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 [1].

One of the most powerful techniques for glycan investigation is the combination of liquid chromatography (LC) with mass spectrometry (MS), however even this method cannot distinguish all the various forms of isomerism. The development of a new robust method is needed to determine the glycan isomer content and guarantee mAb quality.

Our group has recently demonstrated that cryogenic infrared (IR) spectroscopy provides unique vibrational spectra of glycans [2]. Since spectroscopic fingerprints can be extremely sensitive to the slightest differences between molecules, we can distinguish all the various types of isomerism present in glycans.

In this work, we apply ultrahigh-resolution ion mobility separation (IMS) combined with cryogenic IR-spectroscopy to distinguish isomeric glycans with different terminal galactose positions, using G1F N-linked glycan as an example. We performed a selective chemoenzymatic synthesis of the G1(α1-6)F isomer, which is employed as a standard for assigning the mobility-separated positional isomers of G1F based on their unique IR fingerprint spectra. The arrival-time distribution (ATD) of G1F exhibits four peaks, two of which we assign to each positional isomer based on their cryogenic IR spectrum. These doublets typically occur from the two reducing-end anomers, which we have previously shown can be separated by ultrahigh-resolution IMS. Using these results, we then investigated the impact of the host cell line (CHO and HEK-293) on the G1F glycan profile at the isomer level. We find that IgG produced in the CHO cell line exhibits a slightly higher content of G1(α1-3)F [3].

Our results demonstrate that the combination of ultrahigh-resolution IMS with cryogenic vibrational spectroscopy represents a rapid and reliable analytical method to distinguish positional isomers of glycans capable of monitoring subtle differences in galactosylation of N-glycans cleaved from mAbs.

[1] Michihiko Aoyam, Noritaka Hashii, Wataru Tsukimura, et. al., MAbs, 2019, 11, 826-836.
[2] Ahmed Ben Faleh, Stephan Warnke and Thomas R. Rizzo, Anal Chem, 2019, 91, 4876-4882.
[3] Irina Dyukova, Ahmed Ben Faleh, Stephan Warnke, et. al., Analyst (submitted).