Oligomerization and glycosylation modulate therapeutic glycoprotein stability and efficacy. The interplay between these two critical attributes on therapeutic glycoproteins, is however often hard to define. Here, we present a native top-down mass spectrometry (MS) approach to assess the glycosylation status of therapeutic cytokine and hormone assemblies and relate interfacial glycan occupancy to complex stability. We found that interfacial O-glycan stabilizes tumor necrosis factor-α trimer. On the contrary, interferon-β1a dimerization is independent of glycosylation. Moreover, we discovered a unique distribution of N-glycans on the follicle-stimulating hormone α subunit. We found that the interfacial N-glycan, at Asn52 of the α subunit, interacts extensively with the β subunit to regulate the dimer assembly. Overall, we have exemplified a method to link glycosylation with assembly status, for cytokines and hormones, critical for informing optimal stability and bioavailability.
Native top‐down mass spectrometry (MS) reveals subunit composition and glycan heterogeneity of therapeutic glycoprotein assemblies. By comparing the measured oligomer proteoforms with theoretical models, reconstituted from the dissociated subunits, we show that the interfacial glycan influences complex stability.
Keywords: Biopharmaceuticals; Glycan; Glycoprotein; Native Mass Spectrometry; Oligomerization.
© 2022 The Authors. Angewandte Chemie published by Wiley-VCH GmbH.