Protein aggregation is a major concern for delivering biologic-based drug therapies that must journey from the manufacturing pipeline to the patient. Routes along the way that pose challenges in this process include: storage, transportation, drug delivery, and tissue absorption. As such, drug aggregates represent a known, but not well understood, problem in biologic-based product development. This study focused on understanding the mechanistic impact of protein conformational structure, protein-protein interaction, and protein-surfactant interaction on aggregate formation of a pharmaceutical monoclonal antibody (mAb).
Differential scanning calorimetry (DSC) was used to measure thermodynamic stability and assess conformational structure in formulation buffers of pH 4.5 and pH 6.5. Light scattering was used to characterize protein-protein interaction. Size-exclusion chromatography (SEC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were utilized to monitor degradation induced by thermal stress. The cutting-edge technology ofliquid cell electron microscopy (EM) was used to visualize mAb molecules in the formulation buffer environment. In addition, liquid cell EM was used to observe the protein molecule distribution in the presence and absence of polysorbate 80 (PS80) and to characterize protein aggregate morphology in solution without PS80.
DSC results showed that protein molecules were in a partially unfolded conformational state in the formulation buffer of pH 4.5. The results obtained from light scattering and liquid cell EM in the formulation buffer of pH 4.5 were in good agreement and indicated stronger repulsive interactions along with a higher degree of uniformity for molecular distributions compared to that of pH 6.5. The stability results revealed a lower level of aggregation but a higher level of fragmentation at pH 4.5 compared to that at pH 6.5. In addition, liquid cell EM determined a higher degree of uniformity among individual proteins in the formulation containing PS80 in comparison to formulations lacking PS80. Morphological aggregates were also visualized using molecular imaging techniques.
The results obtained from this study revealed new degradation insights associated with protein structure, protein-protein interactions, and protein-surfactant interactions. Repulsive force and homogeneity in the molecular distribution was minimized during aggregation formation. The partially unfolded conformational state at pH 4.5 may induce molecules to be exposed within the acidic buffer, leading to fragmentation. The results obtained from liquid cell EM demonstrated that PS80 improved the degree of uniformity among the molecular distribution, which may be a key factor in the self-assembly process to minimize aggregation. The significance of liquid cell EM imaging provided a real-time view of formulation solutions, bringing forth a new means to visualize protein-based therapeutics at the molecular level.