Advancing O-Glycosylation Analysis of Fc-Fusion Proteins at the Domain Level by HILIC-MS with Supercharging

Fragment crystallizable (Fc)-fusion proteins are formed by the fusion of functional peptides or receptor extracellular domains (ECDs) with Fc. They are normally very heterogeneous with multiple N- and O-glycosylation sites and a high level of sialylation. Characterization of O-glycosylation in Fc-fusion proteins is important for understanding their biological significance and for monitoring process consistency during drug development. However, due to the heterogeneity, close localization, and especially low ionization efficiency of O-glycans, it is challenging to achieve the overall O-glycosylation profile at the protein level. In this study, a supercharging reagent, m-nitrobenzyl alcohol (m-NBA), was applied to mobile phases for hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) analysis of two Fc-fusion proteins at the domain level. The addition of m-NBA mitigated MS signal suppression caused by TFA in mobile phases and enabled the characterization of the overall O-glycosylation profiles of ECDs. While de-N-glycosylation of Fc resulted in only a slight increase in MS signal intensity, the average MS signal intensity of the top three ECD-derived O-glycoforms increased by approximately 11.4- to 13.0-fold and 6.9- to 10.8-fold for these Fc-fusion proteins with and without terminal sialic acid, respectively. This observation indicates that m-NBA facilitates increased MS signal intensity for glycosylated proteins, with a pronounced effect on sialylated species. In addition, the enhanced MS signal intensity of major released N-glycans achieved through supercharging enabled their identification without the need for sample separation, derivatization, and enrichment. Overall, this HILIC-MS method combined with supercharging provides a promising approach for domain-level characterization of O-glycans and simultaneous analysis of released N-glycan without derivatization.