The Effect of Operating Parameters and Matrix Properties on the Productivity of an Expanded Bed Adsorption Column
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THE EFFECT OF OPERATING PARAMETERS AND MATRIX PROPERTIES ON THE PRODUCTIVITY OF AN EXPANDED BED ADSORPTION COLUMN A thesis submitted for the degree of ENGINEERING DOCTORATE by Philippa Jola Gardner September 2005 The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London Torrington Place London WC1E 7JE UMI Number: U592018 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U592018 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 A b s t r a c t Expanded bed adsorption (EBA) combines clarification, concentration and purification into a single processing step reducing processing time and increasing productivity. Much work has been conducted on model proteins but little attention has been paid to the emerging issues of tailoring the operational variables of matrix size, operating flowrate and ligand type so as to maximise process outputs. The aims of this study were to investigate the effects of matrix properties and operating parameters on breakthrough behaviour and productivity. Second generation matrices designed to operate at high flowrates were also examined. Operating at low velocities, beds formed from smaller particles had a shallower breakthrough than beds formed from large particles but the latter were more productive. At the higher velocities typically utilised in EBA, the behaviour of beds formed from either small or large particles was comparable in terms of breakthrough with the beds made up of small particles being slightly more productive than those containing large particles. A prototype 2nd generation EBA matrix consisting of a multi-modal ligand that could operate at high conductivity levels typical of fermentation broths was also examined. It was found that the productivity could be increased 5-fold as compared with commercially available matrices, with no loss of yield or purity. Conventional breakthrough curve analysis is not applicable in situations of variable particle size and operating velocity. Results using a novel dimensionless group to facilitate comparison between unlike systems is presented. 2 Abstract The thesis presents an analysis of the relative productivity of a range of systems and highlights the gains in terms of productivity to be achieved with the development of 2nd generation EBA multi-modal adsorbents. The thesis concludes with an analysis of the commercial and regulatory aspects of EBA. 3 A cknowledgements Firstly, I would like to thank my supervisor Prof. Nigel Titchener-Hooker for all of his support, enthusiasm, advice and encouragement that made this work possible. I also owe a great deal of thanks to Dr Nik Willoughby for his technical expertise and help over the course of my studies. This project would not have been possible without the support from Amersham Biosciences and their generous provision of equipment and matrices. In particular I would like to thank Rolf Hjorth for providing me with the opportunity to visit and work with them in Sweden, but also for his continued advice and guidance throughout this project. The work and support of the technical and pilot plant staff within the Biochemical Engineering department at University College is gratefully acknowledged. Finally, to my friends, colleagues and family that have helped me through this whole experience - I couldn't have done it without you. 4 C o n t e n t s A b s t r a c t ..................................................................................................................................... 2 A cknowledgements .............................................................................................................4 C o n t e n t s ......................................................................................................................................5 List o f T a b l e s ......................................................................................................................... 11 List o f Fig u r e s .......................................................................................................................13 A bbreviations .......................................................................................................................18 1 Introduction .............................................................................................................. 21 1.1 Introduction to the Engineering Doctorate .....................................21 1.2 Introduction to Expanded Bed Adsorption.....................................22 1.3 Traditional Processing Steps...............................................................22 1.4 General Chromatographic Theory.................................................... 23 1.4.1 Ion Exchange Chromatography................................................24 1.4.2 Hydrophobic Interaction Chrom atography........................... 25 1.5 Expanded Bed A dsorption................................................................ 27 1.5.1 STREAMLINE Expanded Bed Products................................. 29 1.5.1.1 STREAMLINE A dsorbents ....................................................30 1.5.1.2 STREAMLINE H ardw are ......................................................32 1.5.2 Operation.......................................................................................34 1.5.2.1 Matrix Preparation..................................................................34 1.5.2.2 Bed Expansion and Equilibration........................................ 34 1.5.2.3 Adsorption of Sample.............................................................35 1.5.2.4 W ashing .....................................................................................36 1.5.2.5 Elution ........................................................................................36 1.5.2.6 Regeneration ............................................................................ 37 1.6 Recent Developments - STREAMLINE Direct Expanded Bed Products ............................................................................................................ 37 1.6.1 STREAMLINE Direct Adsorbents ............................................38 1.6.2 STREAMLINE Direct H ardw are ...............................................38 1.7 Conclusions........................................................................................... 39 5 Contents 2 Materials and M ethods ..................................................................................... 40 2.1 Bacterial Cell Growth.........................................................................40 2.1.1 Cell Culture Media .....................................................................40 2.1.1.1 N utrient Broth N o.2 ............................................................... 40 2.1.1.2 Terrific Broth............................................................................40 2.1.1.3 High Cell Density Media.........................................................41 2.1.2 Glycerol stock preparation and m aintenance........................42 2.1.3 Shake Flask Ferm entation ......................................................... 42 2.1.4 5L Ferm entation ..........................................................................42 2.2 Downstream Processing....................................................................44 2.2.1 Cell D isruption............................................................................44 2.2.1.1 Sonication................................................................................ 44 2.2.1.2 Hom ogenisation......................................................................44 2.2.2 Clarification................................................................................. 44 2.2.2.1 Bench top centrifugation.......................................................44 22.2.2 Lab scale centrifugation .........................................................44 2.3 Biological Systems.............................................................................. 45 2.3.1 Alcohol Dehydrogenase/Yeast................................................ 45 2.3.1.1 Feedstock Preparation...........................................................45 2.3.1.2 Chromatography Details........................................................45 2.3.1.3 Clean-in-Place Procedure .......................................................46 2.3.1.4 Residence Time Determ ination........................................... 46 2.3.2 Glutathione S-Transferase/£. coli........................................... 46 2.3.2.1 Feedstock Preparation...........................................................46 2.3.2.2 Chromatography Details........................................................47 2.3.2.3 Clean-in-Place Procedure .......................................................48 2.3.2.4 Residence Time Determ ination............................................48 2.3.3 Bovine Serum A lbum in/ Buffer .................................................48 2.3.3.1 Feedstock Preparation...........................................................48