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Biotechnology and Applied Biochemistry (2003) 37, (207–218) (Printed in Great Britain)

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Review article

Scaleable processes for the manufacture of therapeutic quantities of plasmid DNA

Parviz Ayazi Shamlou¹

Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.

Key words: biomanufacturing of therapeutic genes, non-viral gene therapy, therapeutic plasmid DNA.

Abbreviations used: cGMP, current Good Manufacturing Practice; chDNA, chromosomal DNA; CTAB, cetyltrimethylammonium bromide; CCC, counter-current chromatography; EFA, effective filtration surface area; EU, endotoxin unit; FDA, Food and Drug Administration; ICH, International Conference on Harmonization; NMMCO, nominal molecular-mass cut-off; PEG, poly(ethylene glycol); pDNA, plasmid DNA; TMP, transmembrane pressure; UF, ultrafiltration.

¹e-mail p.shamlou@ucl.ac.uk

The need for scaleable processes to manufacture therapeutic plasmid DNA (pDNA) is easy to overlook when attention is focused primarily on vector design and establishment of early clinical results. pDNA is a large molecule and has properties that are similar to those of the contaminating chromosomal DNA. These, combined with the low initial concentration of plasmids in the host cell, provide unique process challenges that require significant upfront design to establish robust manufacturing processes that can also comply with current Good Manufacturing Practice (‘cGMP’) and produce milligram-to-kilogram quantities of pDNA product. This review describes promising scaleable processes that are currently being assessed for production of therapeutic supercoiled pDNA. Fermentation strategies for improving supercoiled plasmid yield and reducing contaminant concentrations are reviewed, and downstream processes are assessed for their ability to efficiently remove cellular contaminants, separate the supercoiled form of the pDNA from its open circular and linear forms, and prepare the purified drug substance for formulation. Current strategies are presented for developing stable delivery systems, and approaches to quality assurance and quality control are discussed.

Received 15 January 2003/27 March 2003; accepted 28 March 2003

Published as Immediate Publication 28 March 2003, DOI 10.1042/BA20030011

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