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Formulation studies are performed when a new pharmaceutically active ingredient (API) of interest, such as a protein, is under development in order to find conditions for maintaining the chemical and physical stability of the drug to ensure efficacy and safety for the patient (i.e. prevent any adverse effects). A commonly used approach for controlling physical stability involves the use of excipients also known as co-solvents, which are small molecules added in solution to stabilise the protein against various stresses and in particular minimize any potential aggregation events (Mahler et al., 2009). Excipients are carefully chosen to not interfere with the activity of the protein, to maintain protein chemical stability, and to assure that they are safe to administer to patients (Canchi & García, 2013). Protein-excipient interactions are especially weak and transient and controlled by multiple mechanisms with the different chemical groups exposed on protein surfaces. There is currently a lack of systematic studies which are needed to disentangle the different types of excipient interactions. It is crucial to understand these interactions to improve biopharmaceutical formulations in the future. The protein-excipient interactions determine how changing excipient concentration alters protein conformational and colloidal stability, both of which are key indicators of critical formulation attributes, such as the aggregation propensity and the concentrated solution rheological and phase separation behaviour. Here, a novel size-exclusion chromatography coupled with a multi-angle light scattering detection (SEC-MALS) technique is developed to measure preferential interaction parameters, which quantify the accumulation or exclusion of a co-solvent around the surface of a protein relative to the bulk solution. The method relies on using static light scattering as an indirect approach for measuring the refractive index increments of proteins at constant solvent and co-solvent chemical potential from which the preferential interaction parameters can be derived. In order to benchmark the method, it has been applied to five proteins (BSA, ovalbumin, lysozyme, chymotrypsinogen A and ribonuclease A) in solutions containing either sodium chloride or arginine chloride at two pH conditions. Both salts are found to be preferentially excluded from the surface of proteins, with a significantly greater exclusion for sodium chloride compared to arginine chloride. We then show the method can be applied to obtain preferential interaction parameters of denaturants with the folded and the unfolded state of proteins under the same solution conditions, which has never been done before. Interestingly, except for ribonuclease A, the folded state of the protein interacts more favourably with urea than guanidine hydrochloride. On the other hand, the opposite trend was observed for the unfolded state. The preferential accumulation of guanidine hydrochloride around proteins was reported to be significantly greater than in the corresponding concentration of urea. Such results indicate that urea and guanidine hydrochloride denature proteins through a different mechanism of action. The developed SEC-MALS constitutes a promising tool for probing protein-excipient interactions in formulations. It has been shown to provide a more rapid, consistent and accurate quantification of preferential interaction parameters than previously established methods (densimetric and vapor pressure osmometry measurements). Because SEC-MALS is already common to many research laboratories and formulation development groups, we expect the approach to be straightforward to implement. In the last part of the dissertation, the focus is on understanding how changing the solvent from regular to deuterated water alters the protein colloidal stability characterized in terms of protein-protein interaction measurements. The isotropic solvent substitution effect was investigated on the NIST (National Institute of Standards and Technology) monoclonal antibody using dynamic and static light scattering to probe protein-protein interactions as a function of ionic strength, pH, and temperature. By fitting the results to a simplified protein-protein interaction model, the solvent isotope effects on electrostatic interactions are separated from the effects on other short-ranged attractive contributions to the net interaction potential. Interestingly, we find that electrostatic interactions are enhanced in deuterated versus regular water, while the solvent isotope effect on the other short-ranged interactions follows what would be expected for hydrophobic interactions, which are enhanced in deuterated water.

Protein-based therapeutics are widely used for the treatment of different diseases such as anti-cancer and anti-diabetes drugs. Throughout the manufacturing and formulation process of protein drugs, the protein stability must be sustained for long-term storage and the formulation must be used for efficacious delivery that avoids adverse immunogenic side effects. The main physical degradation process is by protein aggregation, which can be defined as a multi-stage process in which mis-folded proteins irreversibly associate with each other forming a soluble or insoluble aggregate. A key stumbling block to predicting aggregation is that key intermediates in the pathways are partially folded proteins, which occur very transiently and at low concentrations during typical formulation conditions. Traditional approaches to overcome this problem use increased temperature to increase the populations of the aggregate precursors thereby accelerating the aggregation process so that it can be studied on reasonable timescales. A common prediction depends on the Arrhenius relationship between protein degradation and temperature. Unfortunately, this approach is not often successful because many proteins do not follow Arrhenius kinetics due to exhibiting multiple aggregation pathways with different temperature dependencies (Wang and Roberts, 2013). This thesis is directed at understanding the aggregation behaviour of protein solutions under chemically denaturing conditions towards developing an alternative predictive approach for assessing storage stability. As such, ovalbumin solutions were studied over a range of denaturant concentrations in the absence of excipients and in the presence of series of excipients (ArgHCl, ArgGlu, NaCl, Na2SO4, NaSCN, ATP, STPP, trehalose and sucrose), using static and dynamic light scattering to identify and better understand the aggregation and find an alternative for estimating the colloidal stability, which is an important indicator for protein aggregation.

Protein and peptide drugs have gained increasing importance in the portfolio of pharmaceutical companies reflected by the growing number of approved drug products in the US and Europe. One of the key challenges represents physical instability of the drug substance, in particular processes of self-association. Several excipients are available to modulate the underlying protein-protein interactions responsible for self-association processes. Here, Cucurbit[7]uril, a macrocycle able to bind hydrophobic amino acid residues is examined for its potential to improve physical stability of protein and peptide therapeutics. Studies presented here, compare solution behaviour of proteins and peptides with mutated Cucurbit[7]uril binding sites examining whether addition of Cucurbit[7]uril is capable of mitigating or fully suppressing self-association processes. One study determines the effect of Cucurbit[7]uril on the aggregation propensity of a monoclonal antibody. Results provide evidence of specific interaction between Cucurbit[7]uril and aromatic amino acid residues located on an aggregation-prone region resulting in improved solution behaviour. Another study, determines the effect of Cucurbit[7]uril on the kinetic profile and morphology of Enfuvirtide fibrils, a commercially available peptide drug. Modulation of fibrillation onset via Cucurbit[7]uril is monitored by Thioflavin T assays and circular dichroism spectroscopy. In contrast to previous reports, findings presented here, indicate Cucurbit[7]uril's potential to delay rather than fully suppress fibrillation onset through binding of a C-terminal aromatic amino acid residue. Furthermore, this work contains a study evaluating a high-throughput PEG solubility assay for its correlation with the diffusion interaction parameter. Conventional methods used in solubility assessment of proteins have high material consumption and are not amenable to high-throughput screening. A set of 8 mAbs formulated at 15 different solution conditions is used to investigate correlation with observations made from DLS measurements. Results highlight correlation of trends observed in PEG-induced phase separation with averaged protein-proteins interactions as described by the diffusion interaction parameter.

Therapeutic monoclonal antibodies comprise the largest class of biopharmaceuticals on the market. Antibody therapeutics are generally administered intravenously and subcutaneously. Subcutaneous administration is volume limited so antibody concentrations in excess of 100 mg/ml are required for adequate drug potency. This requirement can pose a myriad of problems to the manufacture, storage and administration of antibody therapeutics; at high protein concentrations intermolecular distances between molecules mean that attractive protein-protein interactions can start to dominate. Attractive protein-protein interactions have been linked to undesirable solution behaviour such as opalescence and liquid-liquid phase separation. The overall aim of this project is to study the behaviour of high concentration antibody solutions with a focus on their behaviour as they undergo phase separation. The central goal is to demonstrate the link between protein-protein interactions and highly concentrated protein behaviour. Correlations between dilute solution measurements of protein-protein interactions and concentrated solution properties are explored and compared using interaction models. The complicated behaviour of antibody solutions at high protein concentrations which is often referred to as reversible self-association and leads to the formation of oligomers is discussed in terms of protein-protein interactions. Finally, the use of protein-protein interactions as characterised by the second osmotic virial coefficient and the relationship to liquid-liquid phase separation is explored. The contributions in this thesis provide an initial investigation that suggests some monoclonal antibody solutions exhibit universal behaviour.

