Monitoring of low-molecular-weight protein aggregation by CE-SDS as a complementary method to SE-HPLC

Area of Science:

• Biopharmaceutical Analysis
• Protein Characterization
• Analytical Chemistry

Background:

• Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) is established for therapeutic protein analysis.
• Its application for low-molecular-weight proteins (<10 kDa) and peptides is less explored.
• Accurate characterization of protein aggregates is crucial for drug safety and efficacy.

Purpose of the Study:

• To demonstrate the utility of CE-SDS for purity assessment of low-molecular-weight proteins and polypeptides.
• To evaluate CE-SDS in characterizing insulin glargine aggregates under stress conditions (heat, light).
• To compare CE-SDS with size-exclusion high-performance liquid chromatography (SE-HPLC) for aggregate analysis.

Main Methods:

• Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) was employed.
• Insulin glargine samples were subjected to thermal and light-induced degradation.
• Mass spectrometry was used for aggregate confirmation.
• Size-exclusion high-performance liquid chromatography (SE-HPLC) was used for comparative analysis.

Main Results:

• CE-SDS successfully separated insulin glargine monomers, dimers, and trimers.
• Mass spectrometry confirmed two types of insulin aggregates, while SE-HPLC showed only one.
• Denaturation conditions revealed only covalent aggregates via CE-SDS.
• CE-SDS provided more detailed aggregate information than SE-HPLC.

Conclusions:

• CE-SDS is a powerful tool for characterizing the purity of low-molecular-weight proteins and polypeptides.
• CE-SDS offers superior resolution for detecting and differentiating protein aggregates compared to SE-HPLC.
• CE-SDS serves as a valuable supplementary technique to SE-HPLC in biopharmaceutical analysis.