, donors were treated with Hu IL-10 plasmid, and recipients were treated with one dose of CyA. Rejection was established by histopathology: it revealed 100% rejection in control and 33.3% rejection in study groups II, III, and IV. Human IL-10 gene expression prevented acute rejection with the same efficacy as CyA in the HALT model in rats.