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Nucleic Acids Research 2004 32(16):e129; doi:10.1093/nar/gnh124
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Published online 9 September 2004

Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved

Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed

Michael J. Molloy, Valerie S. Hall, Susannah I. Bailey, Katharine J. Griffin, Jim Faulkner and Mark Uden*

Department of Analytical Sciences, Biopharmaceutical Centre of Excellence for Drug Discovery (Beckenham), GlaxoSmithKline, Beckenham, Kent BR3 3BS, UK

* To whom correspondence should be addressed. Tel: +44 0 20 8639 61; Fax: 44 0 20 8639 618; Email: mark.x.uden{at}gsk.com
Present address: Susannah I. Bailey, Molecular Immunology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK

Received July 8, 2004; Revised and Accepted August 19, 2004

Within the biopharmaceutical industry, recombinant plasmid DNA is used both as a raw material (e.g. in lentiviral and AAV vector production) as well as an active ingredient (e.g. in DNA vaccines). Consequently, many analytical laboratories are routinely involved with plasmid DNA topoisoform qualitative analysis and quantification. In order to reliably determine plasmid topology, one must ensure that the methodology employed can reliably, precisely and accurately measure qualitatively and quantitatively all topological isoforms. Presented here are an anion-exchange high-performance liquid chromatography (AEC) and an agarose gel electrophoresis (AGE)-based method developed for this purpose. The strategies undertaken to overcome the respective typical problems of limited linear range of quantitation (for AGE) and isoform resolution (for AEC) are described. Also presented is a subsequent direct comparison (for assay precision/accuracy) of these two methods, as well as a package of species characterization [by chloroquine-AGE, enzymatic digestion, multi-angle laser light-scattering (MALLS) and electron microscopy] undertaken to confirm the identity of a minor supercoiled dimeric concatamer observed by both approaches.


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