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Development, Functionalisation, and Characterisation of Triply Periodic Minimum Surface Hydrogels for Solid-Tolerant Chromatography

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Development, Functionalisation, and Characterisation of Triply Periodic Minimum Surface Hydrogels for Solid-Tolerant Chromatography Development, Functionalisation, and Characterisation of Triply Periodic Minimum Surface Hydrogels for Solid-Tolerant Chromatography A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemical and Process Engineering University of Canterbury Written by Anne Mary Gordon November 2019 Department of Chemical and Process Engineering Te Whare Wānanga o Waitaha, University of Canterbury Ōtautahi, Christchurch Aotearoa, New Zealand i A. Abstract The work presented in this thesis demonstrates the development of the first chromatography columns for solid-tolerant chromatography produced using 3D-printing methods. This work developed from the rapidly evolving 3D-printing industry, with the hypothesis that 3D-printing methods could be used to produce chromatography columns in a controlled geometry structure. In this application, columns were specifically designed for solid-tolerant chromatography, thus addressing one of the bottlenecks in downstream processing of biological feedstocks, because columns combined primary protein recovery with cell removal from a cell culture or fermentation broth. Columns were made in triply periodic minimum surface structures such as Schwarz diamond and Schoen gyroid from agarose and cellulose hydrogels. These columns had large monolith-type channels (300 µm – 500 µm) allowing cell passage but had the benefit of high surface area for adsorption from the inherent porosity of the hydrogels. Columns were functionalised for three common types of chromatography, demonstrated competitive static adsorption performance compared with commercial resins, and were able to capture protein from a cell/protein mixture, demonstrating solid-tolerant chromatography capabilities. The cellulose CM column was measured to have a binding capacity of 132.8 mg/mL cytochrome c, the agarose DEAE column had a static binding capacity of 229.7 mg/mL BSA, and the agarose HIC column had a static binding capacity of 36.4 mg/mL α-lactalbumin. This work goes through several steps to prove the concept of using 3D-printing methods to produce columns for solid-tolerant chromatography. In the first part of this work, the successful prototyping of TPMS hydrogel structures using a 3D-printed template was demonstrated, with column features on the same order of magnitude as beads used in some protein chromatography applications. TPMS features were shown to be prototyped with good fidelity to their initial design. Next, successful functionalisation was demonstrated through ion exchange and protein binding capacity experiments. The performance of the columns under dynamic conditions was then characterised, showing mass transfer effects reliant on TPMS channel diameter and on-sample velocity. An investigation into limiting parameters of the functionalisation procedure was then presented. Lastly, the effect of yeast cells on protein adsorption was analysed, and cell recovery over a range of velocities on the different columns was characterised. ii B. Contents A. Abstract ............................................................................................................................................................... ii B. Contents ............................................................................................................................................................ iii C. List of Figures .................................................................................................................................................. vii D. List of Tables ................................................................................................................................................. xiii E. Nomenclature ................................................................................................................................................... xv Chapter 1. Introduction ........................................................................................................................................ 1 1.1 Background ........................................................................................................................................................................ 1 1.2 Objectives ........................................................................................................................................................................... 6 1.3 Organisation of Thesis ..................................................................................................................................................... 6 Chapter 2. Literature Review .............................................................................................................................. 8 2.1 Chromatography ............................................................................................................................................................... 8 2.1.1 Downstream Processing ................................................................................................................................... 8 2.1.2 Modes of Chromatography ............................................................................................................................ 11 2.1.2.1 Ion exchange chromatography .......................................................................................................... 11 2.1.2.2 Hydrophobic Interaction Chromatography .................................................................................... 14 2.1.2.3 Reversed Phrase Chromatography ................................................................................................... 15 2.1.2.4 Affinity Chromatography .................................................................................................................... 15 2.1.2.5 Size Exclusion Chromatography ....................................................................................................... 16 2.1.3 Stationary Phases .............................................................................................................................................. 16 2.1.3.1 Natural Hydrogels ................................................................................................................................. 19 2.1.3.1.1 Agarose ............................................................................................................................................... 19 2.1.3.1.2 Cellulose ............................................................................................................................................. 21 2.1.4 Alternatives to Packed-bed chromatography ............................................................................................ 22 2.1.4.1 Monolith Chromatography ................................................................................................................. 23 2.1.4.2 Expanded Bed Adsorption .................................................................................................................. 24 2.1.4.3 3D-Printed Adsorptive Media............................................................................................................ 29 2.1.5 Measuring Chromatographic Performance ............................................................................................... 29 2.1.5.1 Static Performance ................................................................................................................................ 29 2.1.5.2 Dynamic Performance .......................................................................................................................... 31 2.1.5.2.1 Mass Transport in Chromatography .......................................................................................... 31 2.1.5.2.2 Residence Time Distribution ........................................................................................................ 33 2.1.5.2.3 Inverse Size Exclusion Chromatography .................................................................................. 35 iii 2.1.5.2.4 Plate Height ....................................................................................................................................... 36 2.1.5.2.5 Breakthrough Curves ...................................................................................................................... 39 2.2 Solids Removal................................................................................................................................................................ 41 2.3 Functionalisation ............................................................................................................................................................ 45 2.3.1 Matrix Activation ............................................................................................................................................. 46 2.3.2 Ligand Coupling ............................................................................................................................................... 48 2.3.3 Other Functionalisation Methods ................................................................................................................ 49 2.4 3D-Printing .................................................................................................................................................................... 50 2.4.1 3D-Printing in Chromatography ................................................................................................................
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