When developing a new monoclonal antibody, the formulation is key in improving the stability of the concentrated therapeutic. The formulation design ensures the therapeutic protein is conformationally and colloidally stable and maintains a long shelf life. Excipients are added to improve the stability of proteins and are usually added at high concentrations to exert excipient effects. The aim of this thesis is to explore the effects of low concentration additives to improve conformational and colloidal stability of model proteins. Several parameters were used to measure conformational and colloidal stability. The melting temperature (T_m), midpoint of denaturation (C_m) and change in free energy between the native and unfolded state (DeltaG) measurements were conducted to measure conformational stability. Measuring colloidal stability involved static light scattering (SLS) measurements to obtain second virial coefficient (B₂₂) measurements and onset temperature of aggregation (T_{agg}) from SLS and dynamic light scattering (DLS) thermal ramps. DLVO model was applied to obtain the Fuchs stability ratio, which describes the probability of two aggregates sticking together following collision, giving another parameter to assess colloidal stability of different solution conditions. Chapter 3 highlights the effects of low concentration of tripolyphsophate (TPP) and adenosine 5'-triphosphate (ATP) on aggregation propensity. The binding of 5 mM TPP and ATP to an acidic protein, recombinant human serum albumin (rHSA), increased repulsive protein-protein interactions (PPI). This in turn significantly reduced aggregation propensity, which we show was due to an increase in the repulsive barrier upon the approach of two rHSA molecules, as quantified by the Fuchs stability ratio. Chapter 4 explores the effects of low concentrations of arginine and glutamate containing dipeptides on the aggregation behaviour of three model proteins. The effects exhibited by the dipeptides could be explained by electrostatics, the addition of the dipeptides to rHSA and ovalbumin, the most repulsive proteins, increased aggregation propensity. On the other hand, addition of the dipeptides to alpha-chymotrypsinogen, which exhibited attractive PPI, improved colloidal stability. The final chapter uses chemical denaturants to observe excipient effect on conformational and colloidal stability of partially and fully unfolded protein. Colloidal stability of unfolded protein is also defined by the size of aggregates formed following dilution refolding. These experiments provide a new method to predict aggregation propensity. Low concentration excipients are developed in this thesis to provide a novel way to reduce protein aggregation. Excipients are also used to determine effects on colloidal stability of partially unfolded protein.

The success of a monoclonal antibody drug product is predicated on the manufacturability and stability of the concentrated therapeutic protein solution. A key factor in facilitating the processing, packaging and administration of monoclonal antibodies is the formulation design. The development of an optimal formulation maintains the conformational and colloidal stability of the protein, and ensures the viscosity of the solution is minimised. Formulation design is dependent on reliable measurements of nanoscale protein properties and interactions which can be related to macroscopic bulk solution behaviour. Analytical techniques to better facilitate formulation design, especially in relation to solution viscosity, are needed. The aim of this thesis is to develop methods to measure parameters which bridge our understanding of protein-protein interactions to bulk solution properties. The grounding of these experimental techniques in theoretical models of molecular properties and solution structure is an objective of this project. The use of differential viscometry to measure the Huggins coefficient is evaluated as a method for the optimisation of protein solution viscosity. The relationship of high protein concentration solution viscosity to protein-protein interactions is explored by measuring viscosity using a microfluidic rheometer and comparing it with results from static and dynamic light scattering experiments. Correlations between dilute solution parameters and the viscosity of concentrated mAb solutions are evaluated. The applicability of models relating the electrophoretic mobility experimentally determined with electrophoretic light scattering to monoclonal antibody net charge is examined. The contributions in this thesis widen the scope of analytical testing of monoclonal antibodies to complement therapeutic protein formulation development.

Antibodies developed against tumour cell-specific surface antigens often display limited therapeutic activity, hence alternate strategies have been explored to enhance their activity including antibody-drug conjugates (ADCs). ADCs have emerged from the combination of an antibody with a highly potent cytotoxic agent that lacks target specificity, thereby mAb and small molecule limitations can be overcome. As a result, ADCs present a synergic therapeutic effect, while minimising undesired side-effects and off-site toxicity. Despite their immense potential, only six ADCs have been approved by regulatory authorities to date, due mainly to the substantial challenges encountered during their complex development. The interplay between the antibody, the linker and the payload suggests that a fine balance is essential to guarantee efficacy. It becomes, therefore, evident why rational design approaches are required to simultaneously address the issues pertaining to their development. In this work, several approaches were pursued to generate a pool of rationally-designed ADCs by varying several factors, such as the site and amount of drug molecules conjugated to cysteine-engineered Trastuzumab, the linker functionalisation by inclusion of hydrophilic polymeric moieties and the formulation composition. Ad hoc mutations were introduced at the CH2 (i239C), CH1 (A118C) and CH3 (S442C) domains of Trastuzumab, which allowed the sitespecific conjugation of four different Pyrrolobenzodiazepines (PBDs)-payloads from the Spirogen library (Spirogen Ltd, a member of the AstraZeneca Group). This in turn led to the generation of a novel panel of ADCs, which were subsequently characterised in different formulations with respect to their (bio)physical properties. Conformational stability was investigated through differential scanning fluorimetry (DSF) and dynamic light scattering (DLS), whilst colloidal stability was evaluated by static light scattering (SLS). In addition, pre-formulation and formulation stability studies provided useful insights on the effect of different excipients (NaCl, ArgGlu and PEG400) on the physical and chemical stability of the ADCs in the liquid state. The influence of the mutations on the conformational and colloidal properties of Trastuzumab both pre- and post-conjugation were investigated. This revealed significant alterations in the transition of unfolding for the mutants and ADCs that underwent mutation at the CH2 domain and increased self-association propensity for the other mutants and their respective ADCs in the NaCl-containing formulation. In addition, pre-formulation studies enabled the rational design of the formulation, which was composed of histidine at pH 6.8 and also contained either 154 mM NaCl, 120 mM ArgGlu or 7% v/v PEG400. The physical and chemical stability of the in- house ADCs was then investigated in real-time (6 months at 5 °C), accelerated (6 months at 25 °C), and stressed (3 weeks at 37 °C) conditions. Finally, excipient screening studies (NaCl, ArgGlu and PEG400) allowed the identification of the factors involved in the purity reduction and payload deconjugation. Hydrophobic interactions were found to be the driving force for aggregation and the hydrolysis of the succinimide ring at the linker moiety seemed to be the best approach to ensure linker stability in the liquid state. Eventually this work covered all the different stages involved in drug design, generation and development of such a complex system as an ADC, thus enabling a deeper understating of the different behaviours observed in the biophysical characterisation and formulation development. Among the parameters under investigation (e.g., conjugation site, chemical composition of the payload and formulation composition), the modulation of the conjugation site was deemed the most effective at improving the ADC's stability.

