Chemistry - Masters by Research Theses

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Evaluation of dynamic light scattering as a technique for the routine monitoring of therapeutic proteins on stability
(University College Cork, 2023) O'Kennedy, Laura; Moore, Eric; Crowley; Lowney, Declan; Crowley, Stephen
Throughout the course of biotherapeutic development and manufacturing, the detection and quantification of protein aggregates and particles is critical to ensure patient safety. Size Exclusion Chromatography (SEC) is one of the most widely used techniques for the separation of proteins and allows for the detection and quantification of aggregates. In terms of aggregate detection at drug substance (DS) and drug product (DP) release, as well as for stability testing, the ICH guidelines reference SEC as an example. However, it is acknowledged that new analytical techniques are continuously being developed and can be used where appropriate. Dynamic Light Scattering (DLS) is one such technique, that has been described on several occasions as an early indicator of protein aggregation. Some benefits of DLS over other aggregation-monitoring techniques, is its ability to analyse proteins in their native environment, its fast analysis time, and its ability to detect a wide particle size range. Additionally, for high molecular weight species (HMWS) detection, DLS is an exceptionally sensitive technique. This sensitivity and level of detection are particularly important in the biopharmaceutical industry as the presence of even a small number of aggregates can significantly increase upon long-term storage. However, this sensitivity can also be a weakness, as scattering from traces of other large particulates can interfere with results, and overall, the quantification of results can be difficult. Consequently, the technique is typically used in a complementary fashion with more widely used techniques such as SEC. The capabilities and applications of DLS have been improved over time with the invention and incorporation of e.g. multi angle DLS (MADLS), extended size range analysis and diffusion interaction parameter (kD) analysis, into the one instrument and software. The aim of this research was to explore the possible benefits and limitations associated with DLS, in order to determine if the technique has the potential to be used more widely for testing protein therapeutics on stability. A range of biotherapeutics including monoclonal, bispecific and trispecific antibodies were tested, that varied in concentration from 2 mg/mL to 160 mg/mL. Multiple stressed samples were included in the test panel to explore the techniques’ ability to detect large particles and aggregates. The particle size and size distribution results were compared to those generated from SEC, to explore whether DLS could give additional insight into the molecules stability. It was found that the technique showed great sensitivity and, in some cases, detected an increase in large particles where SEC failed to do so. These results were then substantiated by looking at data from sub-visible particle analysis via light obscuration. The zetasizer instrument, used to measure DLS, can also perform various other measurements that are indicators of colloidal and/or thermal stability. These include electrophoretic light scattering (ELS), extended size range analysis, particle concentration, kD determination, osmotic second virial coefficient (B22) determination and molecular weight (Mw) measurements. These measurements were performed to determine their respective abilities to provide additional insight into therapeutic protein stability. For most of the measurements however, the results and trends weren’t as consistent and as reliable as the particle size and particle distribution measurements. Further evaluation of these measurements, protein concentration optimisation and additional knowledge of the sample properties would be needed to provide more accurate results. However, overall, DLS is seen to be a highly sensitive and fast analysis tool for the determination of particle size and can provide additional insight into molecule stability when used in conjunction with SEC.
