Enzymatic assay based platform for the chiral purity assessment of cathepsin-cleavable antibody-drug conjugate linker-drugs

Controlling the chiral purity of cleavable linker-drugs for antibody-drug conjugates (ADCs) is important, but traditional chromatographic methods are hindered by long development timelines, extensive column screening, and the need for difficult-to-synthesize impurity standards. Here, we report an enzymatic digestion assay that uses the protease papain to selectively digest the desired (S)-citrulline epimer of cathepsin-cleavable linker-drugs. This approach allows the undigested (R)-epimer to serve as its own quantifiable surrogate via standard reversed-phase liquid chromatography (RPLC). Optimized for robustness using Design of Experiments, the assay was shown to be highly linear (R² > 0.999), specific, and orthogonal to chiral supercritical fluid chromatography (SFC). The molecule-agnostic nature of the assay was confirmed by its successful application to various linker formats, including Sq-Cit, Val-Cit, and GGFG sequences, while its practical utility was demonstrated by distinguishing between manufacturing lots with different epimer levels (1.9 % vs. 9.9 %). Coupling the assay with mass spectrometry further extends its utility to substrates with poor chromophores. This fit-for-purpose enzymatic assay offers a valuable and efficient analytical tool to accelerate process understanding and optimization in the development of ADC therapeutics, particularly at the earliest stages of process development.