Soft hydrogels based on self-assembling peptides are widely used in tissue engineering because they can mimic different environments of extracellular matrix. Commonly in these hydrogels, the peptide chains mainly contain at least 10 amino acids. It is also found that peptides with short chains (i.e. less than 10 amino acids) can form fibrous hydrogels with secondary structures, α-helical coiled-coil and β-sheet structures. However, such short peptide-based hydrogels normally suffer from the relatively low stiffness, i.e. relatively storage modulus (lower than 100 Pa), limiting their applications. One strategy to address this issue is to employ synthetic polymer to be crosslinked with the peptide chains to prepare the hybrid hydrogels with the improved storage modulus. In this PhD project, β-sheet structure peptide FKFEFKFK (FK) was first explored to form the hydrogel by adjusting the system pH value. Then, the FK peptide was crosslinked with polyethylene glycol (PEG) via non-covalent bonding to give the hybrid hydrogels with tuneable mechanical property, which can be varied by changing the ratios of FK to PEG. Also, natural amino acid (i.e. cysteine) modified peptides of CFKFEFKFK (CFK) and CFKFEFKFKC (CFKC) were crosslinked with maleimide PEG via covalent bonding. PEG with molecular weight 2 kDa, 5 kDa were used to synthesis with peptides to study the effect of chain length of conjugates on the properties including solubility and gel formation ability. It was found that the resulting conjugate, i.e. CFK-PEG5k-CFK (TRI), can form the stiff hydrogel on its own due to its approximate hydrophobicity. Furthermore, hybrid hydrogels with tuneable viscoelasticity can be obtained simply by mixing FK and TRI with different ratios of FK to TRI. Peptide CFKC was crosslinked with PEG with high molecular weight, i.e. PEG 20 kDa, to prepare the conjugate of BC-20k, which can form not only hydrogel but also dry film. We demonstrated that the BC-20k hydrogel has a remarkably enhanced viscoelasticity (200 Pa with peptide concentration 5 mg/ml) and the relevant BC-20k film has a good elasticity, i.e. the strain can reach until 15%.

The purpose of this thesis was to investigate and characterise basic physicochemical characteristics of short-chain SFAs (Semi Fluorinated Alkanes) and various SFA-excipient mixtures, to provide a foundation for the future use and application of these interesting compounds. Excipients are inactive substance that improve the overall effectiveness of a formulation. Only a handful of studies have been carried out on short chain SFA molecules with a majority of the work from molecular models and simulations. Therefore this thesis aims to provide novel analytical studies of SFA solution behaviour. This study uses predominantly nuclear magnetic resonance chemical shift and integral data with respect to excipient concentration to provide ternary phase diagrams, solubility and molecular interaction insights to further understand interactions of SFA molecules with themselves and with other small molecules. The interest in SFAs stems from their inert, hydrophobic and surfactant-like properties. The surfactant properties of SFAs are of interest because they do not contain a hydrophilic segment. NMR chemical shifts have been used to explore the molecular environmental changes in SFA mixtures mainly with respect to various organic excipients/additives, including ethanol, propanol, tetradecane, perfluorooctane, caprylic/capric triglyceride, isopropyl myristate, isopropyl palmitate, ethyl acetate, diethylene glycol monoethyl ether, dimethyl isosorbide and propylene glycol laurates, as well as temperature. These organic compounds were chosen as they provide a varied amount of functional groups and are used regularly as excipients. NMR integrals were used to determine compositions in phase separated samples with and without the addition of water to provide liquid-liquid equilibrium data to be used in order to provide miscibility boundary conditions and extraction behaviour in the presence of water. The chemical shift data indicated SFAs F6H8 and F4H5 exhibit preferential interactions in hydrocarbon and perfluorocarbons mixtures. Pure F6H8 was investigated separately by determining the temperature dependence of NMR chemical shifts and coupling constants, which when complemented with molecular simulations, indicated preferential interactions between the like-segments of SFAs at lower temperatures. The degree of association was less when SFAs were mixed with compounds containing long hydrocarbon chains and polar segments, such as caprylic/capric triglyceride, isopropyl myristate, and isopropyl palmitate, when compared to completely non polar tetradecane and perfluorooctane. In mixtures with smaller polar molecules like ethyl acetate, the NMR chemical shift changes were seen to be proportional to the additive concentration, which indicated close to ideal mixing with little preferential interactions. Furthermore, chemical environments of hydrogen diblocks of F6H8 and F4H5 changed much less with addition of excipients when compared to the perfluorocarbon diblocks suggesting that the driving force of association in the mixtures are the perfluorocarbon SFA segments. Ternary phase diagrams and liquid-liquid extraction data was used to derive empirical relations, to determine the saturated threshold water content reachable before phase separation would occur for F6H8 or F4H5 mixtures with either ethanol or propanol.

Microalgae are attractive platforms for the production of high-value products such as recombinant proteins due to the low-cost and the short timeframe of the production process. Moreover, microalgal culture is scalable to relatively high volumes and densities under controlled conditions facilitating biosafety and their expression systems enable the expression of complex recombinant proteins. However, the lack of reproducible genetic transformation systems for most of the microalgae with potential in biotechnology and the low accumulation levels of recombinant proteins are still major limitations for the progress of this technology. This thesis reports the development and test of new molecular tools for the genetic engineering of commercially important microalgae. For this aim, the chloroplast genome of the haptophyte alga Tisochrysis lutea was sequenced, assembled and characterised in chapter 3. In chapter 4, contrary to previous reports, the stable and uniform expression of the recombinant protein Viral Protein 28 from White Spot Syndrome Virus was achieved in several lines of a plastid engineered Chlamydomonas reinhardtii strain. The accumulation level was estimated at 1% of total cellular protein under the regulation of psbD regulatory regions. Reproducible transformation of marine microalgae is the first step for their use as hosts for high-value products. In this regard, the assembly of transformation constructs suitable for Dunaliella tertiolecta and Tisochrysis lutea was accomplished in chapter 5 and 6 of this research. The limited number of efficient selectable markers available for marine algae has driven the development of herbicide based selection markers. The use of algal herbicide resistant genes is desirable for biosafety and commercialisation regulations since the use of antibiotics is avoided. In section 5 of this thesis, a mutated Dunaliella salina gene encoding phytoene desaturase carrying a F131V amino acid substitution was tested in Dunaliella tertiolecta as selectable marker for norflurazon based selection of chloroplast transformants, whilst a mutated Dunaliella tertiolecta gene encoding phytoene desaturase with a R265C amino acid substitution was explored in nuclear transformation of Dunaliella tertiolecta. Finally, an engineered Tisochrysis lutea phytoene desaturase containing a R268T amino acid substitution allowed the recovery of norflurazon resistant lines carrying the selectable marker. The adaptation of available transformation technologies enabled the nuclear transformation of both marine algae using the bacterial ble selectable marker gene under the control of the Dunaliella tertiolecta rbcS and the Tisochrysis lutea actin regulatory regions and zeocin selection.

