Antibody–Drug Conjugate Stability Probed by Variable-Temperature Electrospray Ionization Mass Spectrometry

Antibody-drug conjugates (ADCs) are effective anticancer biotherapeutics, often referred to as "magic bullets" due to their high specificity and cytotoxicity. This unique drug class consists of cytotoxic drugs coupled to monoclonal antibodies that target antigens on cancer cell surfaces. Different modes of drug conjugation are used to produce ADCs, whereby it has been shown that the employed linkage chemistries influence the drug load distribution as well as the stability of the product. While different methods to assess ADC stability are available, they mostly assess bulk properties and thus fail to assess stabilities at an individual stoichiometric drug-load level. Here, we demonstrate that variable-temperature electrospray ionization mass spectrometry can be used to study the heat stability of antibody-drug conjugates, resolving distinct stabilities for individual drug-loaded variants. As this stability is a key attribute of ADCs, we propose that variable-temperature electrospray ionization mass spectrometry may become an asset in the toolbox of analytical chemistry approaches to characterize ADCs in molecular fine detail.