There have been numerous studies on measuring protein-protein interactions in solution using a variety of techniques including membrane osmometry, sedimentation, and static light scattering. Most of these techniques yield an osmotic second virial coefficient. The osmotic second virial coefficient has been shown to be an important parameter for protein crystallization. To date, there have been few fundamental theoretical studies of estimating second virial coefficient values using conventional models because of the diverse and complex nature of the potential of mean force. In the present study, the variation of equine serum albumin interactions was measured with respect to pH and sodium chloride salt concentration by static light scattering to determine the second virial coefficient and electrophoretic light scattering to determine the electrophoretic mobility. The main aim is to show the effect of the solution conditions such as pH and ionic strength on ESA interactions. In this thesis, I will focus on the classical theory of Derjaguin, Landau, Verwey and Overbeek (DLVO). The experimental data from electrophoretic and static light scattering measurements for equine serum albumin are compared with the DLVO model.

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Virus-like particles (VLPs) are elegant functional architectures formed by the self-assembly of viral structural proteins. VLPs have been developed as vaccines against hepatitis B and cervical cancer, and have recently been shown in animal studies to provide protection against both seasonal and avian influenza following intranasal administration. This new class of vaccines offers unprecedented immunoprotection, inherent safety, and a simple route of administration. To realize the full potential of VLP technology as an efficient and responsive vaccine platform, this project exploits the parallel advancements in recombinant technology, analytical techniques and colloidal science to facilitate the swift and economical delivery of candidate VLP vaccines from laboratory to clinical trials, and ultimately into commercial production. Three areas of VLP production are specifically targeted in this work, i.e., VLP subunit production, particle characterisation and assembly. The major research outcomes in this work are: (i) establishment of a simple and economical VLP subunit production method which eliminates inefficient and complicated purification procedures necessitated by the current in vivo production methods; (ii) development of a high-resolution and high-throughput analytical method for rapid and reliable quality control check of VLP products; and (iii) establishment of the foundation to predict optimal VLP self-assembly conditions through molecular thermodynamics. These research outcomes collectively enhance the quantitative knowledge base in VLP assembly and may ultimately enable the development of a mechanistic and descriptive modelling approach to optimize VLP production. From a fundamental perspective, this work introduces the first experimental technique to measure protein interactions of viral subunits undergoing rapid, irreversible assembly reaction. Such information, when correlated with molecular details and assembly conditions may provide unique insights into the molecular switches responsible for viral assembly, unveiling the fundamental mechanism underpinning viral self-assembly.

The virial equation of state p = ρRT[ 1 + B(T) ρ + C(T) ρ2 + · · ·] for high pressure and density gases is used for computing chemical equilibrium properties and mixture compositions of strong shock and detonation waves. The second and third temperature-dependent virial coefficients B(T) and C(T) are included in tabular form in computer codes, and they are evaluated by polynomial interpolation. A very accurate numerical integration method is presented for computing B(T) and its derivatives for tables, and a sophisticated method is introduced for interpolating B(T) more accurately and efficiently than previously possible. Tabulated B(T) values are non-uniformly distributed using an adaptive grid, to minimize the size and storage of the tables and to control the maximum relative error of interpolated values. The methods introduced for evaluating B(T) apply equally well to the intermolecular potentials of Lennard-Jones in 1924, Buckingham and Corner in 1947, and Rice and Hirschfelder in 1954.

The virial equation of state p = ρRT[ 1 + B(T) ρ + C(T) ρ2 + · · ·] for high pressure and density gases is used for computing chemical equilibrium properties and mixture compositions of strong shock and detonation waves. The second and third temperature-dependent virial coefficients B(T) and C(T) are included in tabular form in computer codes, and they are evaluated by polynomial interpolation. A very accurate numerical integration method is presented for computing B(T) and its derivatives for tables, and a sophisticated method is introduced for interpolating B(T) more accurately and efficiently than previously possible. Tabulated B(T) values are non-uniformly distributed using an adaptive grid, to minimize the size and storage of the tables and to control the maximum relative error of interpolated values. The methods introduced for evaluating B(T) apply equally well to the intermolecular potentials of Lennard-Jones in 1924, Buckingham and Corner in 1947, and Rice and Hirschfelder in 1954.

