Characterization of Therapeutic Monoclonal Antibodies at the Subunit-Level using Middle-Down 193 nm Ultraviolet Photodissociation

Monoclonal antibodies (mAbs) are a rapidly advancing class of therapeutic glycoproteins that possess wide clinical utility owing to their biocompatibility, high antigen specificity, and targeted immune stimulation. These therapeutic properties depend greatly on the composition of the immunoglobulin G (IgG) structure, both in terms of primary sequence and post-translational modifications (PTMs); however, large-scale production in cell culture often results in heterogeneous mixtures that can profoundly affect clinical safety and efficacy. This places a high demand on analytical methods that afford comprehensive structural characterization of mAbs to ensure their stringent quality control. Here we report the use of targeted middle-down 193 nm ultraviolet photodissociation (UVPD) to provide detailed primary sequence analysis and PTM site localization of therapeutic monoclonal antibody subunits (∼25 kDa) generated upon digestion with recombinant immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) followed by chemical reduction. Under optimal conditions, targeted UVPD resulted in approximately 60% overall coverage of the IgG sequence, in addition to unambiguous glycosylation site localization and extensive coverage of the antigen-binding complementarity determining regions (CDRs) in a single LC-MS/MS experiment. Combining UVPD and ETD data afforded even deeper sequencing and greater overall characterization of IgG subunits. Overall, this targeted UVPD approach represents a promising new strategy for the comprehensive characterization of antibody-based therapeutics.