Permanent URI for this collection
Browse
Recent Submissions
Publication A life cycle thinking-based environmental risk framework for screening sustainable feedstocks in early-stage bioeconomy projects(Elsevier, 2025-02-25) Bishop, George; Girón-Domínguez, Carmen; Gaffey, James; Henchion, Maeve; Fealy, Réamonn; Zimmermann, Jesko; Kargupta, Wriju; Styles, David; Department of Agriculture, Food and the Marine (Ireland)Understanding the environmental impacts of bio-based feedstock production is essential for sustainable bioeconomy development. Consequential life cycle assessment (LCA) evaluates environmental sustainability, often identifying “hidden” impacts incurred through market displacements. However, it is often impractical to screen multiple bioeconomy feedstocks and value chains using full consequential LCA early in project conceptualisation, owing to high requirements in terms of time, data, and expertise. As a result, critical environmental risks may not be discovered until too late in project development to redirect investment towards more sustainable options. This paper introduces the Bio-based feedstock Environmental Risk Assessment (Bio-ERA) Framework, designed to support early screening of potential upstream environmental risks associated with increased demand for bio-based feedstocks. The Bio-ERA Framework comprises a decision tree that systematically guides stakeholders through consequential life cycle thinking, elucidating sometimes hidden (indirect) pathways of impact among feedstock sourcing decisions. Seven important environmental aspects are addressed: Finite Resource Inputs, Greenhouse Gas (GHG) Emissions, Air Quality, Water Quality, Ecosystem Diversity, Terrestrial Carbon Storage, and Indirect Land Use Change. Criteria are proposed to structure evaluation of (i) probability and (ii) severity of environmental impact, in relation to four categories of feedstock: primary (determining product), high-value by-product, low-value by-product, and waste. Example applications demonstrate how the framework can generate an environmental risk profile for specific feedstocks sourced in specific contexts. Bio-ERA does not avoid the need for detailed LCA evaluation of full bioeconomy value chains, but promotes deeper interrogation and awareness of potential environmental risks associated with feedstock sourcing, in a manner that is accessible to all stakeholders. This could support earlier screening of strategic investment decisions necessary to develop a sustainable bioeconomy.Publication N terminal protein binding and disorder-to-order transition by a synthetic receptor(American Chemical Society, 2025-02-20) Crowley, Peter B.; Mockler, Niamh M.; Ramberg, Kiefer O.; Flood, Ronan J.We describe the capture and structuring of disordered N-terminal regions by the macrocycle sulfonato-calix[4]arene (sclx4). Using the trimeric β-propeller Ralstonia solanacearum lectin (RSL) as a scaffold, we generated a series of mutants with extended and dynamic N-termini. Three of the mutants feature an N-terminal methionine-lysine motif. The fourth mutant contains the disordered 8-residue N-terminus of Histone 3, a component of the nucleosome. X-ray crystallography and NMR spectroscopy provide evidence for sclx4 binding to the flexible N-terminal regions. Three crystal structures reveal that the calixarene recognizes the N-terminal Met-Lys motif, capturing either residue. We provide crystallographic proof for sclx4 encapsulation of N-terminal methionine. Calixarene capture of intrinsically disordered regions may have applications in regulating protein secondary (and tertiary) structure.Publication Nano- and meso-scale aggregation of poly(N-isopropylacrylamide) below the lower critical solution temperature: A wide-angle dynamic light scattering study(Elsevier, 2025-01-25) de Castro, Matheus A.; Ryder, Alan G.Poly-N-isopropylacrylamide (PNIPAm), a thermorresponsive polymer, highly soluble in water below its lower critical solution temperature (LCST), is widely used in biomedical applications like drug delivery. Being able to measure PNIPAm size and aggregation state in solution quickly, inexpensively, and accurately below the LCST is critical when stoichiometric particle or molecular ratios are required. Dynamic light scattering (DLS) is probably the most widely available, and inexpensive nanoparticle sizing technique, but there are limitations with respect to sample polydispersity. Here, we first investigated factors governing the ability of DLS to accurately measure PNIPAm size in solution at 25 °C as part of a quality study of five different molecular weight, commercially sourced PNIPAm. All samples were polydisperse and accurate particle size distribution (PSD) data was only obtained from distribution fitting, being consistent and accurate down to ∼ 0.1 wt%. In water at 1 wt%, Rh, extracted from distribution fitting: 12.4 ± 0.6 nm (55 kDa), 10.0 ± 