The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA.
The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5alpha-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.
Danquah
Michael K
MK
BEL (Bio Engineering Laboratory), Department of Chemical Engineering, Monash University, Wellington Road, Melbourne 3800, Australia.
Forde
Gareth M
GM
D002852:Chromatography, Ion Exchange; D004983:Ethanolamines / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008689:Methacrylates / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D015394:Molecular Structure; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*; D011108:Polymers / Q000737:chemistry*; D015203:Reproducibility of Results
D004983:Ethanolamines; D008689:Methacrylates; D011108:Polymers; C007369:2-diethylaminoethanol