Evolving and optimising protein stability for enhanced pharmaceuticals

A major challenge for the use of proteins as biopharmaceuticals lies in their inherent instability. Despite the widespread appreciation of this issue, whether instability arises from thermodynamic instability, kinetic instability and/or the propensity of proteins to aggregate has not been explored in any systematic way. Here we propose to investigate the origins of instability of antibody domains using a wide range of biophysical methods, with the overall goal of determining which of these fundamental features is key to determining instability and, thereby, to explore new routes to evolve or enhance biopharmaceutical 'shelf-life'. The proposal, therefore, clearly falls in BBSRC's remit and strategic plans, spanning many target areas as outlined below. We will focus our studies on antibody light chains, including both the constant and variable domains, and sequences known to be stable, or to be aggregation-prone. (i) Thermodynamic parameters will be obtained from equilibrium denaturation experiments (using DSC, fluorescence and/or CD as probes); (ii) kinetic parameters will be measured using stopped flow spectroscopy and amide hydrogen exchange monitored using NMR and/or mass spectrometry and (iii) instability due to self-association will be monitored using light scattering and analytical ultracentrifugation). We will also explore the effect of commonly used small molecule additives to tailor each of these properties for different antibody sequences, and will explore the use of novel reagents as stabilising agents that target the different aspects of instability. In addition, we will explore the possibility of using protein engineering to develop new antibody domain sequences that maintain function, yet are tailored specifically to have enhanced stability and solubility in vivo. Identifying such mutations is immensely challenging, however, because the origins of stability in vivo are complex, multi-factorial and inter-related (depending both on the physicochemical properties of the protein of interest, but also its interactions with the cellular milieu). To achieve this, we will use the power of directed evolution to create new protein sequences selected to possess enhanced stability in vivo, using a novel strategy recently developed in SER's laboratory (with Bardwell, Michigan) in which the in vivo stability of a protein is directly linked to antibiotic resistance (Foit at al, currently under revision at Molecular Cell). The approach requires no prior structural or functional knowledge and permits the evolution of protein stability independently of functional constraints. By combining the sequences evolved, with the additives above, our ultimate goal is to develop new, informed routes to enhance stability that should be of general utility across the biopharmaceutical industry. In addition to being of immense practical utility, the ability to evolve protein stability in vivo may indicate how different evolutionary pressures have interacted to produce today's protein sequences. The project will take advantage of the complementary skills of the academic and industrial collaborators. Prof Radford (Astbury Centre, Leeds) has an established track record in the biophysical characterization of protein stability, folding and aggregation spanning 22 years. Professor Smith (Avacta) brings experience in high throughput analysis of protein stability based on the newly developed instrumentation in Avacta and knowledge of protein additives as stabilizing agents in the biopharmaceuticals industry. The collaboration will thus open new doors for research opportunity that will generate new knowledge of immense value both from fundamental and applied viewpoints.