Role of domain unfolding and silicone oil-water interface in aggregation of a monoclonal antibody

Abstract

Monoclonal antibodies (mAbs) are an important class of therapeutic protein drugs that have been developed by pharmaceutical companies to treat major diseases. However, aggregation of mAbs is of major concern for pharmaceutical industry because it can affect product efficacy and can lead to immune response in patients. Reported incidences of immune response in patients range from 3-100%. Monoclonal antibodies fall under the category of multi-domain proteins where each domain contributes to the overall stability of the protein. In this work we found that partial unfolding of a monoclonal antibody resulted in protein aggregation. Partially unfolded protein species were formed when protein was incubated in sub-denaturing concentrations of Gdn.HCl and they had perturbed tertiary structure but native-like secondary structure. Further, partially unfolded protein species had unfolded Cʜ2 domains. In this work, we also investigated the tendency of a monoclonal antibody to form an interfacial gel upon adsorption to the silicone oil-water interface. Moreover, we also found that rupture of the interfacial gel can result in aggregation and particle formation in the bulk solution. Inclusion of surfactants in protein formulation can presumably inhibit interfacial gel formation and this resulted in reduced aggregation due to interfacial gel rupture. Lastly, we demonstrated that particles formed from interfacial gel rupture have a net neutral zeta potential making them colloidally unstable. Thus, over time, particles can agglomerate when incubated quiescently to form more particles.