Analysis of protein drugs aggregation Using Size Exclusion Chromatography

Importance of the Topic

The analysis of aggregation in therapeutic proteins is critical for ensuring their efficacy and safety. Monoclonal antibodies and antibody drug conjugates can undergo aggregation or fragmentation due to environmental factors, impacting product quality. Reliable monitoring by size exclusion chromatography under challenging mobile phase conditions is essential in biopharmaceutical development and quality control.

Objectives and Study Overview

This study demonstrates an optimized SEC method using a metal-free inert UHPLC system and specialized column to suppress secondary interactions, improve reproducibility, and achieve clear separation of monomers, aggregates, and fragments. Two case studies on mAb and ADC aggregates were conducted to evaluate salt and organic solvent effects on chromatographic performance.

Methodology and Instrumentation

The separation was performed on a Shimadzu Nexera XS inert UHPLC equipped with a Shim-pack Bio Diol-300 column (150 mm×4.6 mm, 2 µm). For mAb analysis, a phosphate buffer (100 mmol/L, pH 6.9) with or without 150 mmol/L sodium chloride was used. ADC analysis employed the same buffer with varying acetonitrile concentrations (0–15 % v/v). The inert flow path tolerated high salt and organic content, and UV detection at 280 nm captured protein peaks.

Main Results and Discussion

For mAb, addition of 150 mmol/L NaCl effectively suppressed electrostatic adsorption, eliminating monomer peak tailing and enhancing aggregate/fragment resolution. In ADC analysis, increasing acetonitrile to 5–15 % reduced hydrophobic interactions, sharpening monomer peaks and resolving aggregate and fragment peaks, though excessive organic content risks protein destabilization.

Applications and Practical Benefits

The optimized method delivers highly reproducible aggregate profiles even in corrosive mobile phases, supporting quality control and formulation screening. The inert UHPLC design prevents metal-induced adsorption, extending column lifetime and ensuring data reliability in protein drug analysis.

Future Trends and Opportunities

Further advancements may include coupling inert SEC with multi-angle light scattering or mass spectrometry for absolute molar mass determination, exploring alternative buffer additives, and integrating AI-driven data analysis for real-time monitoring. Development of novel stationary phases with tailored surface chemistries will enhance specificity and throughput in biopharmaceutical analytics.

Conclusion

Optimizing mobile phase composition and employing an inert UHPLC system with a specialized SEC column enables robust, high-resolution analysis of mAb and ADC aggregates. This approach strengthens quality control processes and accelerates therapeutic protein development.

References

1. Rosenberg AS. Effects of protein aggregates: an immunologic perspective. AAPS J. 2006;8:E501–E507.
2. Striegel AM, Yau WW, Kirkland JJ, Bly DD. Modern Size-Exclusion Liquid Chromatography. 2nd ed. John Wiley & Sons; 2009.
3. Arakawa T, Ejima D, Tsumoto K, Gagnon P. Solvent modulation of chromatography. J Jpn Biochem Soc. 2008;80(1):45–51.