5.1 Rationale choice of sugar as protectant 5.1.1 Sugar size and molecular flexibility As described in Section 3.1, it is critical to ensure adequate contact between sugar and proteins to restrict the protein motion. As a good stabilizer for protein stabilization, sufficient hydrogen bonds between protein and sugar, adequate mobility suppression, and sufficiently high Tg during the drying process is the prerequisites. The size and molecular flexibility of sugars determine their ability to stabilize proteins. As such, disaccharides such as trehalose and sucrose are typically considered the best choices for stabilizing disaccharides for therapeutic proteins. Practically, trehalose with good interaction with protein and reduced local mobility is commonly considered an ideal stabilizer. On the other hand, large molecule weight sugar such as dextran is not selected to use as a sugar stabilizer, especially when used alone. The main drawback is the limited flexibility of the molecular chains which limit their ability to interact with protein and form adequate hydrogen bonding due to their large size. As such, the steric hindrance limits the adequate interaction/hydrogen bonding between protein and sugars and compromises the effect of slowing the protein dynamic in the solid state. The capability of vitrification at the surface of the protein is also compromised. Likely, study has shown that molecularly flexible oligosaccharide inulin 4 KDa provided better stabilization in four different proteins than the similarly sized but molecular rigid oligosaccharide dextran 6 KDa. Recently, Nguyen et al. found an interesting correlation between sugar molecular weight and miscibility and stability. They hypothesized that the protein-sugar miscibility is not a function of the absolute size of the sugar, but instead is a function of the relative size of the sugar to the protein. The order of miscibility was found to be trehalose > dextran 2000 kDa > dextran 6 kDa and dextran 70 kDa shown from Figure and Figure . BSA molecules were dispersed within the polymer chains of dextran 2000 kDa, and hence have a lower tendency to be in contact with other BSA molecules. The proposed model of sugar and protein miscibility was shown in Figure . Aside from the stabilization consideration in formulation design, the drying process efficiency also plays a critical role in the formulation optimization. Though it is well known that sucrose is traditionally utilized as a sugar stabilizer in solid protein formulation, from a process perspective, sucrose has some limitations in the drying steps including low glass transition of 60 ºC and collapse temperatures of -34 to -25 ºC. Consequently, these formulations require low primary drying temperatures making the lyophilization cycle time-consuming and costly. Additionally, vitrification can be problematic when the moisture content cannot be kept low. A combination of larger oligosaccharides or polysaccharides which has higher Tg with disaccharides can be an alternative strategy to mitigate this issue to increase the Tg of dried products. Dextran can function as an amorphous bulking agent and facilitate a rapid lyophilization process with the formation of a strong and elegant cake structure. In a study on a model protein BSA, Haeuser et al. found the influence of dextran with different molecule weights on the thermal properties and cake appearance in the solid state. Such a binary mixture of sucrose and dextran in an adequate ratio might potentially be superior to pure sucrose formulation allowing faster freeze-drying cycles resulting in elegant lyophilizates and good protein stability. Moreover, the 1:1 mixture of sucrose and dextran between 10-500 kDa showed about 5-fold reduced aggregation than pure dextran formulations and stability is inversely proportional to the increase of dextran molecular weight. A similar observation was found in the study using dextran 6 kDa and 70 kDa in insulin stabilization. Remarkably, the insulin formulation with dextran 6 kDa was even less stable than the formulation of insulin only (no sugar). In contrast, the dextran 70 kDa formulation showed a more stabilizing effect than when the smaller dextran was used in freeze-dried protein formulations. The stabilizing capacity of dextran 70 kDa was substantially improved through a 1:1 weight ratio mixture.