The effects of pharmaceutically relevant excipients---sucrose, Tween 20, dextran, and benzyl alcohol---on the physical stability of model protein pharmaceutics

The focus of this thesis is gaining knowledge towards the understanding of the effects of excipients on the physical stability of proteins. Maintaining physical stability is vital to formulating protein pharmaceutics. Two prominent formulation states, lyophilized and liquid, and the effects of the excipients on protein stability in at least one of these states are explored.; For the lyophilization study, the effects of Tween 20 and sucrose on the stability of a recombinant human IgG antibody were studied. We found that both excipients protected the secondary structure of the protein in the lyophilized state. When the lyophilized formulations were reconstituted with water, the samples containing Tween 20 had the highest level of aggregates, even more than an excipient-free formulation. Aggregation was prevented only when the antibody was lyophilized with sucrose and reconstituted with an aqueous Tween 20 solution.; The effect of Tween 20 on the freeze-thaw stability of two recombinant four-helix bundle proteins was also studied. Tween 20 destabilized the native state of both proteins by 2 kcal/mol. However, inhibition of freeze-thaw-induced aggregation was observed for both proteins using a range of Tween 20 concentrations. Prevention required levels of Tween 20 above the CMC. In only one case did Tween 20 aid in refolding.; For one of the four-helix bundle proteins, we also investigated the effect of dextran. Like Tween 20, dextran inhibited freeze-thaw-induced aggregation. However, dextran (4.0%) had no effect on the thermodynamic stability, within a 95% confidence interval. We proposed that dextran inhibited the unfolding via macromolecular crowding during freeze-concentration.; The subject of the final study was benzyl alcohol-induced aggregation of a predominately beta-sheet protein. Benzyl alcohol enhanced the propensity of the protein to precipitate as non-native aggregates. While benzyl alcohol had no effect on the protein's secondary structure, the tertiary structure was perturbed. We determined that benzyl alcohol induced a time-averaged conformational change that increased the accessibility of the protein's hydrogen atoms to be exchanged with deuterium atoms. We concluded that the rate-limiting step to aggregation was a time-averaged expansion of the native state, which was more favorable if benzyl alcohol (0.9%) was present.