Title
Inhibition of glycosylation on a camelid antibody uniquely affects its FcγRI binding activity
Author
Natalie Krahn, Maureen Spearman, Markus Meier, July Dorion-Thibaudeau, Matthew McDougall, Trushar R. Patel, Gregory De Crescenzo, Yves Durocher, Jörg Stetefeld, Michael Butler
Year
2016
Journal
European Journal of Pharmaceutical Sciences
Abstract
Glycoengineering of mAbs has become common practice in attempts to generate the ideal mAb candidate for a wide range of therapeutic applications. The effects of these glycan modifications on the binding affinity of IgG mAbs for FcγRIIIa and their cytotoxicity is well known. However, little is understood about the effect that these modifications have on binding to the high affinity FcγRI receptor. This study analysed the effect of variable N-glycosylation on a human-llama hybrid mAb (EG2-hFc, 80 kDa) binding to FcγRI including a comparison to a full-sized IgG1 (DP-12, 150 kDa). This was achieved by the addition of three glycosylation inhibitors (swainsonine, castanospermine, and kifunensine) independently to Chinese hamster ovary (CHO) cell cultures to generate hybrid and high mannose glycan structures. Biophysical analysis by circular dichroism, dynamic light scattering and analytical ultra-centrifugation confirmed that the solution-behaviour of the mAbs remained constant over multiple concentrations and glycan treatments. However, changes were observed when studying the interaction of FcγRI with variously glycosylated mAbs. Both mAbs were observed to have a decreased binding affinity upon treatment with swainsonine which produced hybrid glycans. Following de-glycosylation the binding affinity for EG2-hFc was only marginally reduced (6-fold) compared to a drastic (118-fold) decrease for DP-12. In summary, our data suggest that the relatively low molecular weight of chimeric EG2-hFc may contribute to its enhanced stability against glycan changes making it a highly suitable mAb candidate for therapeutic applications.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Antibodies, Biochemistry