During the freezing step in the lyophilization, protein molecules tend to adsorb and accumulate at the interface between ice and water, leading to denaturation and unfolding of proteins. As the protein solution undergoes freezing, the process of ice nucleation and crystal growth begins, creating an ice-water interface. The presence of this interface significantly impacts protein stability during freezing, as protein molecules interact and adsorb at the ice-water interface. Consequently, the ice formation can disrupt the interaction between water and the protein's surface, resulting in the loss of stabilizing hydration interactions between water and the protein components. It has been reported by Abdul-Fattah et al. that higher protein stability was seen with lower fractions of protein on the surface comparing different drying processes including freeze drying, spray drying, and foam drying associated with levels of sucrose in the formulation. Similarly, a study of using human growth hormone (hGH), the stability of freeze-dried hGH was found in a good correlation with the surface protein quantity. These experimental results all suggest that the relative fraction of protein on the surface over the bulk needs to be considered as well. Therefore, it is anticipated that the protein at the surface is less stable. Thus, the quantity of protein on the surface will impact the overall stability of the protein.