From a thermodynamic point of view, protein stability is maintained if the equilibrium between native and unfolded states is more shifted towards the native state. The stabilizer can increase the free energy of protein unfolding and thus drive the equilibrium to a more stable native state. In the solution phase, the stabilization of protein is mainly governed by the preferential interaction mechanism. That is, when the protein is formulated with a stabilizing excipient, the protein prefers to interact with water while the excipient is preferentially excluded from the protein surface. The thermodynamic consequence of such exclusion leads to the protein being preferentially hydrated and the chemical potential of the protein is increased. This induces a thermodynamically unfavorable situation, which in turn stabilizes the native over the unfolded protein state as the increment of chemical potential is higher for the unfolded state than those for the native state and thus increasing the free energy barrier for protein unfolding and denaturation as shown in Figure . Recently, Arsiccio et al. also demonstrated a new thermodynamic model of excluded volume mechanism in protein stabilization which is the creation of a cavity within the solvent and considered as a hard-particle fluid through thermodynamic modeling and experimental data. The formation of hydrogen bonds between water and protein contributes significantly to the protein stability in the aqueous solution. During the lyophilization process, the removal of water leads to the loss of hydrogen bonds between protein and water. Such water removal can result in notable alterations of the protein structure, and therefore protein instability. It has been hypothesized that the hydroxyl groups of the sugars can replace the water in forming hydrogen bonds with protein upon drying processes, conferring thermodynamic stability and providing stability against degradation by replacing the stabilization role with water and stabilizing the native protein conformation. Therefore, the free energy of protein unfolding is increased, thus maintaining the protein stability in the solid state. solutes are preferentially excluded from the surface of proteins which stabilizes the native versus the unfolded protein state; and (B) exclusion of solutes from the protein surface increases the free energy barrier for protein denaturation. A direct measurement of sugar and protein interaction and hydrogen bonding formation is challenging due to the lack of high-resolution structural investigation. This hypothesis was first assessed and proposed by Carpenter and Crowe et al. In this original study, FTIR was used to detect the changes in the secondary structure of the protein. The FTIR spectra of freeze-dried lysozyme with and without trehalose were compared to those of fully hydrated lysozyme. It was found that trehalose can prevent the shift of the amide I and II bands of dried lysozyme. Moreover, the presence of trehalose in the formulation results in the appearance of the carboxylate band of lysozyme as shown in Figure , a fingerprint band of hydrogen bond, suggesting that trehalose can substitute water to form hydrogen bonds with protein and help stabilize the protein in a near-native state. However, the validation of water replacement theory from the FTIR findings was challenged by Belton and Gil. In their study, they stated that the stabilization of dried lysozyme could be still due to the water-protein hydrogen bonding when the residual water is trapped at the protein-sugar interface upon drying. Nevertheless, Allison and Carpenter et al. later reinforced the water replacement theory by showing the evidence that the lysozyme sample with a 4% moisture level had no evidence of an actual peak of carboxylate band and could not give rise to the fingerprint band for sugarprotein hydrogen bond, indicating that the dehydration-induced spectral shifts could not be solely claimed for water trapped at the protein surface. This solidified that the hydrogen bond formed between sugar, not the trapped water, and protein is responsible for the stabilization of solid-state protein. Furthermore, the polymeric sugars like dextran didn't form hydrogen bonds well with proteins as disaccharides with less intensity of the carboxylate band observed. The failure of dextran to inhibit lysozyme unfolding can result from the insufficient hydrogen bond formation between polymeric sugar and protein due to steric hindrance.