An impurity such as sulfur reduces the quality of fossil fuels and contributes significantly to air pollution, which has serious harmful effects on the environment. Therefore, this research project focuses on the development of biocatalysts based on plant cell cultures for the desulfurization of crude oil. Microbial biodesulfurization has been investigated for a few decades now. Yet there is no process based on biodesulfurization. There are many reasons for this, the main ones involve the difficulty of separating cells from the crude oil and hence the problem of consumption of the hydrocarbons by the cells leading to the loss of value of the crude oil, and high temperatures of the crude oil not being suitable for microbial cultures. The biogenesis of sulfur compounds in fossil fuels is caused by the death and decomposition under high temperature and pressure in the earth's crust of the large populations of plants and animals over geological time. Plants produce complex sulfur compounds and therefore must have enzymes that can degrade sulfur compounds. Use of plant cell cultures for biodesulfurization was the novelty of this project. One of the pathways of bacterial biodesulfurization was elucidated as the 4S pathway. Using the identified enzymes in the 4S pathway, equivalent enzymes in various plant species were searched using databases such as KEGG, and molecular docking software SwissDock. Specially for Nicotiana tabacum (tobacco) as the chosen plant for this research, feruoyl coenzyme A 3-O-methyltransferase (1sus) was 7.8 times more efficient than 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB) enzyme used in the last step in 4S pathway in a biodesulfurizing micoorganism like Rhodococcus erythropolis. Three plant species, Arabidopsis thaliana, Nicotiana tabacum (tobacco), Armoracia rusticana (horseradish) were cultured. Nicotiana tabacum was the chosen plant for the biodesulfurization experiments due to the good growth characteristics and minimal contamination problems. Dibenzothiophene (DBT) was the model sulfur compound used for testing the biodesulfurisation ability of the cultures. The control experiments and biodesulfurization experiments were performed in aqueous medium in shake flasks using Murashige-Skoog (MS) and sulfur-free Murashige-Skoog (SFM) media supplemented either 100 or 200ppm DBT. The MS with 100ppm DBT experiment had the highest maximum biomass concentration at 24.46 g/l by day 15, 95% of DBT was degraded by day 15 and the average concentration of 2-Hydroxyblphen (2-HBP) produced was 66.3ppm. In advance of adding crude oil to the biodesulfurization experiments some tests were done and confirmed that cells could stay alive after contacting with crude oil, this was achieved by looking the cells treated with fluorescein diacetate staining (FDA) under UV microscope. The cells were allowed to have contact with crude oil for 24 h then washed and grown in MS normal medium for around 2 weeks. For the biodesulfurization experiments involving crude oil, the MS with 100ppm DBT and SFM with DBT at 100, 200, 300 and 400ppm were performed in airtight vessels for safety reasons. The 100ppm DBT concentration in SFM had the highest maximum biomass concentration at 26.7 g/l in day 21 and 97.7% of DBT degradation, and fluctuated concentrations of 2-HBP with an average of 46.31ppm. A kinetic model was used for some of the batch experiments; although preliminary attempt prediction was promising, nevertheless the kinetic model needs further refinement with further experiments in order to obtain the model parameters more accurately.

The overall aim of this PhD project was to use chemical denaturants such as urea and guanidine hydrochloride (Gdn HCl) to increase the relative population of intermediate and/or unfolded states of proteins as known precursors to protein aggregation. Measurements of protein-protein interactions (PPIs) under denaturing conditions of both the model protein lysozyme and a set of five pharmaceutically relevant monoclonal antibodies (mAbs) were made. Light scattering techniques were employed including static light scattering (SLS) to determine the second osmotic viral coefficient, B22 and dynamic light scattering (DLS) to determine the interaction parameter, kD. SLS and DLS was also used to measure aggregate growth rates under denaturant and/or temperature accelerated conditions and were ratified by measuring monomer loss kinetics using size exclusion chromatography couple with multi-angle laser light scattering (SEC-MALLS). The data generated in the first results chapter highlighted the importance of measuring the refractive index increment of the protein under constant solvent chemical potential, to ensure correct values of B22 and molecular weight at infinite protein dilution are obtained as well as excluding short delay times of an autocorrelation decay in solutions of high co-solvent concentrations to similarly calculate correct values of kD and hydrodynamic radius at infinite protein dilution. The data generated in the second and third results chapter showed that PPIs of intermediate, aggregate prone states could be quantified using SLS at high protein concentrations (20 g/L) and were related to the aggregation propensity of the mAbs under both native and denaturing conditions. Furthermore, addition of 0.5 M arginine HCl under denaturing conditions caused a reduction in aggregation rates and concordantly an increase in repulsive PPIs.

Microorganisms normally co-exist with other species in nature, so it is necessary for them to develop their own ability to survive in their habitat competing for the common nutritional resources. Following millions of years of competition with such interspecies interactions, microorganisms would have developed the mechanisms of recognition of the presence of a competitor and the defense mechanisms to produce some bioactive compounds such as antibiotics to fight with other species. As the largest antibiotic-producing genus, Streptomyces species produce most antibiotics that are currently used in medicine and agriculture. Streptomyces coelicolor A3(2) that belongs to Streptomyces species can produce many compounds with bioactivities including four known antibiotics; undecylprodigiosin and actinorhodin which are pigmented, and the other two that are colourless, methylenomycin and calcium-dependent antibiotic (CDA). Because undecylprodigiosin has been found to have immunosuppressive and anti-cancer activities and can be used as a novel drug for breast cancer treatment, it has become an active research topic. However, in laboratory conditions, the production of undecylprodigiosin by Streptomyces coelicolor is very low in pure culture. The hypothesis of this research was that the production of secondary metabolites, especially antibiotics, would increase in the presence of another microbial species as this would create competition. The aim of this research was therefore, to investigate the effect of interaction with live and dead cells as well as cell-free supernatant of E. coli culture on antibiotics production by Streptomyces coelicolor MT1110 and wild-type Streptomyces coelicolor A3(2) cultures. With the elicitation by E. coli C600 (live cells and supernatant), S. coelicolor MT1110 and wild-type S. coelicolor A3(2) produced 3.5-fold undecylprodigiosin concentration than the pure culture and the production level of actinorhodin decreased to one fifth of the pure culture. In the bioreactor experiment of wild-type S. coelicolor A3(2), the elicitation by E. coli (live cells and supernatant) reduced the maximum actinorhodin concentration by two thirds and increased undecylprodigiosin concentration by 2.5-fold. In a set of experiments, phthalic acid was used for the elicitation of S. coelicolor MT1110. When elicited with 15 Î1⁄4M phthalic acid, the maximum actinorhodin concentration was 3.684 mg/L, which was one-third of the concentration obtained in the pure S. coelicolor culture; the maximum undecylprodigiosin concentration was 1.676 mg/L which was 1.5- fold of that achieved in the pure culture. A kinetic model of S. coelicolor was developed in order to obtain quantitative comparisons of experimental results. Furthermore, Metabolic Flux Balance Analysis (MFBA) was applied to some of the experimental results using a previously developed metabolic model and integrating phthalic acid metabolism with it in order to test the feasibility of using MFBA as a computational tool for the development mechanisms of microbial interactions, and the design of the relevant experiments. Indeed, both the kinetic model and the MFBA gave encouraging preliminary prediction of the experimental results. They are however, not perfect and need further development. If they can be refined, they can be additional research tools in the future research in the microbial inter-species interactions and especially for the search for and production of novel antibiotics.

Antibodies and antibody fragments are the largest class of biotherapeutics in development with many products already available in the clinic. Antibodies are promising due to their naturally high affinity and specificity for biological targets. A key stumbling block to biopharmaceutical development compared to small molecule drugs is the general requirement for a stable liquid formulation, which is often difficult to obtain due to issues with aggregation, phase separation, particle formation, and chemical instabilities. Aberrant solution behaviour limits the production, storage and delivery of the monoclonal antibody. Biopharmaceutical solution behaviour is determined by weak, transient protein-protein and protein-solvent interactions. An attractive interaction potential between proteins in solution can lead to association. Irreversible association occurs when proteins undergo large scale structural changes and aggregate. Reversible association is less severe, but can lead to undesirable solution properties such as high viscosity, phase separation and opalescence, which can lead to difficulties throughout the downstream processing and formulation steps. These problems can become exacerbated during formulation of antibodies when trying to achieve high protein concentrations often required for effective antibody dosage. Firstly, we studied the domains of the Fab fragment using statistical models and continuum electrostatic calculations and found that the CH1 domain is more soluble than the other domains and has properties of intrinsically disordered like proteins which is supported by observations in the literature. We then investigated the immunoglobulin superfamily and found 11 proteins which may have a similarly disordered nature. We present a new web server for predicting protein solubility from primary sequence using an in-house algorithm that weighs the contribution of various sequence properties for predicting solubility. Lastly, we conducted physical characterisation of an antibody and human serum albumin in pharmaceutically relevant buffers and found that the interaction potential can be modelled using spherical models from low to high protein concentration. We hope that the work outlined in this thesis will contribute to the theoretical understanding and modelling of protein solution behaviour.

