Aggregation and oligomer formation are critical parameters in the field of protein therapeutics and can lead to loss of drug function or even immunogenic responses in patients. Currently two approaches are used to reduce aggregation: (1) finding a suitable formulation which is labor-intensive and requires large protein quantities or (2) engineering the protein by specific, stabilizing mutations, which requires specific knowledge about the protein aggregation pathway. We present a biophysical characterization of the oligomerization and aggregation process by Interferon alpha-2a, a protein drug with antiviral and immunomodulatory properties. We combine high throughput screening with detailed investigations by small-angle X-ray scattering and analytical ultracentrifugation. To get more insight into the molecular mechanism that drives oligomerization and aggregation, we apply molecular Metropolis Monte Carlo simulations. IFNα-2a forms soluble oligomers, which show a fast pH and concentration-dependent equilibrium. We show that attraction between monomers is mainly driven by molecular dipole-dipole interactions, which becomes more pronounced with increasing pH. Repulsion is dominated by ion-ion interaction leading to the formation of insoluble aggregates around the pI which could be prevented by the addition of salt. This study shows how a combination of several methods can help to understand the formation of aggregates and oligomers more systematically and comprehensively, which can lead to better strategies for avoiding aggregation.
SI - Effects of electrostatics on interferon alpha