Stress-dependent interfacial competition with direct binding enhances IgG4 stabilization.

This study elucidates the stress-dependent stabilization mechanisms of nonionic surfactants, polysorbates (Tween 20/80), and poloxamer 188 (P188) for IgG4 monoclonal antibodies (mAbs), a clinically important subclass with distinct structural vulnerabilities. To investigate these mechanisms, we employed a multi-method approach that integrated isothermal titration calorimetry (ITC), critical micelle concentration (CMC) measurements, and a multi-stress model encompassing orbital shaking, vortex stress, and freeze-thaw cycles. Our results demonstrate that polysorbates bind to IgG4 via entropy-driven hydrophobic interactions, whereas no such interaction was observed for P188. Under mild thermodynamically controlled stresses, both surfactants stabilized the protein through saturable interfacial adsorption once their concentrations reached the respective CMC, despite a pronounced difference in CMC values (Tween 20/80 ≪ P188). In contrast, under intense, kinetically controlled vortex stress, their mechanisms diverge: polysorbates require supra-CMC concentrations (4 × CMC) to act as dynamic monomer reservoirs and provide full protection, whereas P188 provides only limited stabilization because of its inherently slower adsorption kinetics. These findings establish that IgG4 stabilization is governed by distinct, stress-dependent pathways. The resulting framework directly connects fundamental protein-surfactant interactions with rational formulation design, facilitating a paradigm shift from empirical screening to mechanism-guided selection of stabilizers.