An automated microscale chromatographic purification of virus-like particles as a strategy for process development.

Full Abstract

The development of fermentation processes for recombinant vaccines requires optimizing expression while maintaining high product quality. Changes to cell fermentation conditions are typically evaluated following cell disruption, with expression levels quantified by immunoassay, liquid chromatography or enzyme activity. However, assay titres do not always predict the effects that intracellular aggregation, proteolysis, post-translational modifications and differences in relative impurity levels can have on purification yield and product purity. Furthermore, heterogeneity in the size and surface properties inherent in viral particles makes unit operations such as chromatography less predictable. In these cases, the purification procedure (or a mimic thereof) must be carried out to give accurate information on the impact of changes in fermentation conditions on purification process performance. This was demonstrated for the development of a recombinant vaccine against human papillomavirus produced in Saccharomyces cerevisiae, where the most informative feedback on fermentation variables was obtained by completing a multistep chromatographic purification to evaluate process yield and product purity. To increase the purification throughput and reduce labour, the chromatography was miniaturized 1000-fold from the laboratory scale using microlitre volumes of adsorbent in a pipette tip and automated on a robotic workstation. The microscale purification is shown to be predictive of the laboratory-scale purification in terms of yield and purity, while providing over a 10-fold increase in throughput and allowing for increased monitoring of fermentation processes. In addition, by reducing the volume of cells needed for this assessment, the fermentation can be correspondingly reduced in scale and carried out in parallel for additional throughput gains.

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Author information

Author/s: Wenger, Marc D (MD); Dephillips, Peter (P); Price, Colleen E (CE); Bracewell, Daniel G (DG);

Affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, UK. marc_wenger(-atsign-)merck.com

Journal and publication information

Publication Type: Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't

Journal: Biotechnology and applied biochemistry (Biotechnol Appl Biochem), published in England. (Language: eng)

Reference: 2007-Jun; vol 47 (issue Pt 2) : pp 131-9

Dates: Created 2007/05/11; Completed 2007/06/19;

PMID: 17311568, status: MEDLINE (last retrieval date: 12/26/2008)

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Chromatography, Liquid - methods
Microfluidics - methods
Miniaturization
Papillomaviridae - genetics; metabolism
Papillomavirus Vaccines - isolation & purification

Papillomavirus Vaccines - isolation & purification
Recombinant Proteins - isolation & purification; therapeutic use
Saccharomyces cerevisiae - virology
Virion - isolation & purification; metabolism
Virus Cultivation - methods

Associated Chemicals: Papillomavirus Vaccines (0) ; Recombinant Proteins (0)

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