Harnessing metal ion affinity for the purification of plasmid DNA

The recent surge in plasmid DNA (pDNA) vaccine research has generated demand for an efficient large-scale pDNA purification process. In this study, three process intensification strategies harnessing the interactions of pDNA, RNA and endotoxin with immobilised and/or free transition metal ions were investigated for the purification of plasmid pcDNA3.1D, containing dengue fever antigenic gene (NS3), from the alkaline cell lysate. In the first process scheme, alkaline cell lysate was applied to Cu^{2 +}-iminodiacetic acid (IDA) for simultaneous removal of RNA and endotoxin. Subsequent addition of CuCl₂ to the supernatant afforded selective precipitation of pDNA, resulting in substantially purified pDNA at an overall recovery yield of 92% with significant reduction in RNA (96%) and endotoxin (> 99 %). In the second process scheme, pDNA, RNA and endotoxin were first isolated from other impurities in alkaline cell lysate by CuCl₂ precipitation. Addition of EDTA to the precipitated pellets selectively solubilised pDNA and RNA while endotoxin remained insoluble. Subsequent application of solubilised pDNA and RNA to Cu^{2 +}-IDA resulted in highly purified pDNA with almost complete removal of RNA and any residual endotoxin (∼ 100 % pDNA recovery, ∼ 100 % removal of RNA and endotoxin). In the third process scheme, RNA and endotoxin were first removed from alkaline cell lysate by ZnCl₂ precipitation. pDNA in the supernatant was then recovered by CuCl₂ precipitation (64% pDNA recovery, 86% RNA and > 99 % endotoxin removal). In essence, the combination of metal ion (CuCl₂) precipitation, followed by immobilised metal affinity chromatography (IMAC) employing Cu^{2 +}-IDA, provides a great potential to achieve significant intensification of pDNA purification process with improved efficiency and throughput.