Moisture-induced aggregation of lyophilized insulin

Area of Science:

• Pharmaceutical Science
• Protein Chemistry
• Biochemistry

Background:

• Protein aggregation is a major challenge in pharmaceutical protein storage and delivery.
• Elevated temperature and moisture accelerate protein aggregation, impacting stability during accelerated storage and in vivo delivery devices.

Purpose of the Study:

• To investigate the mechanisms of aggregation in lyophilized insulin under simulated storage conditions.
• To identify factors influencing insulin aggregation and explore methods for amelioration.

Main Methods:

• Exposure of lyophilized insulin to elevated temperature and moisture.
• Analysis of covalent and noncovalent aggregation pathways.
• Investigation of the role of water content and pH.
• Assessment of copper(II) ion (Cu2+) effects on aggregation.
• Water sorption isotherm analysis.

Main Results:

• Lyophilized insulin undergoes both covalent and noncovalent aggregation when exposed to heat and moisture.
• Covalent aggregation is driven by intermolecular thiol-catalyzed disulfide interchange following beta-elimination.
• Aggregation is accelerated by increased temperature, water content, and alkaline conditions during lyophilization or dissolution.
• Copper(II) ions (Cu2+) were found to ameliorate aggregation, likely by catalyzing thiol oxidation.
• A direct correlation exists between the extent of aggregation and water uptake by the insulin powder.

Conclusions:

• Protein conformational mobility, influenced by water content, is critical for insulin aggregation.
• Understanding these aggregation mechanisms is crucial for developing stable pharmaceutical protein formulations.
• Copper(II) ions show potential as an additive to inhibit insulin aggregation.