Design and use of novel metal catalysts in organic synthesis
(University College Cork, 2023) Fitzgerald, Deirbhile; Maguire, Anita; Collins, Stuart; Irish Research Council
This project focuses on the design and synthesis of novel dirhodium carboxylate catalysts, and their precursor ligands, for use as enantioselective catalysts in transformations of α-diazocarbonyl compounds. Chapter One focuses on the literature background over the past thirty years in transition metal-catalysed asymmetric transformations of α-diazocarbonyl compounds. This overview aims to give an insight into the use of both copper and rhodium-based catalysts in intramolecular and intermolecular C-H insertion reactions, cyclopropanation and desymmetrisation reactions. The evolution of dirhodium carboxylate design since the 1980s is explored. This summary provides insight into their use as enantioselective catalysts in asymmetric transformations of α-diazocarbonyl compounds, thus providing context for this work. Chapter Two describes the design and synthesis of novel, enantiopure dirhodium carboxylate complexes, drawing inspiration from the rhodium (S)-mandelate skeleton. Initially, two enantiopure rhodium carboxylates were synthesised, incorporating carboxylic acid ligands bearing a fenchyl chiral auxiliary. Synthesis of carboxylic acid ligands bearing a bulky adamantyloxy group or a tert-butoxy group in place of the fenchyl substituent was investigated to simplify the stereochemical features, with just one stereogenic centre in each ligand. Two target carboxylic acids were obtained in racemic form, one bearing an adamantyloxy group and the other bearing a tert-butoxy group adjacent to the carboxylic acid moiety. Resolution of these carboxylic acids was explored through formation of diastereomeric salts or enzymatic kinetic resolution; while enantioenrichment was observed, further work is required to obtain the ligands in an enantiopure form for catalyst preparation. Chapter Three describes the synthesis of both racemic mandelamide and enantiopure (S)-mandelamide. In addition, a pair of diastereomerically pure mandelamides with α-methylbenzylamine were synthesised and characterised. The mandelamides were prepared to enable exploration of their crystal landscape in collaboration with another research group in UCC. Chapter Four contains the full experimental details and spectroscopic and analytical characterisation of all compounds synthesised in this project, while details of chiral stationary phase HPLC analysis are included in Appendix 1.
Primary and secondary emissions from upland fires in Ireland
(University College Cork, 2023-04-26) Felberbauer, Clara; Hellebust, Stig; Venables, Dean; Environmental Protection Agency; Horizon 2020
Emissions from wildfires have detrimental effects on air quality, climate change and human health. Despite frequent occurrence of wildfires and prescribed agricultural burns in upland areas, data on wildfire emissions in Ireland is scarce. This work was conducted as part of FLARES (Fire, Land and Atmospheric Remote Sensing of EmissionS), a project funded by the Irish Environmental Protection Agency (EPA) to improve the understanding of Irish upland fire emissions. Emission factors (EF) of typical Irish wildfire fuels (heather, gorse, purple moor grass) were measured for the first time. Emission factors were measured for a set of gases (carbon monoxide CO, carbon dioxide CO2, methane CH4, sulphur dioxide SO2 and nitric oxides NOx as NO), particulates and particulate fractions (fine particulate matter PM2.5, total particulate matter TPM, black carbon BC, organic carbon OC, elemental carbon EC and total carbon TC) and molecular biomarkers (levoglucosan, mannosan, galactosan, 4-Nitrocatechol 4-NC, 4-Nitrophenol 4-NP and 4-Nitroguaiacol 4-NG). Emission factors of measured gases were as follows: CO: 42 g/kg (heather), 27 g/kg (moor grass) and 80 g/kg (gorse); CO2 1167 g/kg (heather), 1558 g/kg (moor grass) and 1442 g/kg (gorse); SO2: 1.38 g/kg (heather), 1.08 g/kg (moor grass) and 1.68 g/kg g/kg (gorse); NOx as NO: 4.46 g/kg (heather), 5.51 g/kg (moor grass), and 6.85 g/kg (moor grass); CH4: 0.21 g/kg (moor grass) and 0.38 g/kg (gorse). Particulate EFs resulted in EF of 4.71 g/kg (heather), 2.42 g/kg (moor grass) and 10.46 g/kg (gorse) for PM2.5; 3.95 g/kg (heather), 4.26 g/kg (moor grass) and 14.33 g/kg (gorse) for TPM; 0.26 g/kg (heather), 0.21 g/kg (moor grass) and 0.50 g/kg (gorse) for BC; 1.28 g/kg (heather), 1.26 g/kg (moor grass) and 5.43 g/kg (gorse) for OC; 0.80 g/kg (heather), 0.81 g/kg (moor grass) and 2.23 g/kg (gorse) for EC; 2.09 g/kg (heather), 2.07 g/kg (moor grass) and 7.65 g/kg g/kg (gorse) for TC. EFs for biomarkers were 0.09 g/kg (heather), 0.18 g/kg (moor grass) and 0.19 g/kg (gorse) for levoglucosan; 0.01 g/kg (heather), 