Although spray drying has been common place in the pharmaceutical industry for decades, the integration of the technique into continuous manufacturing can offer an extensive array of particle engineering applications. Continuous manufacturing aims to deliver consistent and sustainable drug products of a better and higher quality. Spray drying is a continuous processing technique typically adopted for amorphous solid production. However, the unique conditions of the technique can also can be adapted and applied to crystallisation enabling particle property engineering. The semi-continuous lab-scale Büchi B-290 Mini spray dryer is widely available and has been previously studied extensively for particle engineering and as a development platform for applications including pulmonary drug delivery, sustained release formulations and amorphous solid dispersions. The focus of this work is to engineer and enhance particle properties through the use of the Büchi spray dryer. Particle formation has been investigated, with specific focus in terms of polymorph formation in carbamazepine, to develop a predictive model for crystallisability and for co-spray drying of metformin hydrochloride with mannitol and lactose. Particle formation has been described in terms of theoretical drying kinetics and combined with off line characterisation to determine size and form of product. The metastable polymorph, form IV, of carbamazepine was made reproducibly by spray drying with the combination of rapid evaporation and product isolation shown to be crucial to prevention of solution mediated transformation. The application of non-invasive Raman spectroscopy was also utilised to assess product form. A crystallisability predictive model based on a Random Forest method was successfully produced through combining molecular descriptors with published and experimental outcomes. The model provided up to 79 % accuracy in predicting whether an amorphous or crystalline product would be expected from rapid drying. This shows considerable utility in streamlining process development. Finally, co-spray drying in the Büchi system using a three-fluid nozzle was used to produce multicomponent composite particles comprising of two crystallite phases. The effect of process configuration and material properties on the resultant particles was assessed using particle sizing, SEM, XRPD and Raman mapping. The results were compared on the basis of theoretical drying kinetics to assess the ability to predict the resultant particle morphology. Four multicomponent composite particles were produced by co-spray drying from metformin hydrochloride (MF), mannitol and lactose. MF-mannitol composites produced three-phase physical mixtures with both components present on the particle surfaces. The particle surface compositions were contradictory to the expected particle outcomes from the drying parameters. MF-lactose composite particle also produce three-phase physical mixtures with a relatively equal distribution of components present on particle surface. This is consistent with the expected particle from the drying parameters. The different particle outcomes suggest that co-spray drying of miscible multicomponent feeds using the three-fluid nozzle is highly dependent on the drying parameters for each component due to equal mixing of the feed at atomisation of droplets.

Impurities in active pharmaceutical ingredients (APIs) include volatile and involatile organic compounds and inorganic anions and cations. The impurities in an API are acquired during the manufacturing process and may arise from impurities in reagents, side reactions and may, as in the case of ionic impurities associate with the molecule during purification processes. Impurity levels have to be controlled to conform to pharmacopoeial requirements; although this is less apparent in the case of organic and inorganic counterions. In the work reported in this thesis four applications of impurity profiling in APIs and API intermediates were investigated. In chapter 3 the removal of impurities in lipoic acid prior to crystallisation was investigated using high resolution mass spectrometry in combination with chemometric modelling of the data. Several impurities of lipoic acid were characterised by using MSn and the success of the purification process in removing impurities was verified. In chapter 4 the organic impurities in chlorpheniramine maleate were investigated using high resolution mass spectrometry in combination with chemometric modelling of the data. Several new impurities of chlorpheniramine maleate were characterised by using MSn and it was demonstrated that it was possible to distinguish between different manufacturers’ batches of chlorpheniramine maleate according to their impurity profiles by using high resolution mass spectrometry data in combination with chemometric modelling. In chapter 5 the anionic impurities in different pharmaceutical bases were investigated by using capillary ion chromatography. The different pharmaceutical bases contained a wide range of trace ions as well as the main counter-anion. Aside from the anions being present as impurities they might have an impact of processing procedures such as crystallisation. In chapter 6 a range of techniques including nuclear magnetic resonance spectroscopy, high resolution LC-MSn, headspace gas chromatography mass spectrometry, anion and cation chromatography were applied to the analysis of 6-aminopenicillanic acid (6-APA) samples, provided by GSK, which had of low to high clarity values. It was not possible to find any direct association between clarity readings and the various impurity profiles although a number of new impurities in 6-APA were characterised.