0.22 nm (38 kDa), 6.2 ± 0.15 nm (28.5 kDa), and 9.7 ± 0.14 nm (20–25 kDa) were significantly higher than that expected for single PNIPAm chains in solution. Measurements in different buffers of varying pH (7.4–5.0) yielded similar sizes (Rh of 6–15 nm) and polydispersity indicating that these were stable aggregates. These aggregates could be broken down with Triton-X but not with sodium dodecyl sulphate, ultrasound, or by heating above the LCST and then cooling. We suggest that this nanoscale aggregation and increased polydispersity was caused a variety of factors including by solid-state aging during prolonged storage (>5 years) induced by water adsorption, and/or manufacturing processes. Stirring was found to produce larger, meso-scale (Rh > 150 nm), soluble aggregates and the rate of formation of these meso-particles was linear with stirring time (with a concomitant linear decrease in the faction of original nanoscale aggregates). Meso-particle formation was not correlated with MW, but was inversely correlated to polymer concentration suggesting that aggregation was driven by adsorption at air/liquid interfaces rather than solution phase collisions. In conclusion, PNIPAm particle size and distribution was highly dependent on multiple factors including source, storage conditions, and exposure to air–water interfaces. Standard wide angle DLS is however an effective and rapid method for identifying and quantifying PNIPAm aggregation.Publication Crystal growth and morphology control of needle-shaped organic crystals(Royal Society of Chemistry, 2024-01-05) McArdle, Patrick; Erxleben, Andrea; N/A; Science Foundation IrelandUnderstanding the growth of needle-shaped molecular crystals and the factors that influence the needle morphology is not only a fundamental research question but is also important in the design of industrial crystallization processes. Needle-like crystals have poor flow properties, are difficult to filter and to dry and – in the case of pharmaceuticals – difficult to formulate. While the morphology can often be controlled by changing the solvent, some compounds are persistent needle-formers that grow from all solvents as highly anisotropic crystals. In this highlight article we discuss the structural properties that lead to (persistent) needle growth. We also review strategies to modify the morphology such as the use of tailor-made additives, polymeric growth modifiers and cocrystallization.Publication Auranofin loaded silk fibroin nanoparticles for colorectal cancer treatment(Springer, 2024-10-09) Pérez-Lloret, Marta; Reidy, Eileen; Lozano-Pérez, Antonio Abel; Marchal, Juan Antonio; Lens, Piet N. L.; Ryan, Aideen E.; Erxleben, Andrea; Enterprise Ireland; Horizon 2020; Science Foundation Ireland; Engineering and Physical Sciences Research Council Centre; Galway University Foundation; European Regional Development FundColorectal cancer (CRC) is the second most common cause of cancer related deaths worldwide and the prevalence in young people especially is increasing annually. In the search for innovative approaches to treat the disease, drug delivery systems (DDS) are promising owing to their unique properties, which allow improved therapeutic results with lower drug concentrations, overcoming drug resistance and at the same time potentially reducing side effects. Silk fibroin is a biopolymer that can be processed to obtain biocompatible and biodegradable nanoparticles that can be efficiently loaded by surface adsorption with small-molecule therapeutics and allow their transport and sustained release by modulating their pharmacokinetics. Auranofin (AF) has recently been repurposed for its strong anticancer activity and is currently in clinical trials. Its mechanism of action is through the inhibition of thioredoxin reductase enzymes, which play an essential role in several intracellular processes and are overexpressed in some tumours. Taking into account that AF has a low solubility in water, we propose silk fibroin nanoparticles (SFN) as AF carrier in order to improve its bioavailability, increasing cellular absorption and preventing its degradation or avoiding some resistance mechanisms. Here we report the preparation and characterization of a new formulation of AF-loaded silk fibroin nanoparticles (SFN-AF), its functionalization with FITC for the analysis of cellular uptake, as well as its cytotoxic activity against cell lines of human colorectal cancer (HT29 and HCT116) in both 2D and 3D cell cultures. 