Analyzing insulin samples by size-exclusion chromatography: a column degradation study.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Chromatography

Background:

• Insulin analogs are critical therapeutics, but their stability during analytical testing is a concern.
• Degradation in size-exclusion chromatography (SEC) signal during insulin analysis impacts data quality and method reliability.

Purpose of the Study:

• To identify the root cause of signal degradation observed during SEC analysis of insulin samples.
• To develop strategies to mitigate column degradation and extend the lifespan of SEC columns used for insulin analysis.

Main Methods:

• Multiangle light scattering (MALS) for assessing molecular size and aggregation.
• Nuclear magnetic resonance (NMR) spectroscopy for detailed molecular structure analysis.
• Gas chromatography-mass spectrometry (GC-MS) for identifying volatile components and potential contaminants.

Main Results:

• Zinc ions, originating from the insulin product formulation, were identified as the primary cause of column performance degradation.
• The inclusion of ethylenediaminetetraacetic acid (EDTA), a potent zinc chelator, in the mobile phase effectively prevented column degradation.
• EDTA addition maintained consistent SEC signal and column performance over multiple sample injections.

Conclusions:

• Zinc ions in insulin formulations are detrimental to SEC column integrity.
• EDTA is a viable additive for mobile phases to mitigate zinc-induced column degradation in SEC.
• Implementing EDTA can significantly enhance the longevity and reliability of SEC methods for insulin analysis.