Transient Transfection of Rolling‐Circle Amplified DNA in Biomanufacturing‐Relevant Mammalian Cell Lines: A Comparison of Transfection Conditions for Optimal Protein Expression

Rolling-circle amplification (RCA) is a completely synthetic and rapid mechanism for scaling-up bulk DNA that eliminates requirements for large-scale bacterial fermentation. Several reports have described the surprising ability of large unprocessed (hyperbranched) rolling circle-amplified DNA (RCA DNA) to effectively transfect cultured cells; however, to our knowledge a comprehensive analysis of transfection conditions has not been conducted for this unique type of large synthetic DNA. Herein, we present comparative transfection data for two of the most common mammalian cell types used in biopharmaceutical manufacturing (suspension Chinese hamster ovary CHO cells and adherent HEK293 cells) and a panel of commercial transfection reagents that represent three general compositions (i.e., cationic lipid, cationic polymer, and complex blends). We show the efficiency of RCA DNA delivery is highly influenced by the transfection reagent and cell line, but expression patterns generally track with plasmid controls. Reagents comprising complex blends provide some of the best transfection results for RCA DNA, although additional reagent types work well. We also show it is possible to transfect two different RCA DNA products into CHO cells to produce monoclonal antibodies at high titer. We conclude that large and unique synthetic DNA produced by RCA is capable of being used in place of supercoiled plasmid for downstream transfection workflows.