NMR Reveals Antibody Structure Without Isotope Labeling
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- Researchers from ExCELLS established site-specific assignments of methyl signals in the Fc region of human IgG1 antibodies, combining amino acid-selective isotope labeling, mutagenesis, and multidimensional NMR experiments in a Journal of the American Chemical Society study.
- The method assigned methyl resonances from six residue types — alanine, isoleucine, leucine, methionine, threonine, and valine — creating residue-specific structural probes in the Fc domain.
- The same methyl signals were detectable in antibodies at natural isotopic abundance, meaning isotope labeling is not required for the structural analysis — the key practical advantage of the approach.
- The probes detected structural differences tied to glycosylation patterns in the Fc region, including core fucosylation and terminal galactosylation, glycan features known to influence antibody effector functions.
- Methyl signals originating from the hinge region and receptor-binding interface acted as sensitive reporters of local structural flexibility in functionally important regions of the molecule.
- A companion paper in Analytical Chemistry used integrated NMR and LC-MS to characterize methionine oxidation at conserved Fc residues Met252 and Met428, revealing distinct R- and S-sulfoxide forms that perturb local antibody structure.
- Koichi Kato of ExCELLS said the approach offers "a practical route to residue-level structural evaluation of antibody therapeutics," with direct applications in biosimilar comparability and quality monitoring.
Why it matters: Therapeutic antibody manufacturers gain a label-free analytical tool for residue-level structural monitoring applicable to biosimilar comparability and quality control. The companion methionine-oxidation work addresses degradation at Met252 and Met428 with stereochemical resolution, giving developers a mechanistic handle on stability that global techniques like circular dichroism cannot provide.
