The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.
NISTmAb; chemometrics; comparability; higher order structure; monoclonal antibody (mAb) therapeutics; nuclear magnetic resonance spectroscopy (NMR).
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Antibodies, Monoclonal / chemistry*
Biopharmaceutics / standards*
Laboratories / standards*
Magnetic Resonance Spectroscopy / methods*
Reproducibility of Results
NIST acknowledges the support by NIST Biomanufacturing Program and NIST and W.M. Keck Foundation for support of biomolecular NMR instrumentation. The work at Stockholm University was supported by a grant from the Knut and Alice Wallenberg Foundation. The Brazilian Center for Research in Energy and Materials acknowledge the support by the Brazilian Ministry of Science, Technology, Innovation and Communication (MCTIC). A portion of the research was performed using NMR instrumentation at EMSL, a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. This work at Health Canada was supported by Government of Canada. The work at NRC Canada was supported by the NRC Biologics and Biomanufacturing Program and by the Department of Downstream Processing and Analytics (for Protein NMR instrumentation), for which this paper is assigned the NRCC Publication No. NRC_HHT_53378. The work at the University of Queensland was supported by the Australian Research Council and The National Health and Medical Research Council as well as the University of Queensland. BNSP (Biomolecular NMR Spectroscopy Platform) ETH Zürich, is acknowledged for providing equipment and infrastructure. The NMR facility used for this work is supported by the Office of the Vice President for Research at the Medical University of South Carolina. The NMR maintenance was supported in part by the Nanotechnology Platform Program (Molecule and Material Synthesis) and by the grants (JP25102001, JP25102008, JP15K21708 and JP15H02491 to K.K.; JP17H05893 and 18K14892 to S.Y.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.