3D spheroid models provide a 3D environment which mimics the 3D aspects of CRC observed in vivo and represents an effective 3D environment to screen therapeutics for the treatment of CRC. The loaded nanoparticles showed a spherical morphology with a hydrodynamic diameter of ~ 160 nm and good stability in aqueous solution due to their negative surface charges. FESEM-EDX analysis revealed a homogeneous distribution of Au clusters with high electron density on the surface of the nanoparticles. SFN-AF incubated in phosphate buffer at 37 °C released 77% of the loaded AF over 10 days, showing an initial burst and then sustained release. Flow cytometry analysis showed that FITC-SFN-AF was efficiently internalized by both cell lines, which was confirmed by confocal microscopy imaging. SFN enhanced the cytotoxicity of AF in 2D cultures in both CRC lines. Promising results were also obtained in 3D culture paving the way for future application of this strategy as a therapy for CRC.Publication Small lectin ligands as a basis for applications in glycoscience and glycomedicine(Royal Society of Chemistry, 2024-08-20) Murphy, Paul V.; Science Foundation IrelandGlycan recognition by lectins mediates important biological events. This Tutorial Review aims to introduce lectin–ligand interactions and show how these molecular recognition events inspire innovations such as: (i) glycomimetic ligands; (ii) multivalent ligand agonists/antagonists; (iii) ligands for precision delivery of therapies to cells, where therapies include vaccines, siRNA and LYTACs (iv) development of diagnostics. A small number of case studies are selected to demonstrate principles for development of new ligands for applications inspired by knowledge of natural glycan ligand structure and function.Publication Estimating Poly(N-isopropylacrylamide) size in solution below the LCST using fluorescence correlation spectroscopy with non-covalent bound fluorophores(Elsevier, 2024-06-25) van Zanten, Camila; Ryder, Alan G.Accurate size measurements of nanosized polymer chains in dilute solutions is important for understanding polymer behavior, however, these measurements can be challenging to implement accurately, and are technique dependent. Here we explore the use of Fluorescence Correlation Spectroscopy (FCS) to determine the size of single polymer chains in dilute solutions (<2 wt%). FCS, a technique with single molecule sensitivity, generally requires the use of covalently labelled polymers which can distort physicochemical behavior. Here, FCS based size measurements were based on the non-covalent interaction of fluorophores (Alexa 405, Atto 390, and Atto 425) with PNIPAm in water at 25 °C (below the Lower Critical Solution Temperature). FCS estimated size (hydrodynamic radius) of three different MW PNIPAm samples in water were: 5.0 ± 1.0 nm (28.5 kDa), 5.0 ± 1.0 nm (38 kDa), 3.3 ± 0.5 nm (55.5 kDa), in reasonable agreement with theoretical calculations. Accuracy was directly related to the fraction of PNIPAm-bound fluorophore, which were, for 1 wt% PNIPAm solutions in water: ∼11.5 % (Atto 390), ∼8.1 % (Atto 425), and 4 % (Alexa 405). This method has several advantages in that it does not require a covalent labelling of PNIPAm, it can be implemented on very small sample volumes, and allows for in-situ measurements.Publication Effects of viscosity and refractive index on the emission and diffusion properties of Alexa Fluor 405 using fluorescence correlation and lifetime spectroscopies(Springer, 2021-03-19) van Zanten, Camila; Melnikau, Dzmitry; Ryder, Alan G.; Science Foundation Ireland; European Regional Development Fund; Health Research BoardFluorescence Correlation Spectroscopy (FCS) studies of the interaction of polymers or proteins in solution are strongly affected by the viscosity and refractive index of the medium, and the effects are likely to be more significant with the use of short wavelength excitation (e.g., 405 nm diode lasers). Failing to account for these issues can lead to incorrect measurement of average size, conformational changes, and dynamic behaviour of polymers and proteins. Steady-state, time-resolved, and FCS measurements of Alexa 405 in glycerol:water mixtures were performed to determine its suitability for FCS measurements with 405 nm excitation. The effects of the refractive index and viscosity on the diffusion coefficient and photophysical parameters (lifetime and relative quantum yield) of the fluorophore were determined. Alexa 405 lifetime decreased from 3.55 ns in water to 3.25 ns in a 50:50 glycerol:water mixture, while its diffusion coefficient dropped from 333 ± 16 to 44 ± 1 µm2s− 1. Lifetime data collected from micromolar solutions of Alexa 405 did however also suggest that as solvent polarity decreased, aggregates (excimers) were formed as evidenced by the appearance of a rising edge in the decay plots. The interdependence between lifetime, refractive index, and diffusion coefficient could be accurately fitted by a simple polynomial function indicating that the probe is well behaved and predictable in the glycerol:water model system. Overall, Alexa 405 is a most promising and reliable probe for FCS measurement using violet laser diode excitation sources.Publication Implementation of an ISO 50001 energy management system using Lean Six Sigma in an Irish dairy: a case