Fouling involves the unwanted deposition and build-up of solid material on surfaces within a process. This problem is widely encountered in multiphase and solid phase processing in many industries including oil and gas, pharmaceutical and fine chemical manufacturing sectors. Although it is acknowledged to impact both batch and continuous processing methods it poses a particular challenge to the controlled operation of continuous crystallisation processes where extended operation under non-equilibrium conditions is required. Whilst the factors impacting on fouling have been proposed, there have been only a relatively limited number of studies into fouling mechanisms to date. With increased interest in deploying continuous crystallisation processes for pharmaceutical manufacturing, the motivation for this work was to develop an improved understanding of the influence of material properties and process conditions on fouling processes. In this work, a number of studies were conducted in which key materials and process parameters were investigated. These have included different materials of construction (MOCs), process conditions (flow, supersaturation, temperature gradients (ΔT)) and crystallising solutions (solute and solvent). Primary fouling studies were conducted using a small scale batch crystallisation setup to explore the influence on MOCs, supersaturation and agitation rate upon both bulk crystal nucleation and surface fouling of paracetamol. The prominent fouling mechanism was found to be particle deposition which was influenced by supersaturation, agitation rate, different MOCs and exposure time. Fouling is known to occur on heat exchange interfaces due to the localised supersaturation that can be generated e.g. in a plug flow continuous cooling crystalliser. A novel surface induced continuous crystallisation fouling assessment platform (C-FAP) was developed in conjunction with Cambridge Reactor Design (CRD). The C-FAP was evaluated as an assessment tool by exploring different MOCs and process conditions upon fouling and fouling mechanisms via in situ imaging and temperature measurement. The platform was characterised and used to explore surface induction mechanisms in which initiation and growth was strongly influenced by different MOCs, with stainless steel showing a greater tendency than PTFE, in addition to the degree of supersaturation. The temperature difference across the MOC interface (ΔTMOC) was demonstrated to influence nucleation and growth to varying extents. An ideal scenario would be to be able to predict or rule out unfavourable combinations of solute, solvent and MOC properties early in process design to avoid late stage problems. A screen was carried out to assess the potential to develop a multivariate predictive model for fouling propensity and fouling behaviour. The models provide insight into the most influential parameters comprising MOC, solute, solvent and process descriptors to steer subsequent experiments. The importance of MOC properties and process conditions was highlighted for all models. A variety of assessment tools were demonstrated within this work in which recommendations for fouling evaluation were provided in addition to methods to further develop fouling understanding.

Polypills have been proven to improve the patient compliance to multi-drug treatment. However it is still poorly understood when the drugs of a polypill formulation are manufactured as a mixture. The formations of co-amorphous mixtures are one of the possible results of blending multiple drugs in a single process. This project investigated the co-amorphous mixtures of two model drugs, felodipine and paracetamol prepared by melt-cool method. The rationale of the model drug selections is based on the high interaction potentials (hydrogen bonding) between the two drugs as both contain hydrogen donors and acceptor groups. Three molar ratios of the co-amorphous systems of paracetamol and felodipine (P-F) were prepared and their physical stabilities were studied using SEM, DSC, ATR-FTIR and PXRD. It was noted that there is no clear intermolecular interaction between felodipine and paracetamol was identified. Higher paracetamol content led to poorer physical stability of the co-amorphous system which also led to hindered release of paracetamol. The dissolution rates of felodipine from the co-amorphous systems showed no significant improvement in comparison to crystalline felodipine indicating the rapid recrystallization of felodipine during the dissolution process. However the linear nearly zero order release of paracetamol indicated the entrapment of paracetamol in the recrystallized felodipine lattice. This confirmed the formation of co-amorphous mixture without specific hydrogen bonding between the two species. Soluplus was used as the polymeric excipient with an attempt to further improve the physical stability of the co-amorphous systems. The stability of P-F 1:1 loaded Soluplus HME extrudates showed significant improvement than the co-amorphous sample. However no significant dissolution enhancement was observed. This could be partially attributed to the use of the extrudates in the rod form instead of power form in the dissolution tests. Controlled release of paracetamol and poor dissolution of felodipine were obtained from the HME samples indicating the poor solubility enhancement capability of Soluplus in this formulation. The results of this project provided further understanding of the behaviour of co-amorphous system which non-specific interactions.

