Antibody–drug conjugates are among the fastest growing classes of anti-cancer

With the 5 approved and commercially available agents, and nearly 100 other ADC candidates, researchers continue tweaking approaches in the development these complex agents in an attempt to bring next-generation ADCs to market.

Middle-down mass spectrometry (MD MS) has emerged as a promising alternative to classical bottom-up approaches for protein characterization. MD MS combines the benefits of both bottom-up and top-down approaches by exploiting minimal protease digestion of protein samples and the ability to detect multiple post-translational modifications on a single polypeptide chain.[1]


And while middle-level experiments after enzymatic digestion are routinely used for subunit analysis of monoclonal antibody (mAb)-related compounds. Using such an approach provides scientist with 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. But peptide mapping strategies can be challenging when dealing with more complex and heterogeneous mAb formats such as antibody-drug conjugates.

Different fragmentation techniques
Published in the Journal of The American Society for Mass Spectrometry, scientist from Laboratoire de Spectrométrie de Masse BioOrganique at the Université de Strasbourg, in collaboration with scientists from Thermo Fisher Scientific, Catalent Biologics West, and Centre d’Immunologie Pierre-Fabre (CIPF), for the first time report about MD MS analysis of a third-generation site-specific DAR4 ADCs using different fragmentation techniques, including higher-energy collisional- (HCD), electron-transfer (ETD) dissociation and 213 nm ultraviolet photodissociation (UVPD).

Ultraviolet photodissociation 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, the scientists noted that their approach highlighted the complementarity of ETD and UVPD for both primary sequence validation and drug conjugation/glycosylation site assessment.

Based on these observations, the scientists believe that 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 next-generation mAb-based formats, especially for PTMs and drug conjugation sites validation.

[1] Hernandez-Alba O, Houel S, Hessmann S, Erb S, Rabuka D, Huguet R, Josephs J, Beck A, Drake PM, Cianférani S. A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate Using Middle-Down Mass Spectrometry. J Am Soc Mass Spectrom. 2019 Aug 19. doi: 10.1007/s13361-019-02296-2. [Pubmed][Article]