<div>Proteins are susceptible to physical and chemical degradation in solution, which can lead to the loss of therapeutic activity and increase the potential for immunogenic responses when administered. Many degradation reactions are mediated by water, and therefore the proteins are often formulated as solids in which degradation rates are slowed significantly. Lyophilization is the most common method for producing solid protein formulations, which removes the water by sublimation and desorption under vacuum from the frozen protein solutions. Lyophilization requires excipients to protect the protein from the inherent stresses involved in the process. Degradation can still occur during lyophilization and storage, and needs to be characterized in order to develop a successful formulation with desired storage stability. The analytical techniques to characterize solid-state proteins are limited, however, and many do not provide site-specific information and lack the ability to predict stability beforehand.</div><div>Recently, solid-state hydrogen-deuterium exchange mass spectrometry (ssHDX-MS) has been developed to characterize proteins in solid powders with peptide level resolution. The technique was found to be sensitive to formulation and process changes. The ssHDX-MS metrics are highly correlated to the long-term storage stability, suggesting that the method can serve as a formulation screening tool. This dissertation aims to evaluate the factors affecting ssHDX kinetics and to develop a mechanistic understanding of the exchange process in solid samples, which in turn will support the solid-state protein development and enable it to be conducted in a more a cost and time-effective way. First, the contribution of peptide-matrix interactions to deuterium incorporation kinetics in the absence of higher-order structure was assessed using lyophilized poly-D, L-alanine peptides. Deuterium incorporation depended on excipient type and D₂O_(g) activity in the solid samples. A reversible pseudo-first-order kinetic model was proposed and validated using the experimental data. Second, the reversibility of the hydrogen-deuterium exchange reaction in the solid-state was evaluated to support the ssHDX mechanistic model further. The reaction was found to be reversible irrespective of initial conditions and independent of the excipient type. Pre-hydration of the peptide samples prior to deuterium labeling did not affect deuterium incorporation in amorphous samples compared to the controls not subjected to pre-hydration. Third, the contribution of peptide secondary structure to deuterium uptake kinetics was quantified using structured PDLA analogs. The deuterium incorporation in structured peptides was less than that of the PDLA peptides suggesting that both peptide structure and peptide-matrix interactions contribute to ssHDX-MS. Finally, a quantitative data analysis method was presented that allows the interpretation of ssHDX-MS data of a protein relative to controls. Altogether, the findings present a comprehensive mechanistic understanding of the ssHDX-MS of proteins that is relevant to the industry.</div>