Bacillithiol (BSH) is a recently discovered low molecular weight (LMW) thiol found amongst several Gram-positive bacteria including Bacillus anthracis, Staphylococcus aureus and Bacillus subtilis. It plays a fundamental role in the redox processes within the cell, in addition to many other functions including the detoxification of electrophiles such as methylglyoxal (MG). MG is a reactive dicarbonyl produced as a by-product of glycolysis. It is found to be toxic to the cell as it is capable of modifying macromolecules such as proteins and DNA causing loss of biological activity. The previously established glutathione-dependent glyoxalase pathway comprises of the glyoxalase I and glyoxalase II enzyme, which are found to serve as a major mechanism for the detoxification of MG amongst eukaryotes. There is speculation that BSH follows through this same pathway. Herein, the BSH-dependent glyoxalase pathway in B. subtilis is fully explored. The studies have shown a reaction between BSH and MG to occur spontaneously both in vitro and in vivo. Furthermore, these observations, have lead onto the discovery that, for the first time, BSH has shown to react with other metabolites in glycolysis. These include dihydroxyacetone phosphate, D-glyceraldehyde 3-phosphate and pyruvate. In each case they form a hemithioacetal (HTA). As a result, potentially significant concentrations of BSH may be sequestered in these ‘unknown thiol reservoirs’ which were not previously known to exist in the cell. Essentially, this raises questions regarding the true overall concentration of intracellular BSH.

Supramolecular gels are complex materials which have an expanding scope of industrial and biomedical applications, due to their unique viscoelastic properties, high biocompatibility and possibility of functionalisation. The hierarchical structure of molecular materials combines domains with drastically different degrees of ordering and molecular mobility. This makes their full characterisation a significant methodological and experimental challenge. The focus of this work was therefore the understanding of a variety of supramolecular semicrystalline gels in which very rigid solid components coexist with a dynamic and highly mobile solution phase. Using the examples of amino acid and urea-derivatives gelators, control of the self-assembly processes was successfully gained and tuning of the mechanical properties of the resulting materials by incorporating molecular structure modifications or introducing a variety of structurally diverse additives was achieved. Modification of the structure of the gel fibres was observed, which modulated the dynamic properties of the gel/solution interfaces and dictated the overall behaviour of the system, an aspect which is not commonly investigated in molecular gels. The resulting single and multi-component gels were used as model materials for the development of an NMR-based general strategy capable of probing the several hierarchical levels present. The multiphasic character of molecular gels required the combined use of solid, solution-state and HR-MAS NMR methods. This project has expanded the understanding of saturation transfer difference NMR experiments, with special focus in their applicability and limitations for the study of supramolecular soft systems. This approach was validated using complementary techniques, more specifically, rheology, microscopy, X-ray diffraction and computational methods. By combining molecular level understanding and measurements of the bulk properties, a methodology which can be applied to other soft materials used in pharmaceutical, biomedical and food science applications was developed. Moreover, this approach might have a generic impact in different fields of science and technology, enabling one to direct the recognition and host-guest properties of soft solids, which is essential for their targeted applications.

Background: Individuals with limited health literacy ability have poorer health outcomes compared with individuals with adequate health literacy. Health literacy ability is not assessed in routine healthcare environments in the UK. The objective of the thesis is to assess how healthcare professionals could identify an individual’s health literacy ability in daily practice. Methods: A systematic review of existing health literacy assessment instruments was undertaken to identify the optimal health literacy instrument for use in a clinical setting. The selected health literacy instrument was evaluated in a community pharmacy setting to provide an early indication of the feasibility for regular use. A theory based heuristic assessment instrument was developed and piloted as an alternative instrument for use in routine practice. Results: The systematic review identified the NVS instrument to be the most practical health literacy instrument to use. However, the early findings when used in practice indicated that there were barriers that could limit use. The preliminary findings of a heuristic assessment instrument indicate that recall of written potentially could be used. Conclusions: At present, there is no accepted practice to identify an individual’s health literacy ability in UK healthcare. Further research, with a larger sample size, into the use of heuristic indicators could identify a simple process to accurately assess health literacy ability that can be used in routine healthcare environments. Further work is also required to formulate more structured guidance on how to use the heuristic in consistent way so that the predictive ability demonstrated by the experienced pharmacists can be replicated by all.

Aim: The chemokine system not only coordinates leukocyte migration in immunity and inflammation, but it is also implicated in the pathogenesis of many human diseases, including cancer. The expression of chemokines and their receptors is altered in many malignancies and leads to aberrant chemokine receptor signalling. Emerging evidence indicates that the tumour microenvironment has critical roles in all aspects of cancer biology, including growth, angiogenesis, metastasis and progression. One of the important representatives of this system are the chemokine ligand CXCL12 and its receptor, CXCR4 as they are most commonly found on human and murine cancer cells. Our aims are to study and understand if there are any differences in activation of signalling molecules in the downstream signalling cascades in CXC- chemokine receptors in different cell types, and to identify the importance of different effector proteins in migration of cells; the two proteins of interest include Protein Kinase C (PKC) and arrestins. Methodology: Experimentation was undertaken in MCF-7 breast cancer cells and Jurkat leukemic T-lymphocytes which both naturally express the chemokine receptor CXCR4. Small molecule inhibition and protein overexpression was used in chemotaxis and calcium release assays to measure cellular responses. Immunocytochemistry was used to determine the effect of protein blocking and protein overexpression on receptor internalisation, protein localisation and the formation of cellular structures associated with migration. Results: Inhibition of PKC has no effect on Jurkat cell migration, but it blocks MCF-7 cell migration showing that there is a difference in the usage of PKC in different cell types. Arrestin 3 is important for migration in both suspension Jurkat cells and adherent breast cancer MCF-7 cells. Conclusion: Our study shows that CXCL12-induced migration may be arrestin 3 mediated. We have also shown that activation of signalling molecules needed for CXCL12-induced migration can differ between different cell lines. Overall, the research in this thesis has identified potential signalling molecules that can be targeted to interfere with migration of cells.

