| Protein stability is of great importance for the successful development of protein-based therapeutics, which provide improved and innovative treatment of human diseases. In this research plan, we will investigate the mechanisms of protein instability in two case studies for which understating is currently underdeveloped but relevant to the biopharmaceutical industry. This thesis will focus on (i) stresses associated with PLGA devices for protein controlled-release delivery and (ii) the effect of UV on protein structure and function. We believe that the research will provide a fundamental understanding of the mechanisms of instability in these case studies. In addition, a more thorough appreciation of protein instability in these specific cases will allow for the development of appropriate stabilization strategies.;The first half of this thesis focuses on protein instability associated with PLGA systems. The effects of acidic conditions on a model protein, BSA, were investigated to simulate the microclimate within PLGA delivery systems during release. A comparison of acid- and PLGA-induced degradation revealed a high degree of similarity, which confirmed that acidification within PLGA systems is a dominant stress for BSA and can account for the incomplete release of the protein in vitro. We also evaluated heparin as a specific stabilizer for the controlled-release of FGF2 in the treatment of IHD. In addition to preventing damage during lyophilization, heparin also protected FGF2 from aggregation during simulated release from PLGA millicylinders. Collectively, the research presented herein describes the potential protein stresses encountered during the release from PGLA systems, the consequences of such stresses on protein stability, and also provide stabilization approaches for the successful development of PLGA delivery systems for protein controlled-release applications.;In the latter chapters, the effects of UV-exposure on the biophysical and biochemical properties of ALDH3A1 were characterized to better understand the role of ALDH3A1 as a UV-absorbing molecule in the cornea. Human ALDH3A1 was expressed and purified to first evaluate the metabolic properties of the enzyme. ALDH3A1 was then exposed to UVB-light and changes in protein structure as well as enzymatic activity were investigated. The mechanism of ALDH3A1 inactivation, which is the result of protein aggregation rather than modification to the active site Cys residue, is discussed.;Taken together, the work presented in this thesis investigates two specific cases of protein instability: degradation associated with release from PGLA delivery systems and inactivation arising from UV-exposure. We anticipate that the results will be valuable in increasing protein stability in the development of protein-based therapeutics and, eventually, translate into a greater number of stable products available for the treatment of various human diseases. |
