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Antibody-drug conjugates (ADCs) are an emerging category of biotherapeutic products based on monoclonal antibodies (mAbs) coupled to powerful cytotoxic drugs. The production of ADCs entails the formation of species with different number of conjugates drugs. The heterogeneity of ADC species add to the complexity originating from the mAb microvariability. Sheathless capillary electrophoresis- mass spectrometry (sheathless CE-MS) using complementary approaches was used to perform a detailed characterization of brentuximab vedotin. A sheathless CE-MS instrument used as a nanoelectrospray ionization (nanoESI) infusion platform was involved to perform the intact and middle-up analysis in native mass spectrometry (MS) conditions. The nanoESI infusion approaches enabled estimation of the average drug-to-antibody ratio (DAR) and the drug load distribution. A single injection with a sheathless capillary zone electrophoresis (CZE)-MS/MS method was sufficient to characterize the amino acid sequence with complete sequence coverage. In addition glycosylation and drug-loaded peptides could be identified from MS/MS spectra, which revealed robust information about their localizations and abundances. A drug-loaded peptide fragmentation mass spectra study yielded drug-specific fragments, which reinforced the confidence about the identifications. The results reveal the ability of the sheathless CZE-MS/MS method to characterize an ADCs primary structure in a single experiment. [ABSTRACT FROM AUTHOR]

An innovative coupling of capillary electrophoresis and tandem mass spectrometry (CE-MS-MS) provides excellent results for the characterization of the primary structure of biologies. Using a single 200-fmol injection, it is possible to completely characterize the amino acid sequence and obtain information about the location, structure, and relative abundance of 15 glycoforms. Finally, various post-translational modifications can be characterized and their relative abundances estimated, including asparagine deamidation and aspartic acid racemization. And in a biosimilarity assessment between two monoclonal antibodies, it was possible to distinguish minor differences such as a sole amino acid substitution. This level of characterization is permitted by cumulating the specificities of CE and high-resolution tandem MS using a sheathless interface. [ABSTRACT FROM AUTHOR]