study(Emerald, 2023-12-18) Trubetskaya, Anna; McDermott, Olivia; McGovern, SeamusPurpose This article aims to optimise energy use and consumption by integrating Lean Six Sigma methodology with the ISO 50001 energy management system standard in an Irish dairy plant operation. Design/methodology/approach This work utilised Lean Six Sigma methodology to identify methods to measure and optimise energy consumption. The authors use a single descriptive case study in an Irish dairy as the methodology to explain how DMAIC was applied to reduce energy consumption. Findings The replacement of heavy oil with liquid natural gas in combination with the new design of steam boilers led to a CO2 footprint reduction of almost 50%. Practical implications A further longitudinal study would be useful to measure and monitor the energy management system progress and carry out more case studies on LSS integration with energy management systems across the dairy industry. Originality/value The novelty of this study is the application of LSS in the dairy sector as an enabler of a greater energy-efficient facility, as well as the testing of the DMAIC approach to meet a key objective for ISO 50001 accreditation.Publication Industry 4.0 readiness in west of Ireland small and medium and micro enterprises – an exploratory study(Taylor & Francis, 2023-03-21) McDermott, Olivia; Nelson, Stuart; Antony, Jiju; Sony, MichaelQuality 4.0 is the new paradigm in the field of quality management, due to the advent of technological advancement and digital transformation of many organizations. The field of Quality 4.0 is still in its nascent stage, and there is no self-assessment tool to assess whether an organization is ready or not in implementing Quality 4.0. This study uses a five-phase approach to develop a Quality 4.0 readiness self-assessment tool. Phase 1 of the study was devoted to a literature review to identify the readiness factors and sub-factors. In Phase 2 experts¿ opinions sought sure that none of the readiness factors are missed out in our research. Phase 3 was devoted to questionnaire development. Phase 4 was devoted to data collection. In this phase, we identified 6 packaging companies that were willing to participate in the research. Phase 5 was devoted to data analysis and readiness assessment for each of the factors and sub-factors across the participating organizations. The readiness factors identified in the study were: top management commitment and support, leadership, organizational culture, employee competency, and ISO QMS Standard in place. Further, a methodology was developed to classify the participating organizations into five different quality readiness assessment levels.Publication The misplacement of ISO 18404:2015 in organisational improvement: a point-counterpoint article(Emerald, 2023-09-05) McDermott, Olivia; Antony, Jiju; Sony, Michael; Swarnakar, VikasThis paper gives the background to the ISO 18404:2015 standard and explains its rationale. It aims to rebut the Oudrhiri et al. (2022) paper. Furthermore, this paper adds further evidence of the misplacement and unfitness for use of the standard, as evidenced in the previous work by Antony et al. (2021, 2022). A point-counterpoint methods approach with a literature review of studies available on ISO 18404:2015 to respond to the Oudrhiri et al. (2022) study. The findings indicate that Oudrhiri et al.'s (2022) work is not open minded in relation to ISO18404. Each point raised in the Oudrhiri et al. (2020) study has been answered and counter-argued. Other than Antony et al.'s three studies (2021 and 2022) and Oudrhiri et al.¿s (2022) study empirical studies looking into the impact of the ISO 18404 standard in the literature were limited. As the literature has shown, many companies are not utilising the standard given its current format; hence, a lack of information relating to the practical implementation is sparse. The findings indicate that Oudrhiri et al.'s (2022) work can be answered and counter-argued. This study consolidates and strengthens the findings from the three studies by Antony et al. (2021 and 2022) and acts as a rebuttal to the Oudrhiri et al. (2022) study.Publication Mapping the terrain for the Lean Supply Chain 4.0(Emerald, 2023-10-03) McDermott, Olivia; Antony, Jiju; Sony, Michael; Swarnakar, VikasThis study aims to carry out a systematic literature review (SLR) on the integration of Lean, Industry 4.0 and the supply chain or the Lean Supply Chain (LSC) 4.0. The research analyses the current research on the LSC 4.0 concept in an increasingly digitalised world. The authors present the benefits, motivations, critical success factors and challenges of integrating the LSC with Industry 4.0 technologies within this emerging area of research. Design/methodology/approach An SLR is carried out on how Lean can be integrated with Supply Chain 4.0. Using the search strings of ¿Lean Supply