GOALI: Aggregation of Protein Therapeutics in Aqueous Solutions

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

Therapeutic proteins provide numerous unique treatments for human diseases (e.g., diabetes, and cancer). However, due to their delicate three-dimensional structures, proteins are very difficult to keep stable during the required 18-24 month shelf life of a pharmaceutical product. Furthermore, it is currently not possible to predict which solution conditions will result in sufficient stability. The Principal Investigators (PIs) propose to conduct fundamental studies of critical physical properties of proteins to develop methods for predicting optimal solution conditions for long-term stability. This multidisciplinary project combines efforts from Chemical Engineering and Pharmaceutical Sciences Departments at the University of Colorado with those of Amgen, an industrial partner. The PIs propose to determine the effects of critical solution conditions (pH, ionic strength and presence of stabilizing excipients) on rate and pathway for aggregation of several model proteins. Levels, and kinetics for formation and loss (where applicable) of soluble oligomeric species and insoluble precipitates will be measured, and structure of proteins in aggregates will be determined with infrared and derivative UV spectroscopies. To understand these effects, the PIs will determine the effects of pH, ionic strength and stabilizing excipients on protein structure and thermodynamic properties. Protein secondary and tertiary structures will be studied with circular dichroism, infrared, fluorescence and derivative-UV spectroscopies. The free energy of unfolding and hydrogen-deuterium exchange rates will be measured to test the hypothesis that increasing the thermodynamic stability of the native state and shifting the native state ensemble toward most compact species are important factors for inhibiting protein aggregation. The second osmotic virial coefficient will be measured by light scattering to assess the effects of solution conditions on the colloidal stability of the protein systems. High-pressure spectroscopic techniques will be used to complement these measurements. Finally, the effect of solution conditions on chemical stability (e.g., deamidation, oxidation and non-native disulfide formation) of the model proteins will be determined by the industrial collaborators at Amgen, and linkages between physical and chemical stability of the model proteins will be elucidated.