A mass balance study to assess the extent of contaminant removal achieved in the operations for the primary recovery of plasmid DNA from Escherichia coli cells

  1. Laura A. S. Ciccolini,
  2. Parviz Ayazi Shamlou,
  3. Nigel Titchener–Hooker*

Article first published online: 23 JAN 2002

DOI: 10.1002/bit.10166

Issue

Biotechnology and Bioengineering

Biotechnology and Bioengineering

Volume 77, Issue 7, pages 796–805, 30 March 2002

Additional Information

How to Cite

Ciccolini, L. A. S., Shamlou, P. A. and Titchener–Hooker, N. (2002), A mass balance study to assess the extent of contaminant removal achieved in the operations for the primary recovery of plasmid DNA from Escherichia coli cells. Biotechnol. Bioeng., 77: 796–805. doi: 10.1002/bit.10166

Author Information

  1. The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE UK; telephone: 44-0-207-679-3796; fax: 44-0-207-383-2348

*The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE UK; telephone: 44-0-207-679-3796; fax: 44-0-207-383-2348

Publication History

  1. Issue published online: 23 JAN 2002
  2. Article first published online: 23 JAN 2002
  3. Manuscript Accepted: 16 OCT 2001
  4. Manuscript Received: 17 APR 2000

Funded by

  • Biotechnology and Biological Sciences Research Council

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Keywords:

  • alkaline lysis operation;
  • mass balance;
  • contamination;
  • shear rate;
  • neutralization

Abstract

Mass balances were performed on an alkaline lysis operation for the primary recovery of supercoiled plasmid DNA as part of a process for plasmid gene preparation. Escherichia coli DH5α/pSVβ was cultured in defined medium by fed-batch fermentation and harvested at the end of the exponential phase. Alkaline lysis of the recombinant cells was performed at fixed shear rates ranging between 46 and 461 s−1, with neutralization 100 and 300 s after the initiation of the lysis. Mass balance calculations were used to optimize the operating conditions for carrying out the alkaline lysis operation. The results indicated that a plasmid yield of 75% and purity with respect to total DNA of 60% were achievable during the primary recovery operation. The influences of key contaminants, including the soluble proteins and the suspended solids, as they bear on the subsequent purification operations, were evaluated and discussed. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 796–805, 2002; DOI 10.1002/bit.10166

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