Impact of clarification strategy on chromatographic separations: Pre-processing of cell homogenates
Article first published online: 11 FEB 2008
Copyright © 2008 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 100, Issue 5, pages 941–949, 1 August 2008
How to Cite
Bracewell, D.G., Boychyn, M., Baldascini, H., Storey, S.A., Bulmer, M., More, J. and Hoare, M. (2008), Impact of clarification strategy on chromatographic separations: Pre-processing of cell homogenates. Biotechnol. Bioeng., 100: 941–949. doi: 10.1002/bit.21823
- Issue published online: 12 JUN 2008
- Article first published online: 11 FEB 2008
- Accepted manuscript online: 11 FEB 2008 12:00AM EST
- Manuscript Accepted: 14 JAN 2008
- Manuscript Revised: 10 JAN 2008
- Manuscript Received: 1 NOV 2007
- hydrophobic interaction;
This research focused on how the extent and type of primary solid–liquid separation can affect the performance of guard filtration and chromatography, in this instance hydrophobic interaction chromatography. The system used in the study was yeast (Saccharomyces cerevisiae) with the target molecule being an intracellular protein; alcohol dehydrogenase (ADH). As expected, loading more poorly clarified suspensions (both centrates and primary filtrates) required proportionally larger guard filtration areas. In addition for feed suspensions prepared by centrifugation, increased clarification led to greater column capacity. However, where filtration was used to achieve similar clarification considerably lower column capacity was achieved. These results were attributed to centrifugation leading to the aggregation of lipids and their subsequent removal in this form before application to the column. Clarification by filtration leaves such lipids in their original “soluble” state and hence they are not removed. The importance of the need to examine such interactive effects in bioprocess studies is discussed. This observation was confirmed with further analytical work into the nature of the aggregated material formed in the supernatant under centrifugation conditions. This material was only soluble in an organic solvent, and identified as phophatidylcholine and ergosterol as among the components removed by centrifugation and guard filtration as opposed to filtration and guard filtration. Biotechnol. Bioeng. 2008;100: 941–949. © 2008 Wiley Periodicals, Inc.