Czech Society for Mass Spectrometry

(13^th Czech Mass Spectrometry Conference and 11^th Informal Proteomic Meeting - WeO-03)
Simultaneous mapping of peptides and released N-glycans by HILIC-FLD-MS for quality control of protein biopharmaceuticals

Mykyta R. Starovoit ¹, Juraj Lenčo ^1 *

1. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University

Abstract

Protein biopharmaceuticals, especially monoclonal antibodies, have revolutionized therapeutic regimes. However, their structural complexity presents analytical challenges. Modifications such as glycosylation and deamidation affect efficacy and immunogenicity, demanding precise monitoring of critical quality attributes (CQAs). Current multi-attribute methods, based on reversed-phase (RP) LC-MS of tryptic peptides, struggle with hydrophilic peptides and show limited resolution of deamidated, isoAsp-containing, and glycosylated peptides. Glycosylation assessment often requires separate workflows with enzymatic release, fluorescent labeling, and HILIC-FLD analysis.
We propose a UHPLC method that combines the mapping of tryptic peptides with released, RapiFluor-MS-labeled N-glycans in a single HILIC-FLD-MS run, utilizing convenient sequential injection of peptide and glycan fractions. The approach separates peptides in the initial part of the chromatogram, followed by glycans in later retention windows, with fluorescence labeling enhancing glycan detection. Coupled with tandem mass spectrometry, this approach provides full protein sequence coverage and superior resolution of isomeric glycoforms, deamidated peptides, and isoAsp residues versus RPLC workflows. Analysis of released, labeled glycans further outperforms glycopeptide separation, both by RPLC and HILIC, in preserving terminal sialic acids that are prone to in-column cleavage at acidic pH and high column temperatures.
This integrated HILIC workflow could simplify biopharmaceutical quality control by enabling the simultaneous monitoring of multiple CQAs, which now require distinct methods, thereby improving efficiency, reliability, and the cost per sample.

* Corresponding author: lenco@faf.cuni.cz

Acknowledgement:

This study was supported by the project of the Charles University Grant Agency (GA UK, No. 356325), the SVV Project No. 260782, and the ATEBIO project (Advanced Techniques for Biomedical Diagnostics, Reg. No. CZ.02.01.01/00/23_020/0008535) co-funded by the European Union.