In the realm of biopharmaceuticals, the surge in utilizing monoclonal antibodies (mAbs) has underscored the necessity for novel analytical techniques that can accurately scrutinize the physicochemical attributes of these intricate drugs. One crucial quality attribute (QA) for biopharmaceuticals is the higher order structure (HOS), pivotal for ensuring consistency in manufacturing, detecting variations from process alterations, and establishing comparability between biologic products. To tackle the challenge of measuring HOS, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods have emerged, enabling precise atomic-level comparisons of HOS between proteins, including mAbs.
A recent multi-laboratory comparison involving 26 industrial, governmental, and academic labs worldwide has benchmarked the application of 2D-NMR using the NISTmAb as a standard, facilitating the translation of this method into routine use for biopharmaceutical development. This benchmarking exercise involved acquiring two-dimensional 1H,15N and 1H,13C NMR spectra on the unlabeled Fab domain and a uniformly enriched system suitability sample derived from the NISTmAb. Chemometric analyses of over 400 spectral maps showcased the method’s reliability in assessing HOS, marking a significant step towards establishing 2D-NMR as a standardized, routine technique for precise HOS evaluations of mAb-based therapeutics.
The revenue from prescription drugs derived from biotechnology products, especially mAbs, has soared in recent years, emphasizing the importance of robust analytical techniques. Traditionally, methods like circular dichroism or Fourier transform infrared spectroscopy have been employed for HOS evaluation, but the advent of 2D-NMR has sparked immense interest due to its ability to provide comprehensive structural information at the atomic level. By generating spectral fingerprints that capture unique structural features, 2D-NMR offers a powerful tool for assessing the structural similarity between different protein samples, aiding in biosimilarity evaluations and quality control assessments.
The precision and reliability of 2D-NMR in HOS assessment were validated through an extensive inter-laboratory study, demonstrating high spectral reproducibility across different NMR spectrometers and experimental conditions. Key metrics like Combined Chemical Shift Deviation (CCSD) highlighted the method’s precision, with average deviations in the parts-per-billion range for both 1H,15N and 1H,13C spectra. Principal component analysis (PCA) further elucidated the spectral reproducibility and clustering patterns, showcasing the method’s robustness in distinguishing HOS characteristics across samples.
The ability of 2D-NMR to provide atomic-level insights into protein structures, particularly in the mAb therapeutic class, underscores its significance in ensuring structural comparability between different drug lots, formulations, or biosimilar products. The method’s high sensitivity to even subtle structural perturbations makes it a valuable tool for assessing the impact of manufacturing changes on protein therapeutics. By establishing a standardized approach for HOS evaluation using the NISTmAb as a reference material, this study paves the way for widespread adoption of 2D-NMR in biopharmaceutical development and regulatory processes.
The meticulous production of a system suitability sample, the U-15N, 20%-13C NIST-Fab, and the subsequent inter-laboratory comparisons underscore the method’s practicality and reliability, even on moderate field NMR spectrometers. The study’s outcomes not only validate the feasibility of employing 2D-NMR for HOS assessment but also position it as a pivotal tool for ensuring structural integrity and comparability of mAb-based biotherapeutics. The robustness and precision of the method, coupled with chemometric analyses, offer a pathway for leveraging 2D-NMR as a routine practice in characterizing the HOS of diverse protein therapeutics, thereby enhancing confidence in their quality and consistency across manufacturing processes.
Tags: yeast, monoclonal antibodies, analytical chemistry, downstream, quality control, regulatory, filtration, sports
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