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Characterization of Therapeutic Monoclonal Antibodies at the Subunit-Level using Middle-Down 193 nm Ultraviolet Photodissociation
journal contribution
posted on 2016-03-05, 00:00 authored by Victoria
C. Cotham, Jennifer S. BrodbeltMonoclonal
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.