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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.

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 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.

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.