Automated Multienzyme Digestion Workflows for Confident Characterization of Challenging Posttranslational Modifications in Therapeutic Antibodies by LC–MS/MS

RATIONALE: Non-reduced trypsin digestion of therapeutic antibodies can generate large disulfide-linked peptide complexes that exhibit poor chromatographic and mass spectrometric performance. This limits the confident characterization of posttranslational modifications (PTMs) in specific antibody regions, such as the heavy-chain constant region 1 (CH1) domain, where conventional digestion produces 8-kDa complexes with poor fragmentation. METHODS: We developed automated multistep and multienzyme digestion workflows using immobilized trypsin, chymotrypsin, and proteinase K on magnetic beads. These workflows were implemented on a robotic platform and evaluated for their ability to characterize engineered cysteine residues in the CH1 domain of antibody-drug conjugate (ADC) intermediates. Analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The multienzyme workflows generated highly orthogonal peptide pools; for instance, selected workflows using trypsin only, trypsin combined with chymotrypsin, and trypsin combined with proteinase K yielded 13%, 14%, and 34% unique peptide identifications, respectively. In the challenging CH1 region, the multienzyme approach identified six distinct cysteine adducts at engineered Cys152. These included cysteinylation, homocysteinylation, and an unexpected, putative process-related impurity, thioglycolic acid capping, which was validated with unambiguous MS and MS/MS evidence using selected multienzyme workflows. Relative extracted-ion current-based levels of these cappings were observed and found to be similar across selected workflows; for instance, relative levels of thioglycolic acid capping from 12% to 15% were observed across selected workflows. CONCLUSIONS: The presented automated multienzyme workflows serve as complementary tools in an analytical toolbox, which can be strategically deployed for targeting challenging regions of biopharmaceutical products. We demonstrate significantly improved precursor signal quality and MS/MS sequence coverage compared to trypsin-only protocols. The presented workflows are fast (36-92 min total runtime) and utilize commercially available reagents and instrumentation, making them suitable for direct implementation in biopharmaceutical analytical development and quality control laboratories.