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

Danquah, Michael K. & Forde, Gareth M. (2007) Towards the design of a scalable and commercially viable technique for plasmid purification using a methacrylate monolithic stationary phase. Journal of Chemical Technology and Biotechnology, 82(8), pp. 752-757.

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A 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.

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ID Code: 81567
Item Type: Journal Article
Refereed: Yes
Keywords: Anion-exchange purification, Methacrylate monolith, Plasmid DNA, Stationary phase, Ethylene glycol dimethacrylate, Glycidyl methacrylate, Plasmid, Acrylics, Copolymerization, DNA, Electrophoresis, Ethylene glycol, Free radical polymerization, Purification, 2 diethylaminoethanol, cyclic GMP, ethidium bromide, free radical, methacrylic acid, methacrylic acid derivative, polymer, protein, proteinase, resin, ribonuclease, RNA, unclassified drug, agar gel electrophoresis, anion exchange, article, biotechnological procedures, cell lysate, DNA binding, DNA purification, DNA supercoiling, DNA synthesis, elution, Escherichia coli, nonhuman, particle size, polyacrylamide gel electrophoresis, polymerization, restriction mapping
DOI: 10.1002/jctb.1733
ISSN: 0268-2575
Divisions: Current > Schools > School of Chemistry, Physics & Mechanical Engineering
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2007 Society of Chemical Industry
Deposited On: 05 Feb 2015 23:38
Last Modified: 11 Feb 2015 05:34

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