Plasmid DNA isoforms can be separated by both agarose gel electrophoresis and a variety of chromatographic methods, but both of these approaches have significant shortcomings in terms of scalability, throughput, and/or resolution. This study provides the first demonstration that the supercoiled, linear, and open-circular isoforms of plasmid DNA can be effectively separated based on differences in their elongational flexibility in the highly converging flow field that is established during membrane ultrafiltration. Data were obtained with plasmids from 3 to 17 kbp in size using commercially available cellulose ultrafiltration membranes with pores an order of magnitude smaller than the DNA root-mean-square radius of gyration. High-resolution separations were achieved by controlling the filtrate flux between the critical flux values required for transmission of the individual isoforms. The separation behavior in ultrafiltration was very different than that observed in size exclusion chromatography or agarose gel electrophoresis due to differences in the underlying separation mechanisms. The simplicity of the ultrafiltration process makes this approach attractive for a wide range of applications, including large-scale purification of plasmid DNA for gene therapy. (C) 2011 Elsevier Inc. All rights reserved.