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Monolith Absorbants As a Capture Step for Virus-Like

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Monolith Absorbants As a Capture Step for Virus-Like MONOLITH ABSORBANTS AS A CAPTURE STEP FOR VIRUS-LIKE PARTICLES CLAIRE SUZANNE BURDEN Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London. WC1E 7JE Submitted to UCL for the degree of Doctor of Engineering (EngD) in Biochemical Engineering and Bioprocess Leadership September 2012 1 I, Claire Burden, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed…………………………………………………………..Date……………… 2 ABSTRACT Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. Monoliths provide an open pore structure to improve pressure drops and mass transport via convective flow. The challenging capture of a VLP from clarified yeast homogenate was used to develop a new monolith separation which found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity increased three-fold for the monolith with 90% recovery of the VLP. Confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. The lipid formed a layer on top of the column, even after column regeneration, resulting in increasing pressure drops over a number of cycles. Removal of 70% of the lipid pre-column by Amberlite/XAD-4 beads significantly reduced the fouling process. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11 mg/mL column to 0.25mg/mL column. Control of chromatographic conditions can impact the product concentration during elution. Critical parameters which influenced the concentration of measureable VLP eluted included column contact time, salt concentration in mobile phase, and inclusion of lipid. The parameters were co-dependant with a crude lipid feed loaded at low salt and extra wash time of 40 minutes causing the largest decrease of 40%. Reducing the time of contact between the column and the VLP helped reduce such adverse effects. Increasing the flow rate in the column had no effect on the elution profile with crude or reduced lipid feeds. This informs process development strategies for the future use of monoliths in vaccine bioprocessing. 3 ACKNOWLEDGEMENTS It has been a long and amazing five years since I arrived back at UCL. First of all I would like to thank my supervisor Dan Bracewell, for his guidance and help. Thanks must also go to my sponsor company BIA Separations, my industrial supervisors Aleš Podgornik and Aleš Strancar for their support and the welcome when I came to stay for a few weeks. A huge amount of thanks are due to Gaik Sui Kee and Jing Jin for welcoming me to the VLP group. Your input and guidance was invaluable. A massive thank you must go to my parents for all their support and for putting up with me going back to university once again, after they thought I’d finished. Also thank you to my brother for my very cute nephew Thomas, hopefully one day I can make him an engineer too. Over the four years I had the privilege of sharing a department with some brilliant people. Big thanks to The Colonnades and The Vineyard offices, particularly for the tea and the cake, and to everyone else in Biochemical Engineering. Thanks for the laughs, ULU beers and anyone who has helped me along the way. I mustn’t forget the friends who have had to put up with me reminding them about my student discount, including Abena, Miriam, Lindsey, Vicky, the 7P ladies and the rest from Sheffield, and everyone in New Zealand. Anyone I’ve not mentioned I’m sorry and thank you. 4 TABLE OF CONTENTS ABSTRACT ................................................................................................................ 3 ACKNOWLEDGEMENTS ....................................................................................... 4 TABLE OF CONTENTS ........................................................................................... 5 LIST OF FIGURES ................................................................................................. 11 LIST OF TABLES ................................................................................................... 19 LIST OF SYMBOLS AND ABBREVIATIONS ................................................... 20 1 INTRODUCTION ............................................................................................ 23 1.1 RELAVANCE OF PROJECT ..................................................................... 23 1.2 AIMS OF PROJECT ................................................................................... 25 1.2.1 Development of a monolithic VLP separation ..................................... 25 1.2.2 Interactions of impurities with hydrophobic monoliths ....................... 25 1.2.3 Interactions and loss of product on hydrophobic monoliths ................ 26 1.3 ORGANISATION OF THESIS .................................................................. 27 1.4 VIRUS-LIKE PARTICLES (VLP) AND VACCINES .............................. 29 1.4.1 Traditional vaccines ............................................................................. 29 1.4.2 VLP vaccines ....................................................................................... 30 1.4.3 Hepatitis B virus (HBV) ...................................................................... 31 1.4.4 Hepatitis B surface antigen (HBsAg) ................................................... 32 1.4.4.1 Production ..................................................................................... 32 1.4.4.2 Structure ........................................................................................ 33 1.4.5 Vaccine ................................................................................................. 38 1.5 PURIFICATION OF NANOPLEX MOLECULES .................................... 39 1.5.1 Non-adsorption purification methods - Precipitation ........................... 40 1.5.2 Non-adsorption purification methods - Ultracentrifugation ................ 42 1.5.3 Adsorption purification methods – Porous beads ................................ 45 5 1.5.4 Adsorption purification methods – Monoliths ..................................... 49 1.5.5 Adsorption purification methods – Membranes ................................... 56 1.5.6 Cryogels ............................................................................................... 58 1.6 CHARACTERISTICS OF PROCESS CHROMATOGRAPHY PERFORMANCE .................................................................................................. 62 1.6.1 Fouling of chromatographic resins....................................................... 62 1.6.2 Cleaning-in-place (CIP) ....................................................................... 65 1.6.3 Confocal microscopy ........................................................................... 66 1.6.4 Dynamic Light Scattering .................................................................... 68 1.6.5 Zeta potential ........................................................................................ 70 1.7 HYDROPHOBIC INTERACTION CHROMATOGRAPHY .................... 72 1.7.1 Theory .................................................................................................. 72 1.7.2 HIC columns ........................................................................................ 76 1.7.3 Column resins ...................................................................................... 77 1.7.4 Column ligands .................................................................................... 77 1.7.5 Optimising a HIC step .......................................................................... 79 1.7.5.1 Salt ................................................................................................ 79 1.7.5.2 Temperature .................................................................................. 80 1.7.5.3 pH.................................................................................................. 80 1.7.5.4 Additives ....................................................................................... 80 1.7.6 Protein unfolding on HIC columns ...................................................... 81 1.8 CIM MONOLITHS ..................................................................................... 85 1.8.1 Principles .............................................................................................. 85 1.8.2 Applications ......................................................................................... 88 1.8.3 Scale up ................................................................................................ 89 2 METHODS AND MATERIALS .................................................................... 93 2.1 MATERIALS .............................................................................................. 93 6 2.1.1 Chemicals ............................................................................................. 93 2.1.2 2.1.2 HBsAg Cell line .......................................................................... 93 2.2 FERMENTATION OF SACCHAROMYCES CEREVISIAE ................... 93 2.2.1 Media...................................................................................................
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