Biopharmaceuticals (e.g. monoclonal antibodies (mAbs)) must comply with regulations (i.e. United States Pharmacopeia (USP) ) regarding their characterisation and stability. mAbs contain hydrophilic and hydrophobic areas (the latter normally buried inside the bio-macromolecule). Stress conditions (such as high temperature, freezing, shaking etc.) or high concentrations may lead to the exposure of hydrophobic surfaces (i.e. following unfolding) and subsequently lead to the formation of protein aggregates. Furthermore, high concentrated mAb products (i.e. >100mg/ml) have the increased risk of intermolecular interactions which is correlated with high viscosity. These issues pose significant challenges to the economic manufacture of safe and effective protein therapeutics. Current technologies in characterising protein formulations possess limitations in regards to size ranges, specificity, interacting with solution components and concentration; thus there is a current drive in the development of novel applications. This thesis studies the application of two fluorescence-based techniques in characterising mAb solutions, in the context of downstream processing and formulation: Raster image correlation spectroscopy (RICS) analyses to assess aggregation propensity; and fluorescence correlation spectroscopy (FCS) in retrieving viscosity information. An important step for both methods is the identification of appropriate (non-covalent) fluorescent probes. To validate the application of RICS in charactering mAb aggregates in industrially relevant formulations, particle formation (size and counts) in pre-filled syringes was evaluated as a function of polysorbate-20 (PS-20) concentration, following agitation stress. PS-20 limited agitation-induced aggregation whilst increasing the amount of silicone oil sloughing. Thus no correlation between silicone oil and aggregation was observed. Following extrinsic labelling of aggregates by hydrophobic dye SYPRO Red, RICS demonstrated its high specificity to aggregates in mAb solutions containing surfactant and silicone oil. An improved selectivity was observed in comparison to resonance mass measurement (RMM) and micro-flow imaging (MFI), covering a broader size range and using small sample volumes. Although nonionic surfactants such as PS-20 are widely used, their mechanisms in mAb solutions are poorly understood. This is partly due to analytical limitations of current technologies. The application of FCS with utilising SYPRO Orange is validated in accurately determining the critical micelle concentration of (three) nonionic surfactants, along with the micelle size. Moreover, the FCS/SYPRO Orange application is used to detect polysorbate micelles in the presence of high concentration mAb and thus provides scope to assess micelle behaviour in highly concentrated mAb formulations. As an additional method to measure the microviscosity of mAb solutions, FCS was utilised in measuring the self-diffusion of tracers in a wide range of mAb formulations, over a broad concentration range. The diffusion of different sized tracers was investigated and compared against bulk rheometry measurements. It was established a probe of size equal or larger than the mAb gave relatable information to bulk rheometry. RICS and FCS were (sequentially) applied to mAb solutions subjected to various forms of agitation stress. A correlation was established between aggregation development (size and counts) and changes in solution viscosity. Additionally an inverse relationship of agitation-induced aggregation and protein concentration was observed. The potential of measuring aggregation propensity and solution viscosity using the same system set-up is of great interest to the industry due to small sample material and minimal operating time. Thus the combined application of RICS and FCS has the potential to stand as a tool in the characterisation of mAb (aggregate) solutions, particularly in relation to early formulation development.

The production of steroid and drug metabolites is a key area within the pharmaceutical industry. For a drug to pass safety profiling, large amounts of its metabolites are required to undergo extensive toxicology studies. Steroid compounds can be used in the treatment of chronic inflammatory disorders and also demonstrate efficacy in reducing cancerous tumour mass. A key step in the synthesis of these compounds is the insertion of an oxygen atom into a specific C-H unactivated bond. Cytochrome P450 (P450s) circumvent the limitations of synthetic methods for production of high value oxychemicals. The bacterial fatty acid monooxygenase P450 BM3 (CYP102A1) from Bacillus megaterium has the highest recorded P450 monooxygenase activity and is expressed in high yields in Escherichia coli. A plethora of P450 BM3 variants have demonstrated the proteins’ amenability to mutations that alter its substrate binding profile towards non-natural substrates, leading to generation of valuable metabolites. This thesis provides novel information to add to the P450 BM3 knowledgebase. This work shows how a rational design approach, leading to the generation of a P450 BM3 A82F/F87V/I401P triple mutant (TM), results in the protein having a broad substrate selectivity profile for non-natural substrates. The TM BM3 enzyme catalyses the production of not only proton pump inhibitor (PPI) drug metabolites, such as 5-OH-omeprazole, but also oxidised steroid compounds such as 17-OH-progesterone. This is the first reported production of this key steroid pathway intermediate using bacterial P450 methods. This work demonstrates how only three influential point mutations can lead to activity with a wide range of novel substrates, through the destabilisation of the P450 BM3 protein. Further work details a different approach towards P450 BM3 engineering, where the aim was to produce two P450 BM3 variants that can generate 16α- and 16Î2-OH-testosterone. A multi-step screening effort identified two variants that produced both the desired products at >90% selectivity and conversion. The final piece of work in this thesis demonstrates how inclusion of a L86I mutation in the TM BM3 enzyme generates a quadrupule mutant (QM) (A82F/F87V/I401P/L86I) and leads to an overall increase in relative product formation for several tested substrates (including troglitazole, testosterone and omeprazole) and in some cases a change in product profile compared to the TM enzyme. This work led to the first reported production of OH-artemether, which has potential as an anti-malarial drug.

Loss of solubility and aggregation of proteins are important bottlenecks in modern bioprocessing pipelines, where formulation and large-scale production of therapeutic proteins such as antibodies is achieved. The mechanistic basis of protein aggregation propensity and solubility are actively investigated using experimental and computational techniques. A significant part of research in this field involves efforts to understand how sequence- and structure-based properties enable proteins to remain functional under conditions and conditions relevant to physiology and delivery of biotherapeutic agents. Using sequence-based and structural features as well as physico-chemical properties, a model was developed to study how such descriptors can be used in a predictive capacity to separate soluble and insoluble proteins. Therapeutic protein datasets including antibody derivatives and non-antibody biologics were constructed so that their solubility could be studied using the descriptors. Surface charge, polarity, and sequence composition were tested against established thresholds for solubility of E. coli proteins. Surface non-polarity was verified as a consistent feature for separating soluble and insoluble therapeutics, in line with its established role as a key player for determining aggregation propensity in the broader scientific community. The ratio of lysine to arginine composition emerged as a novel sequence-based feature that contributes to solubility, where higher lysine composition is favourable for the solubility. There is potential to use this as a method for engineering proteins for higher solubility with minimal disruption to functionality. The predictive model was subsequently expanded to include a broad array of sequence-based and 3D structural features. Quantitative proteomics studies with high-throughput data for protein solubility, abundance and concentration were used to construct datasets. Web accessible repositories of protein abundance in several species and plasma protein concentration were used to augment the data used to validate the model. Our findings reiterate previously established studies regarding protein length, charge-based properties and surface non-polarity as important descriptors for discriminating soluble and insoluble proteins. The sequence-level lysine/arginine ratio offers a novel perspective on potentially simple ways of protecting proteins against aggregation, which could prove useful for bioprocessing pipelines. Protein-excipient interactions were studied using a dot product metric to measure the association of a set of crystallisation screen ligands with proteins in the PDB database. Enrichment for predicted small molecule (sugars and buffers) binding sites was observed, although the underlying reasons remain unclear without more sophisticated structure-based techniques.

