Smith, Martin P.; (1997) An evaluation of expanded bed absorption for the recovery of proteins from crude feedstocks. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The initial stages of protein recovery often involve the processing of feedstocks containing particulate matter such as whole cells, or cell debris. Conventional isolation processes are complicated by the need to remove such particulate matter before application to packed chromatography beds. Expanded bed adsorption offers the potential for the direct adsorption of proteins in the presence of particulate material, thereby eliminating the requirement for extensive clarification operations upstream of the adsorption step. This thesis examines the application of the expanded bed approach for the direct recovery of a labile protein in the presence of cell debris and compares this with conventional approaches based on clarification prior to packed bed chromatography. Expanded bed adsorption techniques were evaluated at laboratory scale for the recovery of a generic soluble intracellular protein. Alcohol dehydrogenase from S.cerevisiae cell homogenate suspensions. Several mechanisms of interaction including ion-exchange and hydrophobic interaction were investigated for the recovery of ADH in small packed beds using clarified feedstocks. Results indicated that the hydrophobic matrix Phenyl Sepharosetm™ 6 FF gave the best chromatographic performance. A prototype hydrophobic matrix for use within expanded beds, STREAMLINE™ Phenyl was then investigated for direct adsorption of ADH from unclarified cell homogenates. The expanded beds hydrodynamics were characterized through bed expansion and residence time distribution tests. Results indicated that fluid flow prevailing within the expanded bed approximated to plug flow resulting in good adsorption performance. The expanded bed recovery route was compared at lab-scale with conventional routes based on packed beds which included polyethyleneimine flocculation, ammonium sulphate fractional precipitation and centrifugation to provide clarified material to the adsorption step. Comparative studies indicated that the expanded bed route gave higher process yields and reduced process times. However, the presence of cell debris was found to reduce the capacity of the matrix for ADH resulting in the need for large volumes of matrix and excessive buffer consumption when compared with traditional packed bed routes. These findings were confirmed at pilot-plant scale using processes based on 0.2m diameter columns.

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