0.01 g/kg (moor grass) and 0.01 g/kg (gorse) for mannosan and 0.01 g/kg (heather), 0.01 g/kg (moor grass) and 0.02 g/kg (gorse) for galactosan; 0.78 mg/kg (heather), 0.42 mg/kg (moor grass) and 1.36 mg/kg (gorse) for 4-NC; 0.05 mg/kg (heather), 0.04 mg/kg (moor grass) and 0.07 mg/kg (gorse) for 4-NP and 0.25 mg/kg (heather), 0.15 mg/kg (moor grass) and 0.34 mg/kg (gorse) for 4-NG. Combustion took place under flaming combustion conditions, reflected in high modified combustion efficiencies (MCE) of 0.95 to 0.98. Emissions for species like CO, PM2.5 and volatile organic compounds (VOCs) would be expected to be higher under real-world conditions where MCE is lower. Secondary organic aerosol (SOA) formation from combustion emissions of the three fuels was studied under simulated day-time (UV lights and UV+OH precursor addition) and night-time conditions (dark + O3 addition) in an atmospheric simulation chamber. SOA mass increased by 2-11% and was dependent on fuel type and oxidising conditions. Field and laboratory samples were analysed at the Leibniz Institute for Tropospheric Research (TROPOS) in Leipzig, Germany. Filter samples were analysed for the biomarkers levoglucosan, mannosan, galactosan, 4-nitrocatechol, 4-nitrophenol, and 4-nitroguiacol. Laboratory experiments were complemented by field studies. Ambient levels of PM2.5 and BC were measured during an 8-week field study in Glencree, rural Co. Wicklow, Ireland. Source appointment of BC and analysis of biomarkers found no unambiguous signals that could be associated with wildfires. However, this dataset represents a valuable glance at air quality in a rural setting in Ireland. Despite the remote location and low population density in Glencree Valley, anthropogenic influences on air quality were significant, most notably from residential solid fuel burning. Monthly PM2.5 concentrations in Glencree (4.6 μg m–3) were lower than in nearby urban Tallaght (7.0 μg m–3), but less than expected based on the difference in population and local pollution sources. Other field observations included opportunistic sampling of the plume of a major wildfire at Killarney National Park, Co. Kerry, in April 2022. Findings presented in this thesis contribute to the understanding of wildfire emissions in Ireland and highlight the influence of anthropogenic air pollution from solid fuel burning on rural locations.
Computational analysis and partialsynthesis of resolvin analogues
(University College Cork, 2022-06-02) Daly, Kevin; O'Sullivan, Tim; Irish Research Council
An introduction to the role of resolvins in inflammation is outlined in Chapter 1. In addition, a literature survey of the structure-activity relationships of resolvins, protectins and maresins is also included. Chapter 2 briefly outlines the aims and objectives of this project. Chapter 3 focuses on the computational analysis of the outlined virtual library of Resolvin D2 analogues. The key physicochemical properties of each candidate are calculated and unsuitable candidates are eliminated using standard screening protocols. Chapter 4 describes the attempted synthesis of a key intermediate for the preparation of an aromatic resolvin analogue. This attempted synthesis encompasses a monosilylation of a diol, a selective oxidation of an alcohol to an aldehyde, a Wittig olefination and, finally, a comprehensive trial of acetal deprotection methods for a 1,2 dioxane acetal in the presence of a silyl ether. Chapter 5 summarises the overall findings of this project and outlines possible future avenues for exploration. The full computational data set and experimental procedures, including spectroscopic data, are detailed in Chapter 6.
Scalable solid state synthesis of core-chell Pt@Cu2O nanocubes with controlled size and shape
(University College Cork, 2021-09) Neill, Hazel; Collins, Gillian; Long, Brenda
This works reports a novel solid state synthesis approach for the formation of shape controlled Pt@Cu2O nanoparticles immobilized onto Cu substrates. High nanoparticle loading was achieved by the use of a self-assembled monolayer consisting of a long chain diamine. The SAM enabled immobilization of citrate stabilized Pt nanoparticles onto the Cu substrate. Annealing under a reducing atmosphere led to the formation of core-shell nanostructures with a cubic morphology. Characterisation showed the nanocubes to consist of a Pt core and crystalline oxide shell. A key question that is addressed in this work was determining the composition of the shell material. A combination of detailed materials characterisation including XPS, XRD and high resolution TEM analysis confirmed the composition to be Cu2O. Preliminary measurements have demonstrated the use of the substrates in glucose sensing applications.

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