The fundamental of water replacement theory is the hydroxyl groups of sugars interact with the surface molecules of the protein via hydrogen bonding. To further detect this mechanistically critical interaction between protein and sugar in the solid state, advanced high-resolution techniques that can identify and quantify site-specific interaction are urgently needed. For this purpose, solid-state NMR has shown its capability to identify the hydrogen bonds between proteins and excipients. This is often achieved by observing chemical shift changes, intermolecular correlation, or more quantitatively, interatomic distance measurement. For example, in a recent study, chemical shifts of Arg Cξ peaks were comparable in the mannitol and trehalose formulations as shown in Figure , suggesting that they could have interacted with bovine serum albumin (BSA) in similar hydrogen-bonding patterns. In addition, Pielak's group utilized the liquid-observed vapor exchange (LOVE) NMR technique along with the hydrogen-deuterium method to probe the excipient effect on the dry protein structure at the residue level. They found that increased protection of two proteins of the B1 domain of streptococcal protein G and truncated barley chymotrypsin inhibitor 2 when formulated with sugar, especially residues with a greater number of intramolecular H-bonds are better protected, which suggests that sugars retain protein structure in solid state by strengthening intra-protein H-bonds. Whereas, a decreasing protein stability effect was observed when removing three hydroxyl groups (1,2,3-hexanetriol), which indirectly supports the protein stabilization/protection from the water replacement theory. Figure . Protein-sugar interaction characterized by FTIR and ssNMR. (A) Amide band region for hydrated lysozyme (dotted line), lysozyme freeze-dried alone (dashed line), and lysozyme freeze-dried in the presence of trehalose (solid line); 82 and (B) Arg Cξ peaks of spray-dried BSA without any excipient (black), BSA-mannitol (blue), BSA-trehalose (red). 21 X-ray crystallography structure of the BSA monomer (PDB ID:4F5S) highlighting the Arg residues (cyan) and hydrogen bonds with other residues to maintain the secondary and ternary protein structure (magenta).