Shear-Induced Structural Changes Drive Amorphous Aggregate Formation of Human Insulin.

Abstract

The aggregation of protein-based biopharmaceutical formulations constitutes a major challenge in the pharmaceutical industry, where physicochemical stressors, viz., temperature, pH, shear, and high concentrations, synergistically compromise structural integrity, stability, and therapeutic efficacy. While human insulin (HI) aggregation under pH and temperature variations has been extensively studied, the combined effects of pH, shear, and thermal stress on its conformational behavior remain underexplored. This study assessed the HI aggregation kinetics under varying (1-1000 s^-1) and constant shear rates (50, 100, 300, and 500 s^-1) at four temperatures (25°C, 37°C, 50°C, and 60°C). At 60°C and low pH, HI exhibited non-Newtonian rheological behavior, initially undergoing shear thickening due to higher-order structure formation, followed by shear thinning as aggregates fragmented. Shear-induced dissipation energy exceeded the free energy of unfolding (ΔG_unfold) of HI, catalyzing the unfolding, aberrant β-sheet propagation, and eventual aggregate formation. Fluorometry employing thioflavin-T and intrinsic tyrosine fluorescence indicated a time-dependent effect of shear in insulin unfolding. Thioflavin fluorescence showed an 80-fold reduction in fibrillation lag time, highlighting shear as a potent catalyst of aggregation. Tyr^A19 and Tyr^B26 mediated interchain interactions supported fluorometric findings. Circular dichroism revealed α-helix content plummeting to 16% within 2 min at 500 s^-1 shear at 60°C. Transmission electron microscopic studies showed fibrillar-to-amorphous aggregate transition under shear. Native PAGE and BCA assays confirmed monomer depletion, while cytotoxicity studies indicated 53% cell viability after 10 min of HI incubation at 60°C and 500 s^-1 shear. These findings emphasize the necessity of stringent control of thermomechanical stressors in insulin bioprocessing, transport, and storage to mitigate aggregation-related complications to enhance biopharmaceutical stability.