Protein crystallization is significant in both biotechnology and biomedical applications. In biotechnology, crystallization is essential for determining the structure of both native and synthesized therapeutically important proteins. It can also be used as a final purification step and as a stable form for protein storage. With regard to biomedical systems, protein crystallization appears to be involved in the development and manifestation of certain human diseases. In particular, there exists evidence that L-rich ferritin crystals are involved in Hereditary Hyperferritinemia Cataract Syndrome (HHCS). In the current research a microbatch crystallization apparatus has been introduced that enables (1) multiple batch crystallization experiments at various temperatures and solution conditions in parallel and (2) quantitative monitoring of crystal growth without disturbing the progress of an experiment for observation. The primary application of the apparatus is, but not limited to, screening of protein crystallization conditions, although the system can also be used for other macromolecular and small-molecule crystallization experiments. Multiwell microbatch experiments demonstrated the dependence of apoferritin crystal growth kinetics and final crystal size on temperature and cadmium concentration. Although the solubility of apoferritin might be independent of temperature, the results of this study show that the crystal growth kinetics are affected by temperature, profoundly under some conditions. For apoferritin under near physiological conditions the solution thermodynamics in the form of the second virial coefficient have proofed to be a valuable predictor for the crystallization outcome. Furthermore, the significance of the elevated level of some divalent cations in cataractous lenses has been studied both in dilute solutions and under crystallization conditions and cadmium seems to be sole menace in apoferritin condensation.

There is a strong link between solubility, and thus crystallisation, and the molecular interactions of proteins in dilute salt solutions. Such molecular interactions are governed by the weak interaction forces (electrostatic, hydration and hydrophobic). Such forces can be quantitatively estimated in terms of a second virial self-coefficient (B22) and a second virial cross-coefficient (B23) for a single and a binary protein system, respectively. Previous studies confirmed the relation between a value of the second virial coefficient and a type of interaction (attractive or repulsive). The aim of this thesis is to correlate the second virial coefficient with the solubility and nucleation for single and binary protein systems. Model proteins used in this work are lysozyme and ovalbumin from egg-white, and α-amylase from Bacillus Licheniformis (BLA). The measurements are performed for sodium chloride and ammonium sulphate solutions in an acidic pH at 20 oC. Interaction chromatography is used in this work to estimate the B22 and B23 values for the model proteins in salt solutions. From the measured values of B22 and B23, the type of interaction is generalised as a function of the salt type, salt concentration, pH and protein type. For the single protein systems, in ammonium sulphate solutions (0.1 - 2.4 M) at pH 4.0 and 7.0, repulsion or no interactions are observed below 0.8 M and, as the salt concentrations are increased attractive self-interactions are observed for the model proteins. However, for the sodium chloride solutions (0.1 - 2.0 M) at pH 4.0 and 7.0, the interaction patterns vary with the salt concentration, the pH and the type of protein studied. A common feature of the self-interaction for all the model proteins is the attractive interactions close to the isoelectric point. For the binary protein systems, three distinct regions are observed in the ammonium sulphate solutions (0.1 - 1.6 M) at pH in the range 4.0 - 7.0. Attractive or no cross-interactions are observed at low salt concentrations (< 0.5 M). At the intermediate salt concentrations (0.5 - 1.0 M), the cross-interactions are constant and near zero. This is followed by a sharp increase in the attractive interactions above 1.0 M ammonium sulphate concentrations. However, for sodium chloride solutions (0.1 - 1.6 M) at pH 4.0 - 7.0, two distinct regions are observed. Attraction or no interactions are observed at low salt concentrations (< 0.5 M) and above 0.5 M concentrations of sodium chloride, negligible cross-interactions are observed between model proteins. For the single protein system, an overall increase in the solubility of three model proteins is observed with an increase in the concentrations of ammonium sulphate and also for sodium chloride solutions except for BLA, where a salting-in behaviour is observed. Linear regression is used on the solubility data to determine the parameters of the Cohn equation (β and Ks) where the values of β vary with solution pH, protein type and salt type. The values of Ks vary with protein type and salt type. However, it is insensitive to the solution pH for lysozyme in ammonium sulphate, ovalbumin in sodium chloride and BLA in ammonium sulphate solutions. For the binary protein system, the presence of ovalbumin had a measurable effect on lysozyme solubility at pH < 5.0 in both salts. In low concentration sodium chloride solutions (< 0.3 M), a decrease in the solubility of lysozyme was observed with the presence of ovalbumin at acidic pH < 5.0. However, in ammonium sulphate solutions, the lysozyme solubility increases with the addition of ovalbumin in the salt concentration range 1.6 - 2.0 M and at pH < 4.0. The primary nucleation threshold values are also determined for lysozyme in sodium chloride and ammonium sulphate solutions. In sodium chloride solutions (0.2 - 1.0 M), the critical supersaturation values increase as the solution pH is raised from 4.0 to 7.0; however in ammonium sulphate solutions (1.0 - 2.0 M), the reverse effect is observed. The critical supersaturation required to nucleate lysozyme in ammonium sulphate solutions is approximately three times higher than in sodium chloride solutions. For the single protein systems, the measured values of solubility and B22 were correlated using published models (RSL and HDW). For each protein-salt combination, a reasonable single correlation between solubility and B22 is possible as the salt concentrations and pH are varied. There are separate correlations for sodium chloride and ammonium sulphate solutions. Based on the correlation curve of solubility and B22, it is proposed that the acidic pH range (4.0 - 5.0) is better for crystallising and precipitating globular proteins from these salt solutions. If the values of solubility and B22 are converted into a non-dimensional quantity, the data derived from the different protein-salt systems collapse onto a single curve for the same salt type. The B22 values are also correlated with the critical supersaturation (ln(c*/S)) for the primary nucleation of lysozyme in salt solutions. The values of the critical supersaturation increase as the values of the second virial coefficient become negative or reduce. The ideal critical supersaturation required to create nuclei of lysozyme in salt solutions is between 0.1 and 1.4. For the binary protein systems, B23 values were related to the slope of the lysozyme and ovalbumin plot at same salt concentration and solution pH. Further work is required for binary protein systems to generalise such correlations as a function of the salt concentration and pH. The correlations derived in this thesis are useful generally to predict the solubility and primary nucleation of globular protein in salt solutions. This work reinforces the importance of the second virial coefficient in predicting the crystallisation of protein in salt solutions.

