2D NMR Analysis as a Sensitive Tool for Evaluating the Higher-Order Structural Integrity of Monoclonal Antibody against COVID-19

Abstract

The higher-order structure (HOS) of protein therapeutics has been confirmed as a critical quality parameter. In this study, we compared 2D 1H-13C ALSOFAST-HMQC NMR spectra with immunochemical ELISA-based analysis to evaluate their sensitivity in assessing the HOS of a potent human monoclonal antibody (mAb) for the treatment of coronavirus disease 2019 (COVID-19). The study confirmed that the methyl region of the 2D 1H-13C NMR spectrum is sensitive to changes in the secondary and tertiary structure of the mAb, more than ELISA immunoassay. Because of its highly detailed level of characterization (i.e., many 1H-13C cross-peaks are used for statistical comparability), the NMR technique also provided a more informative outcome for the product characterization of biopharmaceuticals. This NMR approach represents a powerful tool in assessing the overall higher-order structural integrity of mAb as an alternative to conventional immunoassays.

Profiling Enzyme Activity of l-Asparaginase II by NMR-Based Methyl Fingerprinting at Natural Abundance

“…The processed file was read into CARA software, where peak picking and peak intensities were determined for subsequent analysis. [ 1 H- 13 C] ALSOFAST-HMQC spectra were recorded at 800 MHz (with RT probe) with 1.024 scans and 226 × 2048 complex points, corresponding to spectral widths of 29.8 × 19.5 ppm with acquisition times of 18 and 65 ms in the t 1 ( 13 C) and t 2 ( 1 H) domains, respectively. − For this purpose, Topspin 4.1.4 version was used which has an in-built pulse program “afhmqcgpphsf” in the Bruker library. The 1 H and 13 C carriers were placed on water resonance and at 20 ppm.…”

Integration of NMR Spectroscopy in an Analytical Workflow to Evaluate the Effects of Oxidative Stress on Abituzumab: Beyond the Fingerprint of mAbs

“…The two approaches can be considered complementary as the first one takes into consideration chemical shift perturbations, while the second one, variations in signal amplitudes. Further, the average CCSD obtained by comparing the 2D 1 H− 13 C ALSOFAST HMQC spectra before and after oxidative stress is 4.72 ppb, a value based on the current literature 21,29,30 is apparently not indicative of significant differences (this algorithm measures the amount the peaks are shifted between two spectra, and considers 0 as a theoretical value corresponding to no shifts, i.e., identical samples). This result suggests that, globally, the fold of the protein is maintained after the stress condition since most of the cross-peaks have the same chemical shift in spectra acquired before and after the stress test (see also Figure S3).…”

Expression of 2H, 13C, 15N-labeled NIST-Fab fragment in the methylotrophic yeast Komagataella phaffii for nuclear magnetic resonance studies

“…All spectra were normalized to a spectrum from a 200 µM unlabeled yNIST-Fab sample with 100% protonation. 2 H isotope enrichment was estimated by subtracting the peak area of labeled sample from corresponding peak from unlabeled yNIST-Fab with a correction for concentration difference.…”

“…The Fc region, disulfide linked CH2 and CH3 domains with N-linked glycosylation at asparagine residue 297, interacts with antibody receptors on the cell surface. * Corresponding author: kchao@umd.edu Fingerprinting methods using nuclear magnetic resonance (NMR) spectroscopy at natural isotopic abundance is gaining increased acceptance for assessment of the critical quality attributes (CQA) of higher order structure (HOS) for protein-based therapeutics [2][3][4][5][6]. Spectral changes, in the form of shifted cross peaks or the appearance and disappearance of signals, reflect HOS perturbations, can arise from misfolding, aggregation, degradation, and/or chemical modifications of proteins.…”