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

Antibody-antibiotic conjugates (AACs) represent a class of highly potent biopharmaceuticals in which monoclonal antibodies (mAbs) are conjugated to antibiotic payloads via chemical linkers. Characterizing the peak purity and impurity profile of AAC drug-linkers is analytically challenging due to their complex, heterogeneous structures. In this study, a multimodal two-dimensional liquid chromatography (2D-LC) workflow was developed to assess peak purity and profile impurities in an AAC drug-linker. The strategy integrated comprehensive (LC × LC), heart-cutting (LC-LC), and selective comprehensive (sLC × LC) modes to provide comprehensive impurity characterization. Initially, LC × LC was utilized for rapid reference method screening, successfully identifying co-elution regions without extensive 1D-LC optimization. Targeted LC-LC was subsequently employed to resolve impurities co-eluting with the main peak through independent ²D optimization. Finally, sLC × LC improved the resolution of two impurities co-eluting within the fronting and tailing regions of the main peak, facilitating their quantification and identification. Beyond increasing peak capacity, this approach provided a rapid route to a comprehensive impurity profile by bypassing exhaustive method development. It directly improved the reliability of peak integration and facilitated the identification of hidden impurities, ensuring a more rigorous determination of the drug-linker purity. The method coupled a polar-selective SB-Aq column with a Phenyl-Hexyl column, effectively resolving impurities associated with both the hydrophilic linker and the hydrophobic drug moiety through 2D-LC. The developed 2D-LC platform provides a highly effective solution for impurity profiling in AACs and is readily adaptable to other bioconjugates, such as antibody-drug conjugates (ADCs) and antibody-oligonucleotide conjugates (AOCs), requiring high-resolution characterization of complex drug-linker species.