Identification of Mobility-Resolved N-Glycan Isomers.

Glycan analysis has evolved considerably during the last decade. The advent of high-resolution ion-mobility spectrometry has enabled the separation of isomers with only the slightest of structural differences. However, the ability to separate such species raises the problem of identifying all the mobility-resolved peaks that are observed, especially when analytical standards are not available. In this work, we report an approach based on the combination of IMSn with cryogenic vibrational spectroscopy to identify N-glycan reducing-end anomers. By identifying the reducing-end α and ß anomers of diacetyl-chitobiose, which is a disaccharide that forms part of the common core of all N-glycans, we are able to assign mobility peaks to reducing anomers of a selection of N-glycans of different sizes, starting from trisaccharides such as Man-1 up to glycans containing nine monosaccharide units, such as G2. By building an infrared fingerprint database of the identified N-glycans, our approach allows unambiguous identification of mobility peaks corresponding to reducing-end anomers and distinguishes them from positional isomers that might be present in a complex mixture.

A new approach for identifying positional isomers of glycans cleaved from monoclonal antibodies.

Glycosylation patterns in monoclonal antibodies (mAbs) can vary significantly between different host cell types, and these differences may affect mAbs safety, efficacy, and immunogenicity. Recent studies have demonstrated that glycan isomers with the terminal galactose position on either the Man α1-3 arm or the Man α1-6 arm have an impact on the effector functions and dynamic structure of mAbs. The development of a robust method to distinguish positional isomers of glycans is thus critical to guarantee mAb quality. In this work, we apply high-resolution ion mobility combined with cryogenic infrared spectroscopy to distinguish isomeric glycans with different terminal galactose positions, using G1F as an example. Selective enzymatic synthesis of the G1(α1-6)F isomer allows us to assign the peaks in the arrival-time distributions and the infrared spectra to their respective isomeric forms. Moreover, we demonstrate the impact of the host cell line (CHO and HEK-293) on the IgG G1F gycan profile at the isomer level. This work illustrates the potential of our approach for glycan analysis of mAbs.

Combining Cryogenic Infrared Spectroscopy with Selective Enzymatic Cleavage for Determining Glycan Primary Structure.

Given the biological relevance and intrinsic structural complexity of glycans, increasing efforts are being directed toward developing a general glycan database that includes information from different analytical methods. As recently demonstrated, cryogenic infrared (IR) spectroscopy is a promising technique for glycan analysis, as it provides unique vibrational fingerprints of specific glycan isomer ions. One of the main goals of a glycan database is the identification and detailed characterization of unknown species. In this work, we combine enzymatic digestion with cryogenic IR-spectroscopy and demonstrate how it can be used for glycan identification. We measured the IR-spectra of a series of cationic glycan standards of increasing complexity and compared them with spectra of the same species after enzymatic cleavage of larger glycans. We show that the cryogenic IR spectra of the cleaved glycans are highly structured and virtually identical to those of standards after both single and multiple cleavages. Our results suggest that the combination of these methods represents a potentially powerful and specific approach for the characterization of unknown glycans.