Abstract for presentation at Chemeca 2007

Therapeutic plasmid DNA (pDNA) manufacture is evolving from the molecular biologist’s laboratory to become a process engineering challenge in response to the escalating demand for pDNA as a vector in pre-clinical and clinical trials for gene medicine and DNA vaccines. For this reason, current pDNA purification methodologies are geared towards obtaining scalable and commercially viable techniques employing continuous monolithic stationary supports which are widely acknowledged to offer better separation hydrodynamics and scalable feasibilities than the orthodox beaded or particle-oriented adsorbent materials and other membrane-based sorbents. Monolithic stationary phases offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance.
A monolithic sorbent was synthesised via free radical copolymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with 2-Chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) for anion-exchange purification of pDNA from clarified lysate obtained from E. coliDH5α-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 minutes anion-exchange purification was a pure and homogenous SCpDNA with no gDNA, RNA and protein contaminations as confirmed with EtBr-AGE, enzyme restriction analysis and SDS-PAGE. This cost effective and non-toxic technique is cGMP compatible and highly scalable for commercially-viable rapid production of pDNA.

Keywords: Plasmid DNA; Methacrylate monolith; Anion-exchange purification; Scale-up; Pore diameter