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

Area of Science:

• Biopharmaceutical Science
• Protein Chemistry
• Rheology

Background:

• Protein aggregation is a major challenge in biopharmaceutical formulations, impacting stability and efficacy.
• Human insulin (HI) aggregation is well-studied under pH and temperature variations, but combined stressors are less understood.
• Physicochemical stressors like temperature, pH, and shear can synergistically compromise protein integrity.

Purpose of the Study:

• To investigate the aggregation kinetics of human insulin (HI) under combined pH, shear, and thermal stress.
• To elucidate the conformational changes and aggregation mechanisms induced by these stressors.
• To assess the impact of shear-induced aggregation on HI's structural integrity and cytotoxicity.

Main Methods:

• Assessed HI aggregation kinetics at varying shear rates (1-1000 s⁻¹) and temperatures (25-60°C).
• Utilized fluorometry (Thioflavin-T, intrinsic tyrosine fluorescence), circular dichroism, and transmission electron microscopy (TEM).
• Employed native PAGE, BCA assays for monomer depletion, and cytotoxicity studies.

Main Results:

• At 60°C and low pH, HI exhibited non-Newtonian behavior, with shear thickening followed by thinning due to aggregate fragmentation.
• Shear-induced energy exceeded unfolding free energy, catalyzing unfolding, β-sheet formation, and aggregation.
• Shear reduced fibrillation lag time by 80-fold; α-helix content decreased significantly under high shear and temperature; TEM showed fibrillar-to-amorphous transitions.

Conclusions:

• Shear stress acts as a potent catalyst for human insulin aggregation, exacerbating unfolding and fibril formation.
• Combined thermomechanical stressors significantly impact HI's structural integrity and can lead to reduced cell viability.
• Stringent control of temperature and shear is essential during insulin bioprocessing, transport, and storage to ensure biopharmaceutical stability.