(595c) Comparing Interface-Induced Protein Particle Formation in a IgG1 Monoclonal Antibody Vs. a Fusion Protein

Use of biotherapeutic drugs in treatment of critical and life-threatening diseases has seen a rapid expansion in recent years. A major challenge limiting the success of biotherapeutics is maintaining stability of the protein formulations during manufacturing and storage steps. Particularly, many protein-based biologics have a propensity to aggregate, leading to protein particle formation that can cause loss of the biologic drug and/or increased immunogenicity risks. In this study, we compare differences in the tendencies of two biologic drugs with structural differences (an IgG1 monoclonal antibody, M2, and a fusion protein, M3) to form interface-induced protein particle formation, when subjected to interfacial dilatational stress, using a Langmuir trough. The interfacial properties were then correlated with microflow imaging (MFI) in the bulk solution and at the air-liquid interface to characterize subvisible particle size, concentration, and morphology of M2 and M3. Since polysorbate 80 (PS80) is a commonly used nonionic surfactant added to therapeutic drug formulations to increase protein stability and mitigate protein particle formation at the interface, when exposed to various mechanical stresses, we also studied the effectiveness of adding PS80 to the two different biologic formulations. Our results showed that in the absence of PS80, while both M2 and M3 readily absorb to the air-water interface, M2 shows a constant evolution of the surface pressure, suggesting reorganization at the interface. When subjected to interfacial dilatational stresses, M3 (fusion protein) showed lower compressibility and less hysteresis, compared to M2, suggesting that the fusion protein has greater interfacial stability than M2 (mAb). MFI data shows that M2 forms significantly more and larger particles at the interface compared to M3. In the presence of PS80 formulated above the critical micelle concentration, the surface activity in all quiescent studies were surfactant dominated for both proteins, while the addition of interfacial dilatational stress led to co-adsorption for M3. Protein particle analysis showed that PS80 was more effective in mitigating particle formation for M2 than M3 suggesting greater interfacial stability for M2 in the presence and absence of interfacial dilatational stress. For M3, the addition of PS80 led to several smaller particles in the bulk and at the interface that were mitigated for M2, with higher PS80 concentrations leading to greater particle formation for M3. This suggests that, while the primary mechanism of protein particle formation is surface-mediated protein particle formation for M2, this may not be the only mechanism leading to particle formation for M3. Our results for the fusion protein suggests the possible formation of surfactant/protein complexes that serve as nucleation sites for protein particle formation in the bulk solution. Thus, we conclude that 1) structurally different biology drugs have differences in their mechanisms of protein particle formation and must be explored in more detail, 2) PS80, while effective in mitigating particle formation, may not always be the best choice for overall protein stabilization.