Towards the design of a scalable and commercially viable technique for plasmid purification using a methacrylate monolithic stationary phase

Abstract

AbstractA monolithic stationary phase was prepared via free radical co‐polymerization of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalized with 2‐chloro‐N,N‐diethylethylamine hydrochloride (DEAE‐Cl) for anion‐exchange purification of plasmid DNA (pDNA) from clarified lysate obtained from E. coli DH5α‐pUC19 culture in a ribonuclease/protease‐free environment. Characterization of the monolithic resin showed a porous material, with 68% of the pores existing in the matrix having diameters above 300 nm. The final product isolated from a single‐stage 5 min anion‐exchange purification was a pure and homogeneous supercoiled (SC) pDNA with no gDNA, RNA and protein contamination as confirmed by ethidium bromide agarose gel electrophoresis (EtBr‐AGE), enzyme restriction analysis and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. This non‐toxic technique is cGMP compatible and highly scalable for production of pDNA on a commercial level. Copyright © 2007 Society of Chemical Industry