The need to high quality therapeutic proteins has grown significantly in the past 30 years. Recombinant proteins are often produced from vectors inserted into E. coli cell lines for large scale production. However, over-expression of the protein within the cell can lead to the formation of large, insoluble aggregates known as inclusion bodies. Native monomer protein can be isolated from inclusion bodies through a refolding process. This entails disruption of the aggregate structure with high concentrations of denaturant and renaturation in native-promoting solution. Our work characterises protein-protein interactions and aggregation between partially unfolded proteins during the refolding process. The protein-protein interactions are characterized in terms of the osmotic second virial coefficient (B22). A positive value indicates repulsive interactions while a negative value indicates attractive interactions. Measurements are carried out for lysozyme, ribonuclease A and preproinsulin as a function of pH, ionic strength and denaturant concentration, alongside a range of known refolding excipients. Past studies (Ho and Middelberg, 2004; Ho et al., 2003) have shown a link between higher B22 values in denaturant solutions and reduced aggregation during refolding. Our experiments have focused on the effects of urea and GdmHCl upon protein-protein interactions, alongside how ionic strength and refolding additives influence interactions between partially-folded states. At low ionic strength, solutions of urea increase net repulsive interactions compared to GdmHCl solutions through an attenuation of short-range attractive interactions. Electrostatic repulsive interactions are screened in solutions of GdmHCl due to the increased ionic strength of the solution; however short-range attractive interactions are also attenuated in a similar fashion to urea solutions. Protein-protein interactions in low and high concentration denaturant solutions have been shown to be highly sensitive to ionic strength and refolding experiments have shown that this correlates with increased aggregation during refolding. The solubilising additive Arg HCl has been shown to reduce short-range attraction between proteins in urea solutions, while the folding-promotor additives sucrose and hexylene glycol have been shown to have a more complex effect on protein-protein interactions in urea solutions dependent on denaturant concentration. Within the wider context of the field of protein aggregation and refolding, the work conducted here will contribute towards the understanding of how denaturants and solutes influence attractive protein-protein interactions and aggregation behaviour between unfolded or partially folded proteins.

Protein misfolding and aggregation are the cause of many problems within the biopharmaceutical industry and medical fields. Although many experimental studies have been implemented in vivo in order to understand this process, the mechanism occurs in time and length scales inaccessible to conventional experiments. On the other hand, computational studies have shown significant improvement in elucidating key aspects of the aggregation pathways and gain insights to the folding behavior of the proteins. Consequently, this makes computational modeling an ideal complement to experiment in understanding the generic behavior and mechanisms of aggregation. This study is concerned with DynamO, a coarse-grained, off-lattice, general event-driven discontinuous molecular-dynamics simulation package. This simulator offers a unique opportunity to gain insight into the process of protein aggregation by displaying the optimal O(N) asymptotic scaling of the computational cost with the number of particles N, rather than O(NlogN) scaling found in most standard algorithms. The study was split into two loosely related projects: in the first project, a computer model was developed in which the effect of model parameters on folding behavior and characteristics of isolated peptides is investigated. The model parameters include chain stiffness (an overlap parameter defined as the ratio of the hard-core diameter to bond length 'sigma/l'), range of interaction potential 'Gamma', sequence, and chains length 'N '. Based on the model chosen from systems of isolated chains, aggregation in multichain systems is studied. In another project, we simulate various square-well fluid systems with different ranges of interaction potential in order to understand the phase behavior of proteins due to its relevance to aggregation and many bioprocessing events. Changing the model parameters shows different folding behaviors. The model-chains with 64 residues, Gamma equal to 1.1 and sigma/l equal to 1.9 is the least computationally expensive model displaying all the characteristics found in real proteins. We introduce a new order parameter which divides the conformational space into folded and unfolded ensemble-structures, this order parameter corresponds to a transition in the folding behavior of the chains. We define a native state ensemble as an ensemble of structures with small deviation in contact maps for spheres inaccessible to the solvent defined as the core of the chain. This native ensemble corresponds to the structures exhibiting low-temperature fluctuations simulating the 'breathing motions' of real proteins which is considered responsible for their catalytic activities. On the other hand, the non-native ensemble unfolds at higher temperatures, which increases the propensity for aggregation by forming intermolecular contacts, and therefore reproduce the behavior of proteins under severe solution conditions which occurs in bio- processing (this includes high concentration, temperature, pressure, pH ...). The behavior of multichain systems shows that it is possible to correlate the aggregation propensity of chains at room temperature from the behavior of chains in isolated system at the collapse temperature, which in turn correlate with the stability of the low-T ensemble. In the second project, we developed a more efficient way of calculating the critical temperature in SW fluids even for strongly short-ranged systems which are especially difficult to simulate. In the supercritical region, every isotherm obeys the linear equation for the pressure with a high precision within the bounds of uncertainty. The linear equation pm = p0 + Rm with Rm being the constant isothermal rigidity (dp/d)T . The constant rigidity can be used to estimate directly a critical temperature (Tc) and critical pressure (pc), respectively, and also to obtain the pressures and densities of the percolation loci based on an empirical quadratic nature of change in pressure with densities outside the percolation loci. Identifying the critical temperature and how it depends on the pair potential is very important in formulations with a growing need to predict when the solution will go opalescent.

Foam fractionation is an adsorptive bubble separation technology that has shown potential as a replacement to the more costly and non-sustainable traditional downstream processing methods such as solvent extraction and chromatography for biological systems. However biological systems mostly tend to be a mixture of surface active species that complicates the foam fractionation separation. In this thesis a detailed experimental study on the application of foam fractionation to separate a well-defined surfactant-protein mixture was performed with emphasis on the competitive adsorption behaviour and transport processes of surfactant-protein mixtures in a foam fractionation process. Surface tension and nuclear magnetic resonance (NMR) measurements showed that nonionic surfactant Triton X−100 maximum surface pressure, surface affinity and diffusivity were a factor of 2.05, 67.0 and 19.6 respectively greater than that of BSA. Thus Triton X−100 dominated the surface adsorption at an air-water surface by diffusing to the surface and adsorbing at the surface faster than BSA. This competitive adsorption behaviour was observed in foam fractionation experiments performed for Triton X−100/BSA mixtures for different feed concentration ratios and air flow rates. The recovery and enrichment of Triton X−100 were found to increase and decrease respectively with increasing air flow rate for all foam fractionation experiments as expected for a single component system. However the recovery and enrichment of BSA were both found to increase with increasing air flow rate for high feed concentrations of Triton X−100.Bubble size measurements of the foamate produced from foam fractionation experiments showed that at steady state conditions the bubbles rising from the liquid pool were stabilised by BSA. However at the top of the column the recovery of Triton X−100 in the foamate (75% to 100%) was always greater than the recovery of BSA (13% to 76%) for all foam fractionation experiments. In addition, for high feed concentrations of both components and at low air flow rates, the enrichment of BSA remained at almost unity for most experiments and only increased when the recovery of Triton X−100 reached 100%. Thus it was concluded that Triton X-100 displaced the adsorbed BSA from the surface. The foam drainage properties of Triton X−100/BSA mixtures were characterised using two methods; forced foam drainage and from pressure profiles of steady state foam fractionation experiments (pressure method). The drainage data from the forced foam drainage was found not to be compatible with experimental foam fractionation results, by indicating that stable foam was not produced during the foam fractionation experiments. However stable foam was repeatedly produced during foam fractionation experiments. The drainage data from the pressure method was found to be in close agreement to experimental foam fractionation experiments. The work in this thesis takes a significant step beyond the literature experimental foam fractionation studies for multicomponent systems. In addition to investigating the effect of foam fractionation process parameters on the separation of mixed systems, the results from the characterisation studies of surface adsorption and foam properties provided insight and deeper understanding of the competitive adsorption behaviour of surfactants and proteins in a foam fractionation process.

