Unraveling the Role of Formulation Parameters in Protein Particle Formation at Moving Interfaces Using Molecular Dynamics and Experiments

Interfacial stress during peristaltic pumping can lead to particle formation in biopharmaceutical solutions. Since the impact of formulation on protein particle formation is not fully understood, we combined molecular dynamics (MD) simulations with experimental methods to investigate and understand the effects of pH, ionic strength, and protein type during peristaltic pumping. Building on our previous work, we improved the MD model to provide a more accurate representation of a protein solution at the polymer interface. Our results indicate that the pH value affects aggregate formation in a human growth hormone solution, both while protein molecules are adsorbed to the interface and during the detachment of aggregates into the bulk. Both steps were also directly influenced by protein-protein interactions. Studies at high ionic strength suggest that when protein self-interaction is similar, the amount of protein molecules adsorbed to the interface can be decisive of the extent of particle formation. Additional studies employing lysozyme as a second protein confirmed that protein-protein interactions are the key factor in protein aggregation at interfaces, validating the MD model and our findings across different low-molecular-weight proteins. Our study uncovers the specific points of action through which formulation parameters influence protein particle formation upon mechanical interfacial stress. Furthermore, our model enables the prognosis of protein particle formation <i>in silico</i>, potentially saving resources in formulation and process development.