ABSTRACT 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 2 D 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.
Wang et al. (Mon,) studied this question.