Chain 4.0,¿ ¿Lean Supply Chain Management 4.0¿ and ¿Lean Supply Chain Digitalisation,¿ a review of published literature was carried out via searches on academic databases. Findings Industry 4.0 has a synergistic effect on the LSC and, depending on the technology and sector applied in, can complement and enhance the LSC. Similarly, the LSC is a precursor for digitalisation. There are considerable implications in the LSC 4.0 for green and sustainable processes. Practical implications Organisations can use this study to understand what the LSC 4.0 means to industry, the benefits and motivating factors for implementation, the critical success factors (CSFs) to implementation and the challenges for implementation. Originality/value This study adds to state of the art around the LSC 4.0 and future directions in this nascent research area. This study will aid organisations in understanding how Lean, supply chain management and Industry 4.0 can be integrated.Publication Employee's performance and Kaizen events' success: does supervisor behaviour play a moderating role?(Emerald, 2023-12-05) Kharub, Manjeet; Gupta, Himanshu; Rana, Sudhir; McDermott, OliviaThe study's goal was to identify the factors contributing to the practical completion of Kaizen events (KEs). The effect of the work-study man's characteristics, the supervisor's conduct and the autonomy of the Kaizen team are analysed in this study. Data were collected from 249 respondents working in the manufacturing sector in India, mainly those who had been involved in Kaizen projects. Three-step procedures, namely, exploratory factor analysis, confirmatory factor analysis and partial least squares, have been applied to test the research hypotheses through structural equational modelling. The exploratory factor analysis extracted in-role performance, creative performance and human aspect as latent variables explaining work-study man's performance (eigenvalue = 1). The study's findings indicate that the performance of work-study man (in-role, creative and human) and supervisors' conduct is directly related to the success of KEs. It was shown that supervisors might influence the outcomes of KEs only by moderating the human aspects. Additionally, the degree of autonomy of the Kaizen team was found having a significant positive relationship with the success of KEs.Publication Size exclusion chromatography for screening yeastolate used in cell culture media(Elsevier, 2023-09-18) Kyne, Michelle; de Faria e Silva, Ana Luiza; Vickroy, Bruce; Ryder, Alan G.; Science Foundation Ireland; European Regional Development FundYeastolate is often used as a media supplement in industrial mammalian cell culture or as a major media component for microbial fermentations. Yeastolate variability can significantly affect process performance, but analysis is technically challenging because of compositional complexity. However, what may be adequate for manufacturing purposes is a fast, inexpensive screening method to identify molecular variance and provide sufficient information for quality control purposes, without characterizing all the molecular components. Here we used Size Exclusion Chromatography (SEC) and chemometrics as a relatively fast screening method for identifying lot-to-lot variance (with Principal Component Analysis, PCA) and investigated if Partial Least Squares, PLS, predictive models which correlated SEC data with process titer could be obtained. SEC provided a relatively fast measure of gross molecular size hydrolysate variability with minimal sample preparation and relatively simple data analysis. The sample set comprised of 18 samples from 12 unique source lots of an ultra-filtered yeastolate (10 kDa molecular weight cut-off) used in a mammalian cell culture process. SEC showed significant lot-to-lot variation, at 214 and 280 nm detection, with the most significant variation, that correlated with process performance, occurring at a retention time of ~6 minutes. PCA and PLS regression correlation models provided fast identification of yeastolate variance and its process impact. The primary drawback is the limited column lifetime (Publication A route to 1-Deoxynojirimycin and 1-Deoxymannojirimycin derivatives with quaternary centers adjacent to the ring nitrogen from Methyl α-d-Mannopyranoside(Wiley, 2020-02-03) Chadda, Rekha; Murphy, Paul V.; Science Foundation Ireland; European Regional Development Fund6-Alkylated-8-azido-1,6-octadiene derivatives were prepared from methyl α-D-mannopyranoside. The sequence to allylic azide precursors included a Horner-Wadsworth-Emmons reaction with a concomitant epimerization that ultimately enabled synthesis of 1-deoxynojirimycin as well as 1-deoxymannojirimy- cin derivatives. Thermally promoted allylic acid rearrangement followed by triazoline formation, then decomposition to aziridine and finally reaction with acetic acid was used to generate products that