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Biotechnology and Applied Biochemistry (2003) 37, (83–90) (Printed in Great Britain)

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Effects of fermentation strategy on the characteristics of plasmid DNA production

Ronan D. O'Kennedy*¹, John M. Ward† and Eli Keshavarz-Moore*²

*Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K., and †Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.

Key words: downstream processing, fed-batch, gene processing, gene therapy.

Abbreviations used: gDNA, genomic DNA; pDNA, plasmid DNA; OC-pDNA, open circular pDNA; SC-pDNA, supercoiled pDNA; V(T)-pDNA, volumetric (total) pDNA; S(T)-pDNA, specific (total) pDNA; OUR, O₂ uptake rate; CER, CO₂ evolution rate; SDCAS, synthetic defined (SD) medium (for composition, see the text) with casamino acids (CAS); LB, Luria–Bertani; X-Gal, 5-bromo-4-chloroindol-3-yl b-D-galactopyranoside.

¹Present address: GlaxoSmithKline, Beckenham, Kent, BR3 3BS, U.K.

²To whom correspondence should be addressed (e-mail e.keshavarz-moore@ucl.ac.uk).

The synthesis of supercoiled plasmid DNA (SC-pDNA) for therapeutic use will involve large-scale production in bioreactors. The success of these fermentations will be dependent on the interactions between the host organism, the recombinant plasmid vector and the growth environment. In the present study, the recombinant host, Escherichia coli DH5a bearing the recombinant plasmid pSVb, was grown in shake flasks, batch and exponentially fed-batch bioreactors. Specific and volumetric pDNA yields were increased 8- and 25-fold respectively using exponentially fed-batch cultures in comparison with shake-flask cultures. The percentage of SC-pDNA as a proportion of total plasmid DNA decreased over time in batch cultures, but remained relatively constant during fed-batch cultures. The relative merits of different modes of fermentation and their effects on the quality of alkaline lysate extracts of pDNA with respect to genomic contamination and the percentage of SC-pDNA are discussed.

Received 16 October 2002/12 December 2002; accepted 17 December 2002

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