Quantitative Structural
Characterization of Local
N‑Glycan Microheterogeneity in Therapeutic Antibodies by Energy-Resolved
Oxonium Ion Monitoring
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Abstract
Site-specific characterization of glycoform heterogeneity
currently
requires glycan structure assignment and glycopeptide quantification
in two independent experiments. We present here a new method combining
multiple reaction monitoring mass spectrometry with energy-resolved
structural analysis, which we termed “energy-resolved oxonium
ion monitoring”. We demonstrated that monitoring the yields
of oligosaccharide-derived fragment ions (oxonium ions) over a wide
range of collision induced dissociation (CID) energy applied to a
glycopeptide precursor exhibits a glycan structure-unique fragmentation
pattern. In the analysis of purified immunoglobulin glycopeptides,
the energy-resolved oxonium ion profile was shown to clearly distinguish
between isomeric glycopeptides. Moreover, limit of detection (LOD)
of glycopeptide detection was 30 attomole injection, and quantitative
dynamic range spanned 4 orders magnitude. Therefore, both quantification
of glycopeptides and assignment of their glycan structures were achieved
by a simple analysis procedure. We assessed the utility of this method
for characterizing site-specific N-glycan microheterogeneity on therapeutic
antibodies, including validation of lot-to-lot glycoform variability.
A significant change in the degree of terminal galactosylation was
observed in different production lots of trastuzumab and bevacizumab.
Cetuximab Fab glycosylation, previously known to cause anaphylaxis,
was also analyzed, and several causative antigens including Lewis
X motifs were quantitatively detected. The data suggests that energy-resolved
oxonium ion monitoring could fulfill the regulatory requirement on
the routine quality control analysis of forthcoming biosimilar therapeutics