Impurity Profiling of an Antibody-Antibiotic Conjugate (AAC) Drug-Linker Using Multiple 2D-LC Modes

Area of Science:

• Biopharmaceutical analysis
• Chromatographic techniques
• Conjugate chemistry

Background:

• Antibody-antibiotic conjugates (AACs) are potent biopharmaceuticals requiring rigorous purity assessment.
• Characterizing AAC drug-linker purity and impurities is analytically challenging due to structural complexity and heterogeneity.

Purpose of the Study:

• To develop and validate a multimodal two-dimensional liquid chromatography (2D-LC) workflow for comprehensive impurity profiling of AAC drug-linkers.
• To enhance the reliability of peak integration and impurity identification in AAC analysis.

Main Methods:

• Integrated comprehensive (LC × LC), heart-cutting (LC-LC), and selective comprehensive (sLC × LC) modes of 2D-LC.
• Employed a polar-selective SB-Aq column coupled with a Phenyl-Hexyl column.
• Utilized LC × LC for rapid screening, LC-LC for targeted resolution, and sLC × LC for resolving challenging co-elutions.

Main Results:

• Successfully identified and resolved co-eluting impurities without extensive 1D-LC optimization.
• Improved resolution of impurities in the fronting and tailing regions of the main AAC peak.
• Demonstrated enhanced peak capacity and facilitated identification of previously hidden impurities.

Conclusions:

• The developed multimodal 2D-LC platform provides a rapid and effective solution for AAC impurity profiling.
• This approach significantly improves the accuracy of drug-linker purity determination.
• The method is adaptable for characterizing other complex bioconjugates like ADCs and AOCs.