Viscoelastic fluid flow with immersed boundaries of complex geometry is widely found both in nature and engineering processes. Examples include haemocytes moving in human blood flow, self-propulsion of microscopic organisms in complex liquids, hydraulic fracturing with sand in oil flow, and suspension flow with viscoelastic medium. Computational modelling of such systems is important for understanding complex biological processes and assisting engineering designs. Conventional simulation methods use conformed meshes to resolve the boundaries of complex geometry. Dynamically updating the conformed mesh is computationally expensive and makes parallelization difficult. In comparison, Cartesian grid methods are more promising for large scale parallel simulation. Using Cartesian grid methods to simulate viscoelastic fluid flow with complex boundaries is a relatively unexplored area. In this thesis, a sharp interface Cartesian grid method (SICG) and a smoothed interface immersed boundary method (SIIB) are developed in order to simulate viscoelastic fluids in complex geometries. The SICG method shows a better prediction of the stress on stationary boundaries while the SIIB method shows reduced non-physical oscillations in the computation of drag and lift forces on moving boundaries. Parallel implementations of both solvers are developed. Convergence of the numerical schemes is shown and the implementations are validated with a few benchmark problems with both stationary and moving boundaries. This study also focuses on the simulation of flows past 2D cylindrical or 3D spherical particles. Lateral migration of particles induced by inertial and viscoelastic effects are investigated with different flow types. Equilibrium positions of inertia-induced migration are reported as a function of the particle Reynolds number and the blockage ratio. The migration in the viscoelastic fluid is simulated from zero elastic number to a finite elastic number. The inclusion of both inertial and viscoelastic effects on the lateral migration of a particle is the first of its kind. New findings are reported for the equilibrium positions of a spherical particle in square duct flow, which suggest the need for future experimental and computational work.

Protein aggregation is a frequently cited problem during the development of liquid protein formulations, which is especially problematic since each protein exhibits different aggregation behaviour. Aggregation can be controlled by judicious choice of solution conditions, such as salt and buffer type and concentration, pH, and small molecule additives. However, finding conditions is still a trial and error process. In order to improve formulation development, a fundamental understanding of how excipients impact upon protein aggregation would significantly contribute to the development of stable protein therapeutics. The underlying mechanisms that control effects of excipients on protein behaviour are poorly understood. This dissertation is directed at understanding how excipients alter the conformational and colloidal stability of proteins and the link to aggregation. This knowledge can be used for finding novel ways of either predicting or preventing/inhibiting protein aggregation. Experiments using static and dynamic light scattering, intrinsic fluorescence, turbidity and electrophoretic light scattering were conducted to study the effect of solution conditions such as pH, salt type and concentration on protein aggregation behaviour for three model systems: lysozyme, insulin and a monoclonal antibody. Emphasis is placed on understanding the effects of solution additives on protein-protein interactions and the link to aggregation. This understanding has allowed the rational development of stable formulations with novel additives, such as arginine containing dipeptides and polycations.

The project contributes to the general theme of complex chemical systems and strengthens ties with Innospec, a multi-national chemical company. Sodium lauroyl methyl isethionate (SLMI. Trade name “Iselux”) is a newly developed surfactant with attractive product properties for personal care applications. Little is known about the fundamental surface and solution properties of SLMI, and it is not currently possible to use information on available surfactants to predict phase behaviour. We characterise the solution and liquid crystalline phase behaviour of the SLMI/water system using a combination of optical microscopy, X-ray scattering and differential scanning calorimetry techniques. SLMI is synthesised using a batch process that leads to variable component concentrations. Preliminary studies conducted by Innospec indicate that the presence of particular process components has a significant influence on SLMI formulation rheological properties. We investigate the effects of synthesis-derived components on the rheological properties of the SLMI/sodium {(3-(dodecanoylamino)propyl)(dimethyl)ammonio)}acetate/water system using rheology and light scattering (static and dynamic) techniques. SLMI is often formulated into personal care products on mixing aqueous formulation components. Micelle growth occurs via a mechanistic process that is not understood and the equilibrium viscosity is attained at a time after mixing that ranges from seconds to weeks. Developing an improved understanding of the micelle growth mechanism is of both academic and industrial value. We utilise static light scattering and nuclear magnetic resonance techniques to probe a range of samples in the viscoelastic region of the SLMI/(carboxymethyl)hexadecyldimethyl ammonium hydroxide/water system. Experimental findings improve our current understanding of micelle growth process and provide a platform for future research on non-equilibrium mixing kinetics. In the final section we investigate salt-induced cloud point and precipitation phenomena in the SLMI/salt/water system. The cloud point is commonly observed in surfactant and protein systems by increasing the solution temperature above a critical value, resulting in phase separation of solute-rich and solute-depleted layers. Cloud point induced phase separation may also be prompted by addition of salt. The mechanistic process driving electrolyte-induced cloud point phenomena is not understood. We use a combination of turbidimetry measurements and lightscattering (static and dynamic) techniques to measure cloud point curves andcharacterise micellar behaviour prior to clouding.

Many methods are used for screening/characterisation of strong protein-protein interactions (PPIs), but there has been slow progress in understanding weak PPIs due to technical difficulties. In this thesis, combined biophysical and structural approaches were used to study weak PPIs in a model cytochrome P450 (P450) system. P450s are biotechnologically important heme-binding oxidase enzymes involved in lipid, steroid and drug metabolism. Bacillus megaterium P450 BM3 (BM3) was used as a model. BM3 is a high activity P450 with its redox partner (an FAD- and FMN-binding reductase) linked to the P450 in a single polypeptide. Genetic dissection enabled generation of individual P450 (BMP) and P450 reductase (BMR) domains, as well as separate FAD- and FMN-binding domains. Preliminary work on BM3, BMP and BMR used equilibrium optical binding and showed blue (type I) heme Soret band shifts with substrates (N-palmitoylglycine, lauric acid), and red (type II) shifts with inhibitors (imidazole, 4-phenyl imidazole), consistent with typical P450 behaviour. Steady-state kinetic catalytic efficiency of BM3 in reducing cytochrome c was higher than for BMR, suggesting that BMP promotes interactions between BMR/cytochrome c. Interactions between BMP/BMR domains were studied by examining NADPH-dependent electron transfer (e.t.) rate between these domains using stopped-flow absorption spectroscopy, and across a range of ionic strengths. The inter-domain e.t. rate in the reconstituted system was ~1000-fold less than in intact BM3. Elevated ionic strength decreased the e.t. rate in both systems, and particularly for the reconstituted domains. Slow BMP reduction by NADPH in absence of BMR was also observed over longer time scales.Biophysical methods were used in quaternary structural characterization of BM3 and its domains. BM3 reversibly dimerizes (mostly stabilized by electrostatic interactions) with a dissociation constant (Kd) of 0.29 µM. Reversible dimerization was also seen for BMR, but with a weaker Kd than BM3. The FAD domain is near-fully dimerized most likely due to intermolecular disulphide bonds, while FMN and heme domains are monomers. Results indicate that BMR domain interactions regulate BM3 dimerization. The BM3 shape is compact and globular, BMR is compact and ellipsoidal, and the FAD domain is globular with extended ends. Differences between FAD domain crystal and solution structures suggest conformational differences possibly due to disulphide bonds stabilizing solution dimer. 6 different BMR conformations were obtained by rigid body modelling, all showing that BMR exists in an “open” conformation with respect to orientation of its FAD and FMN domains.

