Protein Modality and Bulk Concentration Impact the Evolution of Rheological Properties of IgG mAb and Fc-Fusion Protein Films at an Air-Water Interface.

Therapeutic proteins, such as monoclonal antibodies (mAbs), can form two-dimensional films at the air-water interface, which, when ruptured or stressed, can lead to protein instability in solution. However, the details of the dynamics of mAb film formation, particularly the transitions in the rheological properties of the film, are not well understood. The emergence of novel protein-based modalities also raises the question of whether our understanding of mAb film formation can be extended to other novel biotherapeutic modalities. This work aims to understand the dynamics of film formation in novel therapeutic proteins by correlating protein adsorption kinetics, measured using surface tensiometry, with the evolution of the interfacial rheological properties, measured using a passive microrheology technique, for two protein modalities: IgG mAb and Fc-Fusion protein. Further, to accurately record how differences in packing density at the interface might lead to differences in the rheological properties in these different protein modalities during film aging, the bulk protein concentration was varied over 3 orders of magnitude. Our results indicate that the multistage protein adsorption process seen in both protein modalities can result in transitions in film rheology from purely viscous to viscoelastic and elastic films with time, depending on the bulk concentration and protein modality. Fc-Fusion proteins demonstrated an earlier onset of viscoelastic film transition compared with IgG mAbs for all bulk concentrations studied. Further, polysorbate 80 (PS80), often added to protein solutions to mitigate protein aggregation, prevents this transition in IgG mAb solutions but not in Fc-Fusion protein solutions. Together, our results provide rheological characterization of the protein films during adsorption and film aging in these novel biotherapeutics and will help inform the development of appropriate mitigation strategies to maintain their interfacial stability.