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Anal Chem. 2012 Nov 20;84(22):9655-62. doi: 10.1021/ac3023372. Epub 2012 Oct 2.

Quantitative structural characterization of local N-glycan microheterogeneity in therapeutic antibodies by energy-resolved oxonium ion monitoring.

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1
Life Science Research Center, Shimadzu Corporation, Kanda-Nishikicho 1, Chiyoda-ku, Tokyo, Japan.

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.

PMID:
23004563
DOI:
10.1021/ac3023372
[Indexed for MEDLINE]

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