Glycosylation of monoclonal antibodies (mAbs) critically affects their effector function, stability, pharmacokinetics, and immunogenicity. Glycan structures can vary with cell type and culture conditions and are often considered key critical quality attributes that must be tightly controlled. While most mAbs are glycosylated only in the Fc-domain, cetuximab contains non-human glycans (galactose-α1-3-galactose, α-Gal) in its Fab-region, which can trigger hypersensitivity in patients with α-Gal-specific IgE. Previous studies linked bivalent α-Gal glycans to IgE binding, but the roles of other α-Gal glycans were unclear. Using glycoengineering, we herein incorporate α-Gal glycans found in commercial products and test their binding to patient-derived anti-α-Gal IgE. Unexpectedly, certain monovalent α-Gal glycans bound IgE as effectively as bivalent forms, and molecular modeling suggests that this may be attributed to interactions with the protein backbone. These findings provide important guidance for manufacturers and regulators in the development and evaluation of mAbs, biosimilars, and emerging glycoprotein therapeutics.
Hatfield G, Tepliakova L, Tran J, Lu H, Gilbert M, et al.
mAbs •
Monoclonal antibody (mAb) production using non-human cells can introduce non-human glycan epitopes including terminal galactosyl-α1-3-galactose (α1-3-Gal) moieties. Cetuximab is a commercial mAb associated with causing anaphylaxis in some patients due to the binding of endogenous anti-α1-3-Gal IgE to the Fab (containing bi-α1-3-galactosylated glycans) but not to the Fc region (containing mono-α1-3-galactosylated glycans). Despite being low in abundance in typical commercial mAbs, the inherent sensitivity of cell culture conditions on glycosylation profiles, and the development of novel glycoengineering strategies, novel antibody-based modalities, and biosimilars by various manufacturers with varying procedures, necessitates a better understanding of the structural requirements for anti-α1-3-Gal IgE binding to the Fc region. Herein, we synthesized mAb glycoforms with varying degrees and regioisomers of α1-3-galactosylation and tested their binding to two commercial anti-α1-3-Gal human IgE antibodies derived from a human patient with allergies to red meat (comprising α1-3-Gal epitopes), as well as to the FcγRIIIA receptor. Our results demonstrate that unexpectedly, anti-α1-3-Gal human IgE antibodies can bind to Fc glycans, with bi-α1-3-galactosylation being the most important factor, highlighting that their presence in the Fc region may be considered as a potential critical quality attribute, particularly when using novel platforms in mAb-based biotherapeutics.
