title: The use of rapid on-line monitoring of products and contaminants from within an expanded bed to control separations exhibiting fast breakthrough characteristics and to maximize productivity.
abstract: Conventional control of expanded-bed adsorption (EBA), like that of packed-bed chromatography, is based upon off-line measurements of the column eluant. The relatively high-void volumes in EBA systems means that this approach can lead to significant performance losses caused by the inability to achieve tight control of breakthrough. This problem is made worse if the product has a fast breakthrough characteristic or if it is necessary to operate to low levels of product loss. In this article we examine the utility of constant on-line monitoring from within the expanded bed using stopped-flow analysis (SFA) to provide data for the control of the expanded-bed operation. A modified Streamline 50 column with side ports that enable sampling along the expanded axis of the bed was used. Comparisons between off-line and on-line measurements are presented, showing how the advanced monitoring method can lead to better control and to an analysis of breakthrough development within the bed. The expanded bed was used to purify alcohol dehydrogenase from homogenized suspensions of bakers' yeast. Accurate control of breakthrough to 10% of the target enzyme was achieved using a SFA control system with a response time of 40 seconds. On-line data compared well to assays carried out off-line on the outlet stream for both the product enzyme (ADH), total protein, RNA, and cell debris levels (via UV 650 nm). This information was used to generate a series of graphs with which to track the EBA process in real-time. Results showed that bed utilization was not linear along the bed axis so that, for example, 60% of ADH is bound in the bottom 33% of the column during loading.
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom.
authors.forename: N
authors.initials: N
authors.lastname: Willoughby
authors.forename: G
authors.initials: G
authors.lastname: Habib
authors.forename: M
authors.initials: M
authors.lastname: Hoare
authors.forename: R
authors.initials: R
authors.lastname: Hjorth
authors.forename: N J
authors.initials: NJ
authors.lastname: Titchener-Hooker
mesh_terms: D000426:Alcohol Dehydrogenase / Q000302:isolation & purification; D001709:Biotechnology / Q000379:methods*; D004798:Enzymes / Q000302:isolation & purification*; D005656:Fungal Proteins / Q000302:isolation & purification; D009862:Online Systems; D011506:Proteins / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D004798:Enzymes; D005656:Fungal Proteins; D011506:Proteins; D000426:Alcohol Dehydrogenase