EFFECTS OF PROTEIN DRYING METHOD AND EXCIPIENTS ASSESSED AT THE RESIDUE LEVEL

Drying proteins is an effective solution to preserve their efficacy and increase shelf life. To this end I wanted to know if drying methods protect similarly and if excipients could potentially protect synergistically. I describe the effects of drying methods and excipients on residue-level protein structure using LiquidObserved Vapor Exchange Nuclear Magnetic Resonance spectroscopy (LOVE NMR) and activity assays. In Chapter 1, I review the mechanisms proposed to explain protection in a vitreous matrix at low hydration and the efforts to use these ideas to protect proteins in the dry state. In Chapter 2, I evaluate how drying methods affect the B1 domain of streptococcal protein G (GB1) and the enzyme adenylate kinase (AdK). I collaborated with Dr. Samantha Stadmiller at Lindy Biosciences to implement their microglassification technique on GB1 and AdK. I compare the commonly used lyophilization drying technique to vacuum drying and microglassification. The results show that global unfolding residues are drying-method agnostic because these residues are packed within the hydrophobic core, and that local unfolding residues have the greatest change in protection because they are more solvent accessible. The results also show that lyophilization is inferior to vacuum drying and microglassification. In Chapter 3, I describe the effectiveness of phytoglycogen 13 (PG13) as a protectant of AdK. I then investigate the protective synergy involving three additives: tardigrade cytosolic abundant heat soluble protein D (CAHS D), trehalose, and PG13, and conclude that synergy involving CAHS D is tied to its intrinsic disorder. In Chapter 4, I summarize my findings as well as logically conclude what the mechanisms of protection are for the excipients used in this research.