The number of protein containing therapeutic drugs is growing day by day. Lack of proper storage conditions can cause protein degradation or aggregation. The osmotic second virial coefficient, B22, is a thermodynamic parameter, which can predict protein interaction with other proteins and solvent molecules. B22 has been successfully used as predictor of crystallization conditions for a protein in the solution, and in this study an attempt has been made to relate B22 as a predictor of stability of the protein. Static light scattering was used to measure B22 in our studies. B22 and the solubility of three proteins were measured in several excipient solutions. George et al. in 1997 related the osmotic second virial coefficient with the solubility of protein in a solution. In this study we have attempted to relate solubility with B22 and stability of lysozyme, human serum albumin, and ovalbumin in buffer solutions containing various excipients.

Crystallization of proteins in the human body can lead to the development of diseases such as sickle cell anemia and cataract. Understanding protein crystallization can give insight into such diseases. Furthermore, protein crystallization is necessary for protein structure resolution. This is important since resolution of protein structure is the first step towards establishing structure/function relations, and possibly towards performing specific structural modifications that may change the function in desirable directions. Another important application of protein crystallization is in downstream processing in the pharmaceutical industry where it is used for separation and as a final purification step. The present study increases knowledge of interactions between protein molecules during crystallization and hence the crystallization process. Crystallization of proteins in the human body can lead to the development of diseases such as sickle cell anemia and cataract. Understanding the processes involved in protein crystallization can help us gain a better understanding of such diseases. Crystallization of human gamma D-crystallin (HGD) and apoferritin, two proteins found in the lens, was studied in relation to cataract formation. Crystallization of both proteins was studied in the presence of divalent cations which are found at elevated concentrations in cataractous lenses. Results indicate that the divalent cations studied enhance crystallization of these proteins. A thermodynamic property, the osmotic second virial coefficient, was measured in protein solutions and its value was correlated with the occurrence of crystallization. It was found that the second virial coefficient successfully predicted crystallization of both proteins. A new method was developed for indirect measurement of the second virial coefficient using dynamic light scattering. This new method is more robust and efficient than the traditional static light scattering method. Finally the ability of solubilizers to prevent crystallization in HGD solutions was studied. A commercial solubilizer, NDSB-201, was found to increase the energy barrier to nucleation. Although this did not prevent crystallization, it resulted in fewer and smaller crystals being obtained. The naturally occurring alpha A-crystallin was a superior solubilizer to NDSB-201, as it suppressed aggregation and prevented crystallization of HGD under conditions for which NDSB-201 did not. The findings in the present study provide insight into the processes by which protein crystallization occurs and hence into diseases associated with protein crystallization. The findings in the present study provide insight into the processes by which protein crystallization occurs. Using the second virial coefficient to assess whether a protein will crystallize out of solution, approaches for retardation and prevention of protein crystallization, and implications for future research, are discussed.

