As illustrated in Section 4.2, the miscibility of protein and excipient is crucial in maintaining protein stability. Excipient crystallization during the drying process can compromise the interaction between protein and sugar limiting the protein motion. As such, it is crucial for excipients as amorphous during the freezing and drying process to ensure sufficient hydrogen bonding interaction between protein and stabilizing agents. The capacity of sugar to form intermolecular hydrogen bonding can be diminished and loss of its stabilization function if the sugar crystallizes. For example, though trehalose is considered as best sugar for protein stabilization as amorphous, crystallization can occur during the sublimation process when trehalose is formulated at high concentrations and subsequently, hydrogen bonding with protein is no longer possible. Carpenter et al. showed that the FTIR spectrum of lysozyme with crystalline trehalose was similar to the sugar-free enzyme lyophilized sample with limited frequency of amide II band which indicated decreased interaction between trehalose and protein when trehalose crystallized at high concentration. With this design principle to retain the amorphous form of sugar stabilizer, studies have explored formulation optimization to overcome excipient crystallization during the protein drying process. Suryanarayanan and coworkers investigated the influence of sucrose in trehalose dihydrate crystallization 247 and the effective inhibition of mannitol crystallization using pharmaceutical-acceptable additives, including salts and polysorbate Additionally, buffers also play a role in affecting the crystallization of bulking sugar agents in freeze-dried formulations. For example, potassium phosphate buffer was observed to hinder the mannitol crystallization and inhibit the protein structural changes during freeze-drying. Similarly, the inhibition of mannitol crystallization was found with the presence of sodium phosphate and sodium citrate. Furthermore, the formulation composition including the ratio of trehalose to proteins and the process parameter such as the slow cooling rate was found also play a critical role in inhibiting trehalose crystallization. Similarly, for sugar which has a crystallization tendency, the concentration and percentage of sugar used in the formulation are the critical factors that drive the risk of crystallization and require optimization during the formulation development. For example, leucine used at 50% weight percentage in the BSA formulation was observed to lead to significant crystallization post spray-drying and subsequently worse BSA storage stability. In contrast, no crystallization was found when leucine was only used at 20% and good protein stability was observed. Among the sugars studied by Chen et al, leucine exhibited the highest propensity of crystallization following spray drying observed from 13 C MAS NMR. Overall, the stabilizing capability of sugars varies which largely depends on the sugar types, crystallinity, and drying process. Elucidation of the interplay between sugar type and drying regime would be valuable for formulation optimization.