Willoughby, Nicholas Allen; (2000) Expanded bed adsorption: a study of bed behaviour during the recovery of a typical bioproduct. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The majority of work in this Thesis will investigate the use of expanded beds for different separations in the field of Biotechnology and consider how best to utilise expanded beds most efficiently and effectively. The aims of the project were to develop a novel affinity purification from initial method scouting to pilot plant expanded bed level, and then to consider and develop a more effective and faster method of monitoring and control of and expanded bed than traditionally used off-line manual techniques. A protocol was successfully developed for the purification of yeast alcohol dehydrogenase (ADH) from unclarified homogenate using immobilised metal ion affinity chromatography in a STREAMLINETM expanded bed adsorption system, demonstrating high levels of recovery of the target protein. A novel system of breakthrough monitoring and control was developed using a combination of in-bed sampling and a rapid stopped flow analysis system. This enabled accurate control of product breakthrough at different levels when purifying ADH from crude homogenate feedstock. In addition this monitoring technique demonstrated variation in matrix utilisation with axial position, providing scope for the final section of work noted here. In-bed sampling also allowed the physical properties of the matrix to be studied and experimental radial and axial particle size distributions during expansion were obtained, along with the development of a method for measuring voidage in short sections of the bed under expansion. Finally, matrix utilisation was studied using segregated beds of varying particle size ranges in order to establish dynamic binding capacities and hence matrix productivity. Determination of dynamic capacities at different breakthrough levels for beds consisting of smallest and largest particles showed that, while smaller particles have more than double the total binding capacity of larger particles, at low breakthrough levels the larger particles have a higher dymanic binding capacity and hence a higher productivity.

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