A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate Using Middle-Down Mass Spectrometry

Abstract

Middle-down mass spectrometry (MD MS) has emerged as a promising alternative to classical bottom-up approaches for protein characterization. Middle level experiments after enzymatic digestion are routinely used for subunit analysis of monoclonal antibody (mAb)-related compounds, providing information on drug load distribution and average drug-to-antibody ratio (DAR). However, peptide mapping is still the gold standard for primary amino acid sequence assessment, post-translational modifications (PTM) and drug conjugation identification and localization. However, peptide mapping strategies can be challenging when dealing with more complex and heterogeneous mAb formats, like antibody-drug conjugates (ADCs). We report here, for the first time, MD MS analysis of a third-generation site-specific DAR4 ADC using different fragmentation techniques, including higher energy collisional-(HCD), electron-transfer (ETD) dissociation and 213 nm ultraviolet photo-dissociation (UVPD). UVPD used as a standalone technique for ADC subunit analysis afforded, within the same liquid chromatography-MS/MS run, enhanced performance in terms of primary sequence coverage compared to HCD-or ETD-based MD approaches, and generated substantially more MS/MS fragments containing either drug conjugation or glycosylation site information, leading to confident drug/glycosylation site identification. In addition, our results highlight the complementarity of ETD and UVPD for both primary sequence validation and drug conjugation/glycosylation site assessment. Altogether, our results highlight the potential of UVPD for ADC MD MS analysis for drug conjugation/glycosylation site assessment, and indicate that MD MS strategies can improve structural characterization of empowered nextgeneration mAb-based formats, especially for PTMs and drug conjugation sites validation.

Comprehensive Middle-Down Mass Spectrometry Characterization of an Antibody–Drug Conjugate by Combined Ion Activation Methods

“…The characterization of antibody subunits by tandem mass spectrometry (MS/MS), typically coupled with reversed phase liquid chromatography, has been extensively explored. ,,− Multiple MS/MS methods, including combinations of 213 and 193 nm ultraviolet photodissociation (UVPD), electron-transfer dissociation (ETD), collisionally activated dissociation (CAD), higher-energy collisional dissociation (HCD), electron capture dissociation (ECD), and hybrid ETD/HCD (EThcD), have been used to enhance characterization, with coverages of 50–80% typically reported. ,,, Fraction collection of subunits followed by MALDI-insource decay has also been utilized to obtain sequence coverages as high as 90% for each subunit. , While many MS1-based top-down and middle-level characterization methods for ADCs have been developed, ,− MS/MS analysis of ADCs is still in the early stages of development. Two recent studies have demonstrated the initial application of middle-down MS/MS strategies for the analysis of ADC subunits. , One expansive study reported the use of ETD to effectively identify payload locations on heterogeneous antibodies by focusing on specific diagnostic fragment ions, even attaining localization of conjugation sites of positional isomers as well as oxidation sites . Another investigation compared ETD, 213 nm UVPD, and HCD for analysis of a next-generation ADC .…”

“…Two recent studies have demonstrated the initial application of middle-down MS/MS strategies for the analysis of ADC subunits. , One expansive study reported the use of ETD to effectively identify payload locations on heterogeneous antibodies by focusing on specific diagnostic fragment ions, even attaining localization of conjugation sites of positional isomers as well as oxidation sites . Another investigation compared ETD, 213 nm UVPD, and HCD for analysis of a next-generation ADC . For the antibody in this aforementioned study, the payload is fixed on C213 for the Fc/2 subunit and C163 for the Fd′ subunit via modified amino acids .…”

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry

“…Also, follow-up studies could focus on more targeted topics related to TD/MD MS of mAbs, for example ADC analysis. 105,106…”

Recent advances in mass spectrometry based clinical proteomics: applications to cancer research

“…One such application is the study of engineered antibodies which have shown potential to be used as cancer therapies [68]. ETD-based MS methods have been reported in the literature to characterize monoclonal antibodies (mAb) and antibody drug conjugates (ADC) [69][70][71][72][73][74].…”