Complete and Rapid NMR Characterization of Therapeutic Monoclonal Antibodies via Differential Labeling of Fab Fragments in Escherichia coli

Characterization of therapeutic monoclonal antibodies (mAbs) structure and dynamics using NMR spectroscopy has benefited from recent advances in sample preparation methods for isotopically labeled Fab and Fc fragments. Here, we propose a simple and accessible method using E. coli to produce the heavy and light chains separately, thereby allowing differential labeling of either chain of the refolded fragment. This was demonstrated on the Fab fragments of adalimumab, bevacizumab, infliximab, rituximab, and trastuzumab. The new labeling scheme produced NMR spectra with almost no resonance overlap, allowing near-complete assignments (>90%) of backbone resonances in less than an hour. In addition, a near-complete assignment of side-chain methyl groups was carried out that allowed the assignments of Avastin. The total time from Petri dish to final backbone assignment was 5 weeks per chain.