Characterization of Glycosylated Proteins at Subunit Level by HILIC/MS

Valentina D'Atri; Davy Guillarme

doi:10.1007/978-1-0716-1241-5_6

Characterization of Glycosylated Proteins at Subunit Level by HILIC/MS

Methods Mol Biol. 2021:2271:85-95. doi: 10.1007/978-1-0716-1241-5_6.

Authors

Valentina D'Atri^{1

2}, Davy Guillarme^{3

4}

Affiliations

¹ School of Pharmaceutical Sciences, University of Geneva, CMU, Geneva, Switzerland.
² Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU, Geneva, Switzerland.
³ School of Pharmaceutical Sciences, University of Geneva, CMU, Geneva, Switzerland. davy.guillarme@unige.ch.
⁴ Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU, Geneva, Switzerland. davy.guillarme@unige.ch.

PMID: 33908001
DOI: 10.1007/978-1-0716-1241-5_6

Abstract

Hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (MS) is considered as the reference analytical technique for glycans profiling, especially for the characterization of glycosylated protein therapeutics such as monoclonal antibodies (mAbs) and mAbs-related products. Although HILIC/MS is mainly known to profile enzymatically released and fluorescently labeled N-glycans, the recent commercialization of new widepore HILIC amide bonded stationary phases packed with sub-2 μm particles has allowed for remarkable separations also at the subunit level. Here, we describe a simple protocol to perform the mAb glycans profiling at subunit level by HILIC/MS.

Keywords: Biopharmaceutical proteins; Hydrophilic interaction chromatography; Mass spectrometry; Monoclonal antibodies; N-glycosylation; Protocol.

MeSH terms

Biological Products / analysis*
Chromatography, Liquid*
Glycosylation
Hydrophobic and Hydrophilic Interactions
Protein Processing, Post-Translational*
Research Design
Rituximab / analysis*
Spectrometry, Mass, Electrospray Ionization*
Trastuzumab / analysis*
Workflow

Substances

Biological Products
Rituximab
Trastuzumab