Poloxamer 188 Promotes Severe Aggregation of Insulin under Shaking Conditions

Abstract

Insulin is an indispensable cornerstone in the management of diabetes mellitus, yet its extreme susceptibility to aggregation under mechanical stress remains a formidable challenge for long-term formulation stability. This study investigates the potential of Poloxamer 188 (P188) as a chemically stable alternative to the industry-standard Polysorbate 20 (PS20) for stabilizing insulin. Formulations containing graded concentrations of P188 (0.005%−0.15%, w/v), 0.05% (w/v) PS20, or no surfactant were subjected to 24 h shaking stress at 300 rpm. Our results reveal a striking concentration-dependent profile for P188, where 0.05% PS20 consistently maintains robust protection, whereas low-to-moderate concentrations of P188 (0.005%−0.05%) fail to stabilize insulin and instead exert a pronounced “pro-aggregatory” effect. At 0.05% P188, this pro-aggregatory behavior leads to severe macroscopic turbidity, a massive surge in particle concentration (≥2 μm), and the near-total disappearance of detectable insulin monomers in the supernatant. Crucially, elevating the P188 concentration to 0.15% effectively prevents macroscopic turbidity and preserves high soluble protein recovery. Mechanistically, the instability observed at low-to-moderate P188 concentrations stems from its sluggish interfacial adsorption kinetics compared to PS20, leaving nascent air-liquid interfaces temporarily unprotected. Under such conditions, P188 functions as a macromolecular scaffold and nucleation core, cross-linking interface-denatured proteins with native molecules to drive rapid co-assembly. These findings serve as a critical warning for the biopharmaceutical industry that the use of P188 in insulin-based products must be approached with extreme caution to avoid inadvertent protein aggregation.