Retention of glycopeptides analyzed using hydrophilic interaction chromatography is influenced by charge and carbon chain length of ion‐pairing reagent for mobile phase

Characterization of the glycansof glycoproteins is essential for the development and production of biologics. Numerous methods are available for analyzing the glycansof glycoproteins directly and labeled glycans. Nevertheless, glycopeptidesare difficult to resolve because of their exceptional complexity and the microheterogeneity of glycans. These properties represent technical challenges to efforts to insure the accurate characterization of biopharmaceuticalsto comply with regulatory requirements. Therefore, we investigated the retention behavior of peptides and glycopeptidesin hydrophilic interaction chromatography-mode HPLC in the presence of ion-pairing reagents. Anionic ion-pairing reagentsdecreased the retention times of glycopeptidesand improved resolution in the presence of higher concentrations or hydrophobicities of ion-pairing reagent. Anionic ion-pairing reagentsincreased retention times of larger glycansbecause of their increased hydrophilicity. In contrast, in the presence of cationic ion-pairing reagents, the retention times of glycopeptideswith greater numbers of sialic acid residues decreased. It is appropriate to add an anionic ion-pairing reagentto the mobile phase for good separation of glycopeptides. The collision cross-sectional area values of glycopeptidesdetermined using electrospray ionization- ion mobility spectrometry-mass spectrometrycorrelated with retention times. These findings support the implementation of hydrophilic interaction chromatography-mode HPLC to improve the characterization of glycosylated biopharmaceuticals.