Protein aggregation is commonly encountered during the manufacture of protein-based bioproducts in processing such as protein expression, purification, refolding, shipping and storage (Volkin and Middaugh, 1992; Brange, 2000). Aggregation may shorten the shelf-life of pharmaceutical proteins (Frokjaer and Otzen, 2005) and induce severe hypersensitivity (Rosenberg, 2006). In addition, several diseases ranging from Alzheimer’s disease to cystic fibrosis are associated with protein aggregation in the form of amyloid fibrils and plaques (Dobson, 1999; Luheshi et al., 2008). Hence, studies on protein aggregation, especially those dealing with high concentrations of proteins, are highly demanded in both academic and industrial laboratories. To address the aforementioned issues, physicochemical factors affecting protein aggregation were investigated systematically in this project. Strategies were developed to inhibit protein aggregation during renaturation and to enhance protein stability against aggregation during and after production, especially when dealing with high protein concentrations. ∆5-3-Ketosteroid isomerase (KSI) was used as a model for aggregation studies during protein renaturation due to its intrinsic aggregation properties. KSI was overexpressed as inclusion bodies (IBs) in Escherichia coli (E. coli). Cost- and time-efficient combination of chemical extraction and one-step affinity purification ensured the production of denatured KSI with high purity at high yield. Several key factors, including protein concentration and ionic strength, were determined to greatly influence KSI aggregation during renaturation. Polymer addition (PEG 3000 and Eudragit S-100) was found to alter KSI aggregation behaviour in a polymer-specific manner, as quantified using reversed phase-high performance liquid chromatography (RP-HPLC) analysis. Light scattering for second virial coefficient (SVC) measurement, surface plasmon resonance (SPR), and microfluidics were applied to study the fundamental mechanism of protein aggregation. Lysozyme was further introduced as a control protein for comparison with KSI. A rapid lumped method was established to measure specific refractive index (∂n/∂c) and SVC values for KSI and lysozyme, which provided quantitative and qualitative information on thermodynamic interactions of molecules in solution. SPR and microfluidics were also used to explore protein aggregation properties. To our best knowledge, it is the first time SPR and microfluidics have been used to investigate protein aggregation behaviour. Both SPR and microfluidics present significant potential for assessing protein aggregation and diagnosis or drug screening of protein aggregation related diseases. The chemical and physical stability of proteins needs to be maintained after successful refolding to ensure an acceptably long shelf life, especially at high protein concentration (Chang and Hermsdorf, 2002). The pharmaceutical effects of lectins on cell growth provided incentive for studies to improve their stability. Human galectin-2 (hGal-2, a homodimeric lectin) was used as a study model in this project. Mutations were introduced at one of the two Cys residues (C57A, C57M, and C57S). Only the C57M variant was highly expressed in bacteria in soluble form. No aggregate of this mutant was detected during 3 weeks of storage. hGal-2 C57M also facilitated site-directed introduction of poly(ethylene glycol) (PEG) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one PEG chain per protein subunit in homodimer. Neither secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results document the feasibility of tailoring a human galectin for enhanced stability against aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as a growth regulator and the serum clearance rate of hGal-2.