Vancomycin is a member of an important antibiotic group known as the glycopeptide antibiotics, which mainly act as bactericidal agents against Gram positive bacteria. Vancomycin acts by destroying the integrity of the bacterial cell wall and thereby exerts its detrimental effect on the most virulent gram-positive bacteria such as MRSA. Emergence of resistant strains against vancomycin has drawn scientists’ curiosity to find new analogues to restore its powerful activity or to avoid loss of its efficiency against infectious bacteria. One of the routes that has been followed to find a novel vancomycin analogue is via chemical modification to its structure relying on its well-known mechanism of action. Our approach was to synthesise the constituent amino acids of the backbone of vancomycin in Fmoc-protected form to be suitable for conjugation using solid phase peptide chemistry (SPPS). The SPPS procedure potentially ensures rapid synthesis and modification of a peptide through concise and less demanding chemistry. The central amino acid of vancomycin was synthesised in Fmoc-protected form at the α-amine terminal with a free carboxylic acid group. The synthesis proceeded through chemistry modification of the route developed by Nicoloau and co-workers and involved a series of reactions such as Sharpless AD reaction, Mitsunobu reaction and a TEMPO oxidation reaction. The Fmoc protected amino acid was successfully obtained and was potentially suitable for the conjugation with other amino acids using Fmoc chemistry and SPPS. Several trials were performed for the conjugation of the central unit on SPPS. Although different coupling reagents and solid supports were used, the results were unsuccessful. To confirm that peptide coupling with this amino acid was possible, it was demonstrated that solution phase coupling led to the successful generation of a tripeptide containing this unit. The suggestion here is that the triazene unit on the central amino acid is too unstable for incorporation into the SPPS protocol. Finally, the successful synthesis of the Fmoc-protected ethyl ester analogues of the two tyrosine isomers, amino acid 2 and amino acid 6, in the vancomycin structure was completed. Several trials were attempted to hydrolyse the ethyl ester to release the carboxylic acid but were unsuccessful. Due to the time constrains, further efforts for hydrolysis reaction attempts were halted. 1H NMR, 13C NMR, IR, and mass-spectrometry were used throughout for assignments and characterisation of all the products and intermediates of the chemical reactions.

Saturation transfer difference (STD) NMR spectroscopy is a powerful NMR technique extensively used to obtain epitope maps of ligands binding to biologically relevant protein receptors. This allows to reveal semi-quantitative structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. In this thesis, the main effort has been put to develop two novel implementations of STD NMR, aimed at elucidating the surroundings of the ligand (i.e., the amino acids lining the binding pocket, or an adjacent bound ligand) in biologically relevant complexes. First, we report the development of the novel “DiffErential EPitope mapping STD NMR” (DEEP‐STD NMR), a method producing differential epitope maps through i) differential frequency and/or ii) differential solvent (D2O/H2O) STD NMR experiments. These two approaches provide complementary information on the architecture of the binding pocket. The second novel method we propose is “Inter-ligand STD NMR” (IL-STD NMR), which relies on on-ligand differential frequency STD NMR to detect contacts between ligands bound to adjacent sites of a receptor. These novel STD NMR methodologies, in combination with traditional STD NMR and computational tools, have been applied to the study of two systems: the interactions of Cholera Toxin subunit B (CTB) with a set of promising inhibitors; and the interactions of an intramolecular trans-sialidase from Ruminococcus gnavus, a gut microbiota symbiont, with a set of mucin-related sialylated ligands. In the first study, we discovered the existence of a hitherto unknown binding subsite in the GM1 binding site of CTB. In the second study, we provided the first 3D molecular model of a Michaelis complex for an IT-sialidase. In both cases, we demonstrate that our newly developed approaches increase the level of resolution of STD NMR, widening its potential to impact the field of ligand design for biologically relevant receptors.