Comparison of Protein Particle Formation in IgG1 mAbs Formulated with PS20 Vs. PS80 When Subjected to Interfacial Dilatational Stress.

Polysorbates (PS) are nonionic surfactants that are commonly included in protein formulations to mitigate the formation of interfacial stress-induced protein particles and thus increase their long-term storage stability. Nonetheless, factors that dictate the efficiency of different polysorbates in mitigating protein particle formation, especially during the application of interfacial stresses, are often ill defined. Here, we used a Langmuir trough to determine the surface activity of two IgG1 monoclonal antibodies formulated with two different polysorbates (PS20 and PS80) when subjected to interfacial dilatational stress. Interfacial properties of these formulations were then correlated with characterization of subvisible protein particles measured by micro-flow imaging (MFI). Both mAbs, when formulated in PS20, demonstrate faster adsorption kinetics and higher surface activity compared to PS80 or surfactant-free formulations. Compression/expansion results suggest that when exposed to interfacial dilatational stresses, both mAb/PS20 formulations display interfacial properties of PS20 alone. In contrast, interfacial properties of both mAb/PS80 formulations suggest mAbs and PS80 are co-adsorbed to the air-water interface. Further, MFI analysis of the interface and the bulk solution confirms that PS20 is more effective than PS80 at mitigating the formation of larger particles in the bulk solution in both mAbs. Concomitantly, the efficiency of PS to prevent interface-induced protein particle formation also depended on the protein's inherent tendency to aggregate at a surfactant-free interface. Together, the studies presented here highlight the importance of determining the interfacial properties of mAbs, surfactants, and their combinations to make informed formulation decisions about the choice of surfactant.

Developability Assessments of Monoclonal Antibody Candidates to Minimize Aggregation During Large-Scale Ultrafiltration and Diafiltration (UF-DF) Processing.

Therapeutic proteins are subjected to a variety of stresses during manufacturing, storage or administration, that often lead to undesired protein aggregation and particle formation. Ultrafiltration-diafiltration (UF-DF) processing of monoclonal antibodies (mAbs) is one such manufacturing step that has been shown to result in such physical degradation. In this work, we explore the use of different analytical techniques and lab-scale setups as methodologies to predict and rank-order the aggregation potential of four different mAbs during large-scale UF-DF processing. In the first part of the study, a suite of biophysical techniques was applied to assess differences in their inherent bulk protein properties including conformational and colloidal stability in a PBS buffer. Additionally, the inherent interfacial properties of these mAbs in PBS were measured using a Langmuir trough technique. In the next part of the study, several different scale-down lab models were evaluated including a lab bench-scale UF-DF setup, mechanical stress (shaking/stirring) studies in vials, and application of interfacial dilatational stress using a Langmuir trough to assess protein particle formation in different UF-DF processing buffers. Taken together, our results demonstrate the ability of a Langmuir-trough methodology to accurately predict the mAb instability profile observed during large scale UF-DF processing.

Evaluating the Combined Impact of Temperature and Application of Interfacial Dilatational Stresses on Surface-mediated Protein Particle Formation in Monoclonal Antibody Formulations.

Formation of submicron and subvisible protein particles (0.1-100 µm) present a major obstacle during processing and storage of therapeutic proteins. While protein aggregation resulting in particle formation is well-understood in bulk solution, the mechanisms of aggregation due to interfacial stresses is less understood. Particularly, in this study, we focus on understanding the combined effect of temperature and application of interfacial dilatational stresses, on interface-induced protein particle formation, using two industrially relevant monoclonal antibodies (mAbs). The surface activity of Molecule C (MC) and Molecule B (MB) were measured at room temperature (RT) and 4 °C in the absence and presence of interfacial dilatation stress using a Langmuir trough. These results were correlated with Micro-flow imaging (MFI) to characterize formation of subvisible protein particles at the interface and in the bulk solution. Our results show that the surface activity for both proteins is temperature dependent. However, the extent of the impact of temperature on the mechanical properties of the monomolecular protein films when subjected to dilatational stresses is protein dependent. Protein particle analysis provided evidence that protein particles formed in bulk solution originate at the interface and are dependent on both application of thermal stresses and interfacial dilatational stresses. In the absence of any interfacial stresses, more and larger protein particles were formed at the interface at RT than at 4 °C. When mAb formulations are subjected to interfacial dilatational stresses, protein particle formation in bulk solution was found to be temperature dependent. Together our results validate that mAb solutions maintained at 4 °C can lower the surface activity of proteins and reduce their tendency to form interface-induced protein particles both in the absence and presence of interfacial dilatational stresses.

Impact of Polysorbate 80 Grade on the Interfacial Properties and Interfacial Stress Induced Subvisible Particle Formation in Monoclonal Antibodies.

Polysorbate 80 is a nonionic surfactant that is added to therapeutic protein formulations to mitigate protein particle formation when subjected to various mechanical stresses. Variations in the PS80 grade has recently sparked questions surrounding the effect of oleic acid content (OAC) on surfactant's ability to mitigate interface-induced protein particle formation when stressed. In this work, a Langmuir trough was used to apply interfacial dilatational stress to two IgG molecules (mAb1 and mAb2) in formulations containing Chinese pharmacopeia (CP) and multicompendial (MC) grades of PS80. The interfacial properties of these mAb formulations, with and without interfacial dilatational stresses, were correlated with subvisible particle count and particle size/morphology distributions as measured by Micro-flow imaging (MFI). Overall, differences in interfacial properties correlated well with protein particle formation for both molecules in the two PS80 formulations. Further, the impact of grade of PS80 on the interfacial properties and interfacial stress-induced protein particle formation depends on the adsorption kinetics of the IgG molecules as well as the concentration of the surfactant used. This study demonstrates that measuring the interfacial properties of mAb formulations can be a useful tool to predict interfacial stress induced protein particle formation in the presence of different excipients of varying quality.