have quaternary anomeric centers adjacent to the piperidine ring nitrogen atom (cyclic α-tertiary amines). The stereoselectivity is accounted for based on minimization of steric interactions in the transition state structure, favouring the product where the larger methyl substituent is equatorial and the vinyl group prefers to be axial.Publication Advanced spectroscopy and APBS modeling for determination of the role of His190 and Trp103 in mouse thymidylate synthase interaction with selected dUMP analogues(MDPI, 2021-03-06) Prokopowicz, Małgorzata; Jarmuła, Adam; Casamayou-Boucau, Yannick; Gordon, Fiona; Ryder, Alan; Sobich, Justyna; Maj, Piotr; Cieśla, Joanna; Zieliński, Zbigniew; Fita, Piotr; Rode, WojciechA homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the abortive reaction inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.Publication Modelling Förster resonance energy transfer (FRET) using anisotropy resolved multi-dimensional emission spectroscopy (ARMES)(Elsevier, 2020-11-16) Gordon, Fiona; Elcoroaristizabal, Saioa; Ryder, Alan G.; Science Foundation Ireland; European Regional Development FundBackground Förster Resonance Energy Transfer (FRET) is widely used to study the structure and dynamics of biomolecular systems and also causes the non-linear fluorescence response observed in multi-fluorophore proteins. Accurate FRET analysis, in terms of measuring changes in donor and acceptor spectra and energy transfer efficiency is therefore critical. Methods We demonstrate a novel quantitative FRET analysis using anisotropy resolved multidimensional emission spectroscopy (ARMES) in a Human Serum Albumin (HSA) and 1,8-anilinonaphathalene sulfonate (ANS) model. ARMES combines 4D measurement of polarized excitation emission matrices (pEEM) with multivariate data analysis to spectrally resolve contributing fluorophores. Multivariate analysis (Parallel Factor, PARAFAC and restricted Tucker3) was used to resolve fluorophore contributions and for modelling the quenching of HSA emission and the HSA-ANS interactions. Results pEEM spectra were modelled using Tucker3 which accommodates non-linearities introduced by FRET and a priori chemical knowledge was used to optimise the solution, thus resolving three components: HSA emission, ANS emission from indirect FRET excitation, and ANS emission from direct excitation. Perpendicular emission measurements were more sensitive to indirectly excited acceptor emission. PARAFAC modelling of HSA, donor emission, separated ANS FRET interacting (Tryptophan) and non-interacting (Tyrosine) components. This enabled a new way of calculating quenching constants using the multi-dimensional emission of individual donor fluorophores. Conclusions FRET efficiency could be calculated using the multi-dimensional, resolved emission of the interacting donor fluorophores only which yielded higher ET efficiencies compared to conventional methods. General significance Shows the potential of multidimensional fluorescence measurements and data analysis for more accurate FRET modelling in proteins.Publication Analyzing protein conjugation reactions for antibody-drug conjugate synthesis using polarized excitation emission matrix spectroscopy(Wiley, 2022-09-07) de Faria e Silva, Ana L.; Ryder, Alan G.; Science Foundation Ireland; European Regional Development FundAntibody-drug conjugates (ADCs) are promising anticancer therapeutics, which offer important advantages compared to more classical therapies. There are a variety of ADC critical quality attributes (CQAs) such as the protein structure, aggregation, and drug-to-antibody ratio (DAR), which all impact on potency, stability, and toxicity. Production processes can destabilize antibodies via a variety of physical and chemical stresses, and or by increased aggregation after conjugation of hydrophobic drugs. Thus, a proper control strategy for handling, production, and storage is necessary to maintain CQA levels, which requires the use of in-process quality measurements to first identify, then understand, and control the variables which adversely affect ADC CQAs during manufacturing. Here, we show how polarized excitation emission matrix (pEEM) spectroscopy, a sensitive, nondestructive, and potentially fast technique, can be used for rapidly assessing aggregation and DAR in a single measurement. pEEM provides several sources of information for protein analysis: Rayleigh scatter for identifying aggregate/particle formation and fluorescence emission to assess chemical and structural changes induced by attachment of a linker and/or a small molecule drug payload. Here, we used a nontoxic ADC mimic (monoclonal antibody with linker molecule) to demonstrate efficacy of the measurement method. Emission changes caused via light absorption by the attached linker, allowed us to predict DAR with good accuracy using fluorescence signal from the final purified products (6% relative