560. Optimization of E. coli Fermentation for Plasmid DNA Production

Abstract

Bacterial plasmids are the vectors of choice for DNA vaccines and gene therapeutics. Growing plasmid DNA by microbial fermentation (E. coli) is usually combined with alkaline lysis/chromatography methods of purification. As gene therapy and DNA vaccines advance to FDA approval, it is essential to devise industrial processes whereby DNA can be economically manufactured not just at the gram scale, but at the kilogram scale and beyond. To date, optimal plasmid fermentation media and processes result in yields of 100-200mg plasmid DNA/liter of culture medium, using standard high copy pUC origin containing plasmids. In order to address this initial and yield-limiting upstream step, we developed plasmid media, and identified novel fermentation control parameters, for both batch and fed-batch fermentation. The resulting batch and fed-batch fermentation strategies significantly increase specific plasmid yield with respect to cell mass while enhancing plasmid integrity and maintaining supercoil content. Fed-batch fermentation productivity exceeding 1000mg plasmid DNA/L fermentation media has been obtained with pUC origin containing plasmids. This five to ten fold increase in plasmid yield dramatically decreases plasmid manufacturing costs, and improves the effectiveness of downstream purification by reducing the fraction of impurities.