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Mapping Monoclonal Antibody Structure by 2D 13C NMR at Natural Abundance

Published

Author(s)

Luke Arbogast, Robert G. Brinson, John Marino

Abstract

Monoclonal antibodies (mAbs) represent an important and rapidly growing class of biotherapeutics. Correct folding of a mAb is critical for drug efficacy, while misfolding can impact safety by eliciting unwanted immune or other off-target responses. Robust methods are therefore needed for the precise measurement of mAb structure for drug quality assessment and comparability. To date, the perception in the field has been that NMR could not be applied practically to mAbs due to the size (∼150 kDa) and complexity of these molecules, as well as the insensitivity of the method. The feasibility of applying NMR methods to stable isotope- labeled, protease-cleaved, mAb domains (Fab and Fc) has been demonstrated from both E. coli and Chinese hamster ovaries (CHO) cell expression platforms; however, isotopic labeling is not typically available when analyzing drug products. Here, we address the issue of feasibility of NMR-based mapping of mAb structure by demonstrating for the first time the application of a 2D 13C NMR methyl fingerprint method for structural mapping of an intact mAb at natural isotopic abundance. Further, we show that 2D 13C NMR spectra of protease-cleaved Fc and Fab fragments can provide accurate reporters on the domain structures that can be mapped directly to the intact mAb. Through combined use of rapid acquisition and nonuniform sampling techniques, we show that these Fab and Fc fingerprint spectra can be rapidly acquired in as short as approximately 30 min.
Citation
Analytical Chemistry
Volume
87
Issue
7

Keywords

monoclonal antibodies, NMR Fingerprinting, HSQC, Non-uniform sampling

Citation

Arbogast, L. , Brinson, R. and Marino, J. (2015), Mapping Monoclonal Antibody Structure by 2D 13C NMR at Natural Abundance, Analytical Chemistry, [online], https://doi.org/10.1021/ac504804m (Accessed April 16, 2024)
Created February 28, 2015, Updated October 12, 2021