Superporous agarose anion exchangers for plasmid isolation

Abstract

Superporous agarose beads have wide, connecting flow pores allowing large molecules such as plasmids to be transported into the interior of the beads by convective flow. The pore walls provide additional surface for plasmid binding thus increasing the binding capacity of the adsorbent. Novel superporous agarose anion exchangers have been prepared, differing with respect to bead diameter, superpore diameter and type of anion-exchange functional group (poly(ethyleneimine) and quaternary amine). The plasmid binding capacities were obtained from breakthrough curves and compared with the binding capacity of homogeneous agarose beads of the same particle size. Significantly, the smaller diameter superporous agarose beads were found to have four to five times higher plasmid binding capacity than the corresponding homogeneous agarose beads. The experimentally determined plasmid binding capacity was compared with the theoretically calculated surface area for each adsorbent and fair agreement was found. Confocal microscopy studies of beads with adsorbed, fluorescently labelled plasmids aided in the interpretation of the results. Superporous poly(ethyleneimine)-substituted beads with a high ion capacity (230 μmol/ml) showed a plasmid binding of 3–4 mg/ml adsorbent. Superporous quaternary amine-substituted beads had a lower ion capacity (81 μmol/ml) and showed a correspondingly lower plasmid binding capacity (1–2 mg/ml adsorbent). In spite of the lower capacity, the beads with quaternary amine ligand were preferred, due to their much better plasmid recovery (70–100% recovery). Interestingly, both capacity and recovery was improved when the plasmid adsorption step was carried out in the presence of a moderate salt concentration. The most suitable superporous bead type (45–75 μm diameter beads; 4 μm superpores; quaternary amine ligand) was chosen for the capture of plasmid DNA from a clarified alkaline lysate. Two strategies were evaluated, one with and one without enzymatic digestion of RNA. The strategy without RNase gave high plasmid recovery, quantitative removal of protein and a 70% reduction in RNA.