HPLC Method Development For Monoclonal Antibody Aggregates With SEC

By Brian Rivera

This content is brought to you by Phenomenex, a Danaher Operating Company.

Size exclusion chromatography may look straightforward for monoclonal antibody analysis, but mAbs rarely behave like ideal SEC analytes. True size-based separation depends on minimizing electrostatic and hydrophobic interactions with the stationary phase, and that can be challenging when working with complex, surface-active biomolecules. Even a standard buffer such as 1X PBS may not prevent secondary interactions that distort peak shape, reduce recovery, or compromise quantitative aggregate analysis. For teams relying on SEC to monitor mAb purity, stability, or aggregation, method conditions cannot be treated as an afterthought.

Understanding where non-ideal behavior comes from and how buffer composition, column chemistry, and method parameters influence results can help turn a frustrating method-development exercise into more reliable, reproducible data.