With this method, a single injection 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. 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 ADC’s primary structure in a single experiment.
An analytical methodology for the characterization of the primary structure of biotherapeutic proteins using sheathless CE–ESI-MS-MS instrumentation is presented. For the first time, complete sequence coverage can be achieved using a bottom-up proteomic approach from a single analysis of a tryptic digest. In a biosimilarity assessment, a single amino acid substitution was detected.