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.