Tuning plasmid DNA amounts for cost-effective transfections of mammalian cells: when less is more

Transient gene expression (TGE) in mammalian cells is a well-known approach to the fast expression of recombinant proteins. The human cell line HEK (human embryonic kidney) 293F is widely used in this field, due to its adaptability to grow in suspension to high cell densities in serum-free media, amenability to transfection, and production of recombinant proteins in satisfactory quantities for functional and structural analysis. Amounts of plasmid DNA (pDNA) required in transfections for TGE remain high (usually 1 µg pDNA/mL, or even higher), representing a noticeable proportion of the overall cost. Thus, there is an economic need to reduce amounts of coding pDNA in TGE processes. In this work, amounts of both pDNA and transfecting agent used for TGE in HEK 293F cells have been explored in order to reduce them without compromising (or even improving) the productivity of the process in terms of protein yield. In our hands, minimal polyethyleneimine (PEI) cytotoxicity and optimum protein yields were obtained when transfecting at 0.5 µg pDNA/mL (equal to 0.5 µg pDNA/million cells) and a DNA-to-PEI ratio of 1:3, a trend confirmed for several unrelated recombinant proteins. Thus, carefully tuning pDNA and transfecting agent amounts not only reduces the economic costs but also results in higher recombinant protein yields. These results surely have a direct application and interest for the biopharmaceutical industry, always concerned in increasing productivity while decreasing economic costs. KEY POINTS: • Mammalian cells are widely used to produce recombinant proteins in short times. • Tuning DNA and transfecting agent are of great interest to optimize economic costs. • Reducing DNA and transfecting agent amounts result in higher protein yields.