In this study the heat-induced gelation of two (readily available) proteins, which contain disulphide bonds, has been investigated over a range of protein concentrations in the presence and absence of the presence of the reductant, dithiothreitol at neutral pH. The proteins selected in this study were: β-Lactoglobulin and bovine serum albumin. These proteins have different number of disulphide bonds and possess different protein secondary structures. The influences of the reductant and protein concentration on their heat-induced gelation were explored to see whether the proteins were able to form protein hydrogels and that the mechanical properties of the resulting protein hydrogels were controllable. The tilting test tube method revealed that both proteins formed macroscopic hydrogels, at protein concentrations above the critical gelation concentration and that the critical gelation concentration was constantly lower in the presence of the reductant. Micro-DSC revealed that both proteins had completely denatured upon heating and that the denaturation temperature and enthalpy were significantly lower in the presence of the reductant. IR spectroscopy revealed that both proteins undergo major secondary structure transitions that resulted in the formation of fibers that are rich in β-sheet structure upon heating and that the protein lost some secondary structure before any heating and gained more β-sheet structure in the presence of the reductant. Both proteins had partially denatured before any heating in the presence of the reductant and that β-LG underwent aggregation that was accompanied by the loss of native β-sheet structure and the formation of intermolecular β-sheet structure, while that BSA underwent aggregation that was accompanied by the loss of native α-helix structure and the formation of intermolecular β-sheet structure. Cryo-TEM revealed that both proteins formed fibers (10 nm in diameter) that exist as single entities at low protein concentrations and become entangled into macroscopic networks, at protein concentrations above the critical gelation concentration and that more fibers and denser macroscopic networks were formed in the presence of the reductant. Oscillatory rheology revealed that both proteins formed macroscopic networks exhibit viscoelastic behaviour and that their elastic modulus had increased in the presence of the reductant and with increasing protein concentration.

A clear and predictive understanding of the propensity for crystallisation of one polymorph over another is lacking, and in this regard glycine is a model system due to difficulties in crystallisation of the thermodynamically stable gamma polymorph. The preferential crystallisation of gamma-glycine in the presence of micellar CTAB (Cetyltrimethylammonium bromide) as opposed to the alpha form commonly crystallised from pure solution was observed. A rationale for this result was sought through the observation of the nucleation and growth kinetics of the alpha and gamma polymorphs of glycine (and DL-alanine) using in situ microscopy, the measurement of induction times and following the solution mediated phase transformation of alpha-glycine. These observations help explain the dominant crystal form produced in a number of solutions. The nucleation and growth rates of alpha-glycine were shown to be orders of magnitude greater than those of gamma-glycine in pure solution. Also, the addition of a cationic surfactant (such as CTAB) or modification of the solution pH were shown to dramatically accelerate the nucleation and growth of polar gamma-glycine and DL-alanine, a rarely reported phenomenon. In addition, the growing (00-1) faces of gamma-glycine and DL-alanine, at which growth was accelerated, were shown to be macroscopically rough, indicating a growth mechanism dominated by nucleation rather than the growth of layers. The most likely cause of the inhibited kinetics of gamma-glycine and DL-alanine is water bound electrostatically at the negatively charged (00-1) faces, while the growth acceleration inferred by the additives is related to their ability to release water from these surfaces. Other mechanisms which may play a role include the adsorption of adventitious impurities, strong electrostatic repulsion between like-charged carboxylate groups at the (00-1) surface resulting in structural disorder, and the effect of surface energy on the rate of surface nucleation. This research provides an important example of nature’s complexity in selecting crystal form in polymorphic systems, gives further insight into the causes of the asymmetric growth of polar crystal structures, and introduces the possibility that the crystallisation kinetics of ‘difficult’ slow growing compounds may sometimes be modified through the use of additives.

An understanding of the interactions between polyelectrolytes and proteins is vital to determine structure and functionality of materials constructed of these two components. Possible applications for the protein-polyelectrolyte composites are ranging from materials used to deliver drugs to the methods of protein stabilisation for storage of therapeutics, biosemsors, and encapsulation of medicines for triggered release. The binding of globular proteins to the polyelectrolyte chains can prevent undesired protein aggregation and may help to extend the shelf-life of the protein-containing food. The aim of this project is to study the mechanism of non-covalent binding between proteins and polyelectrolytes, responsiveness of the proteinpolyelectrolyte composites to external stimuli such as changing pH, presence of salt of different types and concentrations or influence of enzyme on the integrity of protein-polyelectrolyte multilayer film. Our study was focused on the effects of different mono- and multivalent salts on binding affinitybetween a negatively charged polyelectrolyte - poly(styrene sulfonate) PSS and bovine serumalbumin BSA or myoglobin. The complex formation between these polymers was examinedusing the static light scattering (SLS), turbidimetric and potentiometric titrations, differentialscanning calorimetry (DSC) and theoretical studies based on molecular dynamics simulations. We established that the inter- and intramolecular interactions between proteins and polyelectrolytes are primarily driven by the electrostatic forces at the conditions when thepolymers are in low ionic strength solutions and attractive or repulsive relations are based upon the charge density and its distribution. When proteins are interacting with polyelectrolytes in solutions of high ionic strength the electrostatic interactions are screened by the salt originated co-ions. In these conditions there is a competition between salting-out effect on proteins leading to protein aggregation or protein-polyelectrolyte complex formation, which can prevent undesired protein-protein association. The forces driving the attractive interactions at high ionic strength are of non-electrostatic origin, these are mainly hydrophobic forces. The computer simulation study shows that more flexible polyanionic chains are stronger binders to the positive patches on protein surface than these of a more rigid backbone. Also a total energy of binding depends on a sum of electrostatic and non-electrostatic energies. The formation of multilayers composed of a protein and a polyelectrolyte, where componentswere: poly-L-lysine – a positively charged homopolypeptide and polygalacturonic acid - apolysaccharide was examined using a quartz crystal microbalance with dissipation monitoring. A 10 and 11 layer film, deposited on the charged surface, exhibited the linear growth pattern for first 5 layers and exponential growth for a flowing 5 (or 6) layers. The influence of pectinase enzyme on digesting the polygalacturonic acid component of the multilayer was most effective for 1 AU/mL concentration of pectinase. After the enzyme was applied the multilayer film was fully disintegrated within the period of 20 minutes for pectinase at 1 AU/mL and the time of disintegration was extended to 120 minutes for pectinase at 0.1 AU/mL.Silk fibroin aqueous solutions were tested rheologically for their structural properties involvingthe existence of fibroin aggregates. We examined the process of ageing of fibroin solutions and solid-liquid transformations taking place within the fluid. The transitions between viscous and elastic behaviour of the fibroin’s semi-dilute solutions were initiated by strain, shear frequency and temperature. We highlighted that the irreversible change in secondary structure of the silk fibroin in aqueous solutions are taking place after the 48 hour period of time since the preparation of protein fluids. We recommend that further processing of silk fibroin such aselectrospinning should be completed within the 48 hour after dissolution.