Multilevel Characterization of a Chemoenzymatic Conjugated ADC by icIEF‐UV/MS and RP‐HPLC‐MS EAD Fragmentation Peptide Map

This study includes the synthesis of monomethyl auristatin E (MMAE) payload conjugated Trastuzumab (TRA) by an enzyme-mediated glycan-remodelling reaction, followed by comprehensive multilevel characterization of conjugation end products. Intact proteoforms of TRA and TRA-MMAE antibody-drug conjugates (ADC) were separated, quantitated, and identified by microfluidic chip-based ultraviolet imaged channel isoelectric focusing mass spectrometry (icIEF-UV/MS). Isoelectric point and deconvoluted intact mass shifts observed with TRA-MMAE allowed for the determination of the drug-to-antibody ratio (DAR) and detection of trace levels of enzymatic and conjugation intermediates. Parallel analysis by reversed-phase high-performance liquid chromatography (RP-HPLC) peptide mapping with electron-activated dissociation (EAD) fragmentation was also performed. Peptide level results corroborated the putative intact identifications, localized posttranslational modifications (PTMs) on the underivatized TRA structure, and validated site-specific conjugation of the MMAE payload to the Asn-300 attached glycan structure of the ADC. Combined icIEF-UV/MS and RP-HPLC peptide map with EAD fragmentation effectively confirmed the high yield of TRA-MMAE DAR 2 produced by the evaluated chemoenzymatic conjugation reaction. Overall, these results establish a streamlined production and analytical workflow capable of providing well-characterized, highly homogeneous ADC structures for downstream preclinical screening and eventual scale-up.