Differential interactions of plasmid DNA, RNA and endotoxin with immobilised and free metal ions

Separation of negatively charged molecules, such as plasmid DNA (pDNA), RNA and endotoxin forms a bottleneck for the development of pDNA vaccine production process. The use of affinity interactions of transition metal ions with these molecules may provide an ideal separation methodology. In this study, the binding behaviour of pDNA, RNA and endotoxin to transition metal ions, either in immobilised or free form, was investigated. Transition metal ions: Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺ and Fe³⁺, typically employed in the immobilised metal affinity chromatography (IMAC), showed very different binding behaviour depending on the type of metal ions and their existing state, i.e. immobilised or free. In the alkaline cell lysate, pDNA showed no binding to any of the IMAC chemistries tested whereas RNA interacted significantly with Cu²⁺-iminodiacetic acid (IDA) and Ni²⁺-IDA but showed no substantial binding to the rest of the IMAC chemistries. pDNA and RNA, however, interacted to varying degrees with free metal ions in the solution. The greatest selectivity in terms of pDNA and RNA separation was achieved with Zn²⁺ which enabled almost full precipitation of RNA while keeping pDNA soluble. For both immobilised and free metal ions, ionic strength of solution affected the metal ion-nucleic acid interaction significantly. Endotoxin, being more flexible, was able to interact better with the immobilised metal ions than the nucleic acids and showed binding to all the IMAC chemistries. The specific interactions of immobilised and/or free metal ions with pDNA, RNA and endotoxin showed a good potential, by selectively removing RNA and endotoxin at high efficiency, to develop a simplified pDNA purification process with improved process economics.