Design and Functionalization of Alumina Monoliths for Protein Purification by Chromatography

Abstract

This thesis is about the development of multimodal porous cellular alumina structures (monoliths) by an emulsion-gel casting technique using eco-friendly and inexpensive lipids such as corn oil, castor oil, margarine and their mixtures as the dispersed phase. The monoliths obtained showed good mechanical stability (in terms of compressive strength) despite being porous (up to 60%). The formation of the porous networks was interpreted based on combined droplet coalescence, flocculation and Ostwald ripening effects. The presence of such effects along the emulsion storage time led to changes in their viscoelastic and morphological properties, which were found to correlate with structural descriptors of the monoliths after sintering (e.g. average pore sizes and porosity). Furthermore, the fact that these monoliths had hierarchically distributed pores supposes that there would be paths or channels for fluids to flow through them. Experimental and computational studies were performed to understand the behaviour of fluid flow through the monolith. As per literature, several modelling approaches have been applied to describe experimentally observed flow behaviour in such materials. Morphology plays a key role in determining their hydrodynamic and mass transfer properties. Therefore, a direct computational fluid dynamics (CFD) modelling approach was applied to simulate flow behaviour in these columns. The morphological structure of a fabricated alumina monolith was first reconstructed using 3D X-ray tomography data and, subsequently, OpenFOAM, an open-source CFD tool, was used to simulate the essential parameters for monoliths’ performance characterisation and optimisation, i.e. velocity and pressure fields, fluid streamlines, shear stress and residence time distribution (RTD). Moreover, the tortuosity of the monolith was estimated by a novel method, using the computed streamlines, and its value (~1.1) was found to be in the same range of the results obtained by known experimental, analytical and numerical equations. Besides, it was observed that fluid transport was dominated by flow heterogeneities and advection, while the shear stress at pore mouths was significantly higher than in other regions. The proposed modelling approach, with expected high potential for designing target materials, was successfully validated by an experimentally obtained residence time distribution (RTD). However, alumina itself is a relatively non-reactive material. Therefore, to explore a potential application for the produced monolith a simple, single stage sol-gel synthesis method was used to functionalize the monoliths with (3-Aminopropyl)triethoxysilane (APTES) in an aqueous environment. The nature of the attachment of APTES to the alumina and its distribution through the monolith column was evaluated using characterization methods involving FTIR-ATR, SEM-EDS and XPS measurements. Furthermore, the reaction conditions in terms of catalyst used (acid or base) and temperature were adjusted and, separately, a factorial experimental design was applied to elicit the interdependent influence of humidity, number of APTES coating layers and precursor concentration on the silanization of alumina. The reaction was found to be optimum at basic pH and a temperature of 80˚C. Optimally functionalized monoliths with highest amine density of 166 μmol/g of the column were obtained with a single coat using 2M APTES solution, and at 100% humidity. Finally, experiments were carried out to understand the protein interactions with the produced amine functionalized alumina monolithic columns. Bovine Serum Albumin (BSA) was used as the model protein. Studies were carried out at varied BSA concentrations (0.5 to 10 mg.mL-1) to understand the interaction behaviour between the protein and the column. It was found that at lower concentrations there appeared to be stronger binding. At higher BSA concentrations, due to the formation of aggregates, the interaction appears to be a multi-layered physical adsorption. Dynamic light scattering measurements further confirmed the presence of protein aggregation phenomena at higher protein concentrations due to the contact of the protein solution with the column. From these inferences, appropriate strategies were used to bind Protein G to the column - a maximum of 1.43 mg Protein G/g of monolith (29% by mass of column) was bound. Finally, a binding-elution experiment using bovine immunoglobulin G was conducted and it was found that 73.4% (IgG/Protein G) could bind to the column and 86% of the bound IgG could be eluted using an appropriate buffer. This proved the potential of the amine functionalized monolith for further application as an Affinity Chromatography medium.