Subvisible particle formation under mechanical and interfacial stress: A case study using bovine serum albumin as a...

The stability of biotherapeutic proteins is critical for drug efficacy and patient safety, with protein aggregation and subvisible particle (SVP) formation posing significant challenges. Mechanical, thermal, and interfacial stresses encountered during manufacturing, storage, and administration can destabilize proteins and increase immunogenicity. While shear stress has traditionally been viewed as a primary cause of aggregation, recent evidence suggests that interfacial dynamics and extensional stresses may play a more significant role. However, the relative effects of these factors remain poorly understood. To address this gap, we developed an experimental process to exert controlled stresses on Bovine Serum Albumin (BSA) samples and measured SVP formation using optical methods. We examined the effects of acoustic- and laser-induced cavitation, shear stress, extensional stress, and air/solution interfacial stress, alongside variations in surfactant and protein concentrations. Our results demonstrate that extensional stress and vapor-solution interfaces induce significantly greater protein aggregation and SVP formation than shear stress and air-solution interfaces. Cavitation proved particularly harmful, generating localized increases in pressure and temperature. The addition of surfactants mitigated the impact of all stress types, reducing both particle count and size. These findings challenge the conventional emphasis on shear stress as the primary driver of protein aggregation, underscoring the need for stress-specific strategies in biopharmaceutical manufacturing. By highlighting the critical roles of extensional stress and cavitation-induced interfaces, this study provides valuable insights for optimizing formulation, manufacturing, and delivery processes to enhance the stability, efficacy, and safety of protein therapeutics while minimizing immunogenicity.