Plasmid DNA purification using divalent alkaline earth metal ions and two anion exchangers

摘要

A process is provided for large scale purification of plasmid DNA to obtain plasmid DNA of pharmaceutical grade. Steps of the process include lysing cells such as by alkaline lysis to obtain a lysate containing plasmid DNA, chromosomal DNA and RNA, precipitating most of the RNA and Chromosomal DNA by adding divalent alkaline earth metal ions such as provided by calcium chloride, removing the precipitated RNA and chromosomal DNA, and contacting the lysate with an anion exchange matrix to obtain purified plasmid DNA. The anion exchange matrix may be cross-linked agarose containing anion exchange groups bound to the matrix via an extender such as dextran. The anion exchange groups may be quaternary ammonium groups. The purified plasmid DNA may be further contacted with an anion exchange matrix such as hydrophilyzed polystyrene/divinyl benzene substituted with quaternary ammonium groups to obtain purified plasmid DNA of gene therapy grade.