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. 2019 Oct;11(7):1191-1196.
doi: 10.1080/19420862.2019.1636602. Epub 2019 Aug 2.

Glycoform-resolved FcɣRIIIa affinity chromatography-mass spectrometry

Steffen Lippold  1 , Simone Nicolardi  1 , Elena Domínguez-Vega  1 , Anna-Katharina Heidenreich  2 , Gestur Vidarsson  3 , Dietmar Reusch  2 , Markus Haberger  2 , Manfred Wuhrer  1 , David Falck  1
Affiliations

Glycoform-resolved FcɣRIIIa affinity chromatography-mass spectrometry

Steffen Lippold et al. MAbs. 2019 Oct.

Abstract

Determination of the impact of individual antibody glycoforms on FcɣRIIIa affinity, and consequently antibody-dependent cell-mediated cytotoxicity (ADCC) previously required high purity glycoengineering. We hyphenated FcɣRIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies. The approach enabled reproduction and refinement of known glycosylation effects, and insights on afucosylation pairing as well as on low-abundant, unstudied glycoforms. Our method greatly improves the understanding of individual glycoform structure-function relationships. Thus, it is highly relevant for assessing Fc-glycosylation critical quality attributes related to ADCC.

Keywords: FT-ICR-MS; FcɣRIIIa affinity chromatography; IgG Fc glycosylation; Monoclonal antibody (mAb); immunoglobulin glycans; mass spectrometry; structure-function relationships.

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Figures

Figure 1.
Figure 1.
FcɣRIIIa affinity chromatography for a therapeutic mAb. (a) UV chromatogram using reported non-MS compatible conditions. (b) AC-MS under MS-compatible conditions represented by extracted ion chromatograms of detected glycoforms. (c and d) Deconvoluted mass spectra and charge state distribution (inserts) of (c) 2x fucosylated (18– 34 min) and (d) remaining glycoforms (34– 42 min). In case of multiple possibilities (asterisk), the most probable glycoform is presented, based on reference data (Supplemental Table 7); all possible glycoforms and their structures are listed in Supplemental Table 1 and 4.
Figure 2.
Figure 2.
Extracted ion chromatograms of AC-MS with glycoengineered mAbs containing a high level of bisected and afucosylated glycoforms (a) and high level of bisected and fucosylated glycoforms (b). Increased affinity is observed for afucosylation compared to 1x (a)fucosylation. Bisected fucosylated glycoforms showed also increased affinity compared to non-bisected fucosylated glycoforms. Positive effect of galactosylation is represented for each glycosylation feature.
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