error of prediction [REP]) and also from unpurified alkylation intermediates (11% REP). pEEM changes could also be correlated with size (hydrodynamic radius, Rh) and aggregate content parameters obtained from dynamic light scattering and size exclusion chromatography (SEC). For the starting material and purified product samples, pEEM correlated better with Rh (R2¿=¿0.99, 6% REP) than SEC determined aggregate content (18% REP). Combining both fluorescence and light scatter signals also enabled in-process size quantification (6% REP). Overall, combining polarized measurements with EEM and Rayleigh scatter provides a single measurement, multi-attribute test method for ADC manufacturing.Publication Development of a rapid Polarized Total Synchronous Fluorescence Spectroscopy (pTSFS) method for protein quantification in a model bioreactor broth(Wiley, 2021-01-27) Boateng, Bernard O.; Elcoroaristizabal, Saioa; Ryder, Alan G.; Science Foundation Ireland; European Regional Development FundProtein quantification during bioprocess monitoring is essential for biopharmaceutical manufacturing and is complicated by the complex chemical composition of the bioreactor broth. Here we present the early-stage development and optimization of a polarised Total Synchronous Fluorescence Spectroscopy (pTSFS) method for protein quantification in a hydrolysate-protein model (mimics clarified bioreactor broth samples) using a standard benchtop laboratory fluorometer. We used UV transmitting polarizers to provide wider range pTSFS spectra for screening of the four different TSFS spectra generated by the measurement: parallel (||), perpendicular (¿), unpolarized (T) intensity spectra and anisotropy maps. TSFS|| (parallel polarised) measurements were the best for protein quantification compared to standard unpolarized measurements and the Bradford assay. This was because TSFS|| spectra had a better analyte signal to noise ratio (SNR), due to the anisotropy of protein emission. This meant that protein signals were better resolved from the background emission of small molecule fluorophores in the cell culture media. SNR of > 5000 was achieved for concentrations of BSA/YST 1.2/10 g L-1 with TSFS|| . Optimisation using genetic algorithm and interval Partial Least Squares based variable selection enabled reduction of spectral resolution and number of excitation wavelengths required without degrading performance. This enables fast (Publication Evaluating the interaction of human serum albumin (HSA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes in different aqueous environments using anisotropy resolved multi-dimensional emission spectroscopy (ARMES)(Elsevier, 2022-01-07) Gordon, Fiona; Casamayou-Boucau, Yannick; Ryder, Alan G.; Science Foundation Ireland; European Regional Development FundStudying the interaction between plasma proteins and liposomes is critical, particularly for their use as drug delivery systems. Here, the efficacy of anisotropy resolved multidimensional emission spectroscopy (ARMES) for investigating the interaction of human serum albumin (HSA) with liposomes was explored and compared to conventional spectroscopic techniques. Dynamic Light Scattering (DLS) and absorbance spectroscopy (with Multivariate Curve Resolution (MCR) modeling) indicated that the highest degree of liposome rupturing, and aggregation occurred in water, with less in ammonium bicarbonate buffer (ABC) and phosphate buffered saline (PBS). Fluorescence emission spectra of HSA-liposome mixtures revealed significant hypsochromic shifts for water and ABC, but much less in PBS, where the data suggests a non-penetrating protein layer was formed. Average fluorescence lifetimes decreased upon liposome interaction in water (6.2¿5.2 ns) and ABC buffer (6.3¿5.6 ns) but increased slightly for PBS (5.6¿5.8 ns). ARMES using polarized Total Synchronous Fluorescence Scan measurements with parallel factor (PARAFAC) analysis resolved intrinsic HSA fluorescence into two components for interactions in water and ABC buffer, but only one component for PBS. These components, in water and ABC buffer, corresponded to two different HSA populations, one blue-shifted and penetrating the liposomes (¿ex/em = ~ 280/320 nm) and a second, similar to free HSA in solution (¿ex/em = ~ 282/356 nm). PARAFAC scores for water and ABC buffer suggested that a large proportion of HSA interacted in an end on configuration. ARMES provides a new way for investigating protein-liposome interactions that exploits the full intrinsic emission space of the protein and thus avoids the use of extrinsic labels. The use of multivariate data analysis provided a comprehensive and structured framework to extract a variety of useful information (resolving different fluorescent species, quantifying their signal contribution, and extracting light scatter signals) all of which can be used to discriminate between interaction mechanisms.