High-Performance Membrane Chromatography of Supercoiled Plasmid DNA

Abstract

Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the three-albeit isolated-bands in the agarose gel. Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (BioScale Q2, 7 x 52 mm, 10-micron porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 x 35 mm). The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.

An Introduction to Monolithic Disks as Stationary Phases for High Performance Biochromatography

“…The analysis of large plasmid molecules by HPMDC has been discussed in a recent paper by Giovannini et al [69]. A 7.2-kb predominantly supercoiled plasmid was taken as example.…”

Membrane chromatography of DNA: Conformation‐induced capacity and selectivity

“…They found that increasing the flow rate by fourfold did not affect the membrane efficiency; however, increasing the flow rates caused a loss in resolution. Further, the authors were not successful in resolving various isoforms of DNA (Giovannini et al, 1998).…”

“…These reviews discuss almost exclusively the separation of proteins on membrane adsorbents. By contrast, the body of literature addressing the chromatography of much larger DNA and virus-like molecules using membranes is scarce (Giovannini et al, 1998).…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…Together with monoliths (Bencina et al 2004;Branovic et al 2004;Jungbauer and Hahn 2004;Urthaler et al 2005), other new supports have been developed to overcome the diffusion limitation as well as to improve the binding capacity of the support for the target molecules. Such superporous supports (Gustavsson et al 1999;Tiainen et al 2007b), and adsorptive membranes (Giovannini et al 1998;Teeters et al 2003) are advanced approaches that have been shown to be able to partially solve the problems associated with low capacity.…”