As discussed in Section 3.1, from a thermodynamic perspective, the hydroxyl groups of sugar molecules form sufficient hydrogen bonds with protein substituting the hydrogen bonds that were previously formed between water and protein to preserve the native conformation of proteins. Straight probing the sugar-water replacement and sugar interaction with protein is challenging in analytical characterization. FTIR was utilized to probe changes in the protein secondary structure as an indirect way to investigate the excipient stabilization effect on protein structure after drying. Carpenter and Crowe demonstrated that sugars can hinder changes in the amide II band through FTIR, which indicates alternations in protein secondary structure and hydrogen after drying as the first evidence of water replacement theory. However, quantitatively characterizing the relationship between alterations in FTIR spectra and the extent of protein structure disruption can be challenging, particularly when there are only slight changes in peak positions. More advanced solid-state characterization technique, such as ssNMR, was further applied to understand the role of protein-excipient interaction in the physical stability of protein after drying. The changes in chemical shifts of NMR spectra can indicate the intermolecular interactions between proteins and excipients. In a recent study of BSA with trehalose and mannitol, found that arginine side chain -NH and -NH2 are largely involved in hydrogen bonding as H donors to maintain the helical structure and tertiary folding of BSA. The signal assigned to the side chain 𝐶 𝜉 of arginine in the mannitol and trehalose was shifted from that of BSA without any excipient, indicating the disruption of intramolecular hydrogen bonds in BSA and possible formation of new intermolecular bonds with sugar molecules. (Figure )