Antibody-based pharmaceuticals are the leading biologic drug platform (> $75B/year).[1] Despite a wealth of information collected on them, there is still a lack of knowledge on their inter-domain structural distributions, which impedes innovation and development. To address this measurement gap, we have developed a new methodology to derive biomolecular structure ensembles from distance distribution measurements via a library of tagged proteins bound to an unlabeled and otherwise unmodified target biologic. We have employed the NIST monoclonal antibody (NISTmAb) reference material as our development platform for use with spin-labeled affinity protein (SLAP) reagents. Using double electron-electron resonance (DEER) spectroscopy, we have determined inter-spin distance distributions in SLAP complexes of both the isolated Fc domain and the intact NISTmAb. Our SLAP reagents offer a general and extendable technology, compatible with any non-isotopically labeled immunoglobulin G class mAb. Integrating molecular simulations with the DEER and solution X-ray scattering measurements, we enable simultaneous determination of structural distributions and dynamics of mAb-based biologics.
Keywords: Double electron-electron resonance spectroscopy; biopharmaceutical development; molecular dynamics; monoclonal antibody biologics; solution X-ray scattering.
© 2025 Chemistry Europe and Wiley-VCH GmbH. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.