Chemically Defined Non-human Glycans Comprising Galactose-α1-3-Galactose (α-Gal) Epitopes Glycoengineered into the Fragment Antigen-Binding (Fab) Domain of Cetuximab Differentially Affect Human Anti-α-Gal Immunoglobulin E (IgE) Binding.
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.