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Biotechnology and Applied Biochemistry (2003) 37, (225–234) (Printed in Great Britain)

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The influence of physico-chemical and process conditions on the physical stability of plasmid DNA complexes using response surface methodology

Clare N. Mount*, Li K. Lee*, Ahmad Yasin†, Andrew Scott‡, Tom Fearn§ and Parviz Ayazi Shamlou*¹

*Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K., †GSK (GlaxoSmithKline), South Eden Park Road, Beckenham, Kent, BR3 3BS, U.K., ‡GSK, Park Road, Ware, Herts., SG12 OPD, U.K., and §Department of Statistical Science, University College London, Gower Street, London WC1E 6BT, U.K.

Key words: factorial design, plasmid DNA condensation, response surface methodology, therapeutic gene processing.

Abbreviations used: RSM, response surface methodology; OFAT, one-factor-at-a-time; pDNA, plasmid DNA; PLL, poly-L-lysine; Re, Reynolds number; PCS, photon correlation spectroscopy; PDI, polydispersity index; PSD, particle size distribution.

¹e-mail p.shamlou@ucl.ac.uk

Research is progressing fast to find safe and effective methods of delivering therapeutic genes to patients afflicted with a range of genetic and acquired diseases that either do not respond at all, or respond poorly, to treatment with small-molecule drugs or protein-replacement therapy. A technical barrier that remains relates to the need for scalable operations that can consistently and reproducibly make large quantities of the therapeutic gene vectors under the current Good Manufacturing Practice (‘cGMP’). The present investigation focuses on these issues and introduces a new method of assessing the engineering effects of process and material factors on the colloidal properties of plasmid-DNA delivery systems based on response surface methodology (RSM) and experimental techniques. Previously, experiments have shown that several factors can reduce the physical stability of non-viral delivery systems. Specifically, it has been demonstrated that the mean size and charge of plasmid DNA condensed by cationic agents are affected by many factors, including the pH and ionic strength of the buffer, and the method of preparation. For example, the method and intensity of mixing of the DNA with condensing and conjugating agents have been shown to be important. Using RSM to analyse new experimental data in the present paper, we report on the impact of these factors and, more crucially, the effects of interaction between the factors on the colloidal properties of the DNA–vector complexes. Specifically, for plasmid DNA condensed by poly-L-lysine, interactions between ionic strength, pH and DNA concentrations play a critical role. Whether poly-L-lysine should be used as a condensing agent in the final delivery system remains to be demonstrated. However, the use of RSM combined with the scaleable experimental approach described in this paper may be applied to other delivery systems.

Received 15 January 2003/27 March 2003; accepted 9 April 2003

Published as Immediate Publication 9 April 2003, DOI 10.1042/BA20030012

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