One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs). APs have been shown to cause aggregation of pharmaceutical proteins. Previous work on a model protein demonstrated that different APs destabilize and aggregate proteins to different extents and in a specific order. The purpose of this investigation is to test the validity of these results by investigating the effect of APs on a pharmaceutically relevant protein, interferon alpha-2a (IFN). Using temperature scanning and isothermal studies, the aggregation and the stability of IFN in the presence of different APs and at varying AP concentration was determined. The apparent aggregation temperature in each case was deduced by monitoring increase in optical density due to light scattering. Denaturant melts were used to determine the structural and stability changes in IFN as a function of AP. NMR was utilized to monitor structural changes in IFN as a function of AP concentration. With increasing concentration of benzyl alcohol, the apparent aggregation temperature of IFN linearly decreased. The slope determined from this relationship is a qualitative measure of the effectiveness of benzyl alcohol in aggregating the protein. The value of the slope obtained in the case of IFN exactly matched with the value obtained from earlier studies on modal proteins. Isothermal incubation experiments indicated that the APs induce protein aggregation in a similar order as observed earlier with model proteins. NMR titration experiments demonstrated chemical shift and volume changes in IFN as a function of benzyl alcohol concentration. As observed earlier in studies on model proteins, the preservatives induced the aggregation by destabilizing the protein. The extent of destabilization was specific to the AP used, and was similar to model studies. Additionally, the preservative used in formulation, benzyl alcohol, caused significant destabilization of the therapeutic protein under formulation conditions. These results on IFN and model proteins suggest that the effect of APs on protein unfolding and aggregation may be independent of the nature of protein.

Antimicrobial preservative-induced aggregation of Interferon alpha-2a

247th National Meeting (2014)

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