Elucidating the Mechanism of Protein Particle Formation under Mechanical Stress at Different Compressible Interfaces...

Mechanical stress of protein solutions in contact with a compressible interface can cause protein aggregation. This is a known problem for air-liquid and silicone-liquid interfaces, which occur during processing and handling of biopharmaceuticals. A systematic study comparing and unraveling the mechanism of particle formation at different compressible interfaces is lacking. To this end, we combined novel molecular dynamics simulations and established experimental setups that isolate and precisely define compression-decompression stress to elucidate and compare the mechanism of protein particle formation at the silicone-liquid interface, reflecting tubing used in pumping, and air-liquid interface. Simulations revealed that protein molecules bind rather loosely to the air-liquid interface and show high mobility. During interfacial compression, protein molecules therefore move from the air-liquid interface toward the bulk, reducing protein aggregation. At the silicone-liquid interface, strongly bound protein molecules are forced together upon compression of the adsorbed protein film, promoting particle formation already at little compression. Aggregates detach easily from the air-liquid interface, and compression further facilitates detachment. This enhanced detachment from the air-liquid interface renders similar particle counts in the bulk for both interface types at high interfacial compression, although simulations indicate less aggregate formation directly at the air-liquid interface. Clusters at the silicone-liquid interface break up during relaxation, whereas clusters at the air-liquid interface persist. This, in combination with more easy detachment leads to the formation of smaller particles at the air-liquid interface compared to the silicone-liquid interface. The simulations indicate that at high compression speed, the highly mobile protein molecules at the air-liquid interface do not have sufficient time to interact during compression and form fewer particles. Additionally, strong repulsive protein self-interaction resulting from high charge at low pH values reduced particle formation at the air-liquid interface more strongly due to the high molecular mobility at this interface as compared to the silicone-liquid interface. Our findings provide insights into the mechanisms of protein aggregation at different compressible interfaces, which is essential for developing strategies to mitigate particle formation in biopharmaceutical manufacturing and handling.