Protein-protein interactions and self-association of etanercept and immunoglobulin G1 in the bulk phase and at the air-water interface: Effects of NaCl and sucrose.

Hypothesis Self-association of therapeutic proteins remains a major challenge in biopharmaceutical development, as it can impair drug efficacy and trigger immunogenic responses. Such protein-protein association processes can occur both in the bulk solution and at interfaces. Excipients such as salts and sugars are commonly employed to modulate protein stability; however, their effects vary with protein identity and solution conditions. Experiments In this study, we investigated and compared protein-protein interactions and self-association behavior of Enbrel, a fusion protein, and the structurally similar IgG1 monoclonal antibody in the presence of two excipients, sucrose and NaCl, both in the bulk solution and at the air-water interface. Protein stability and interactions in these two domains were characterized using surface tension isotherms, interfacial rheology, and concentration-dependent dynamic light scattering. Findings Our results demonstrate that the effects of excipients are strongly protein- and domain-dependent. NaCl shifts the interactions between Enbrel molecules toward weaker attraction in the bulk solution, while showing a trend toward enhanced attractive interactions in IgG1 solutions. In addition, sucrose reduces attractive interactions between proteins in the bulk phase but promotes enhanced intermolecular association of Enbrel at the air-water interface. Taken together, these findings offer a mechanistic understanding of how excipients influence protein interactions and contribute to the rational design of formulations for structurally complex biologics.