Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches
The higher order structure (HOS) of monoclonal antibodies (mAbs) is an important quality attribute with strong contribution to clinically relevant biological functions and drug safety. Due to the multi-faceted nature of HOS, the synergy of multiple complementary analytical approaches can substantial...
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Taylor & Francis Group
2024-12-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2023.2292688 |
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| author | Mingyue Li Victor A. Beaumont Shahajahan Akbar Hannah Duncan Arch Creasy Wenge Wang Kelly Sackett Lisa Marzilli Jason C. Rouse Hai-Young Kim |
| author_facet | Mingyue Li Victor A. Beaumont Shahajahan Akbar Hannah Duncan Arch Creasy Wenge Wang Kelly Sackett Lisa Marzilli Jason C. Rouse Hai-Young Kim |
| author_sort | Mingyue Li |
| collection | DOAJ |
| description | The higher order structure (HOS) of monoclonal antibodies (mAbs) is an important quality attribute with strong contribution to clinically relevant biological functions and drug safety. Due to the multi-faceted nature of HOS, the synergy of multiple complementary analytical approaches can substantially improve the understanding, accuracy, and resolution of HOS characterization. In this study, we applied one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) spectroscopy coupled with chemometric analysis, as well as circular dichroism (CD), differential scanning calorimetry (DSC), and fluorescence spectroscopy as orthogonal methods, to characterize the impact of methionine (Met) oxidation on the HOS of an IgG1 mAb. We used a forced degradation method involving concentration-dependent oxidation by peracetic acid, in which Met oxidation is site-specifically quantified by liquid chromatography-mass spectrometry. Conventional biophysical techniques report nuanced results, in which CD detects no change to the secondary structure and little change in the tertiary structure. Yet, DSC measurements show the destabilization of Fab and Fc domains due to Met oxidation. More importantly, our study demonstrates that 1D and 2D NMR and chemometric analysis can provide semi-quantitative analysis of chemical modifications and resolve localized conformational changes with high sensitivity. Furthermore, we leveraged a novel 15N-Met labeling technique of the antibody to directly observe structural perturbations at the oxidation sites. The NMR methods described here to probe HOS changes are highly reliable and practical in biopharmaceutical characterization. |
| format | Article |
| id | doaj-art-c60148f3693e491c9f2b5b111def14a8 |
| institution | Kabale University |
| issn | 1942-0862 1942-0870 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
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| series | mAbs |
| spelling | doaj-art-c60148f3693e491c9f2b5b111def14a82025-01-31T04:19:38ZengTaylor & Francis GroupmAbs1942-08621942-08702024-12-0116110.1080/19420862.2023.2292688Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approachesMingyue Li0Victor A. Beaumont1Shahajahan Akbar2Hannah Duncan3Arch Creasy4Wenge Wang5Kelly Sackett6Lisa Marzilli7Jason C. Rouse8Hai-Young Kim9Pfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Analytical Research and Development, Andover, MA, USAPfizer, Inc. Pharmaceutical Sciences Small Molecules, Analytical Research and Development, Sandwich, United KingdomPfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Analytical Research and Development, Andover, MA, USAPfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Analytical Research and Development, Andover, MA, USAPfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Bioprocess Research and Development, Andover, MA, USAPfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Bioprocess Research and Development, Andover, MA, USAPfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Analytical Research and Development, Andover, MA, USAPfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Analytical Research and Development, Andover, MA, USAPfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Analytical Research and Development, Andover, MA, USAPfizer, Inc. BioTherapeutics Pharmaceutical Sciences, Analytical Research and Development, Andover, MA, USAThe higher order structure (HOS) of monoclonal antibodies (mAbs) is an important quality attribute with strong contribution to clinically relevant biological functions and drug safety. Due to the multi-faceted nature of HOS, the synergy of multiple complementary analytical approaches can substantially improve the understanding, accuracy, and resolution of HOS characterization. In this study, we applied one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) spectroscopy coupled with chemometric analysis, as well as circular dichroism (CD), differential scanning calorimetry (DSC), and fluorescence spectroscopy as orthogonal methods, to characterize the impact of methionine (Met) oxidation on the HOS of an IgG1 mAb. We used a forced degradation method involving concentration-dependent oxidation by peracetic acid, in which Met oxidation is site-specifically quantified by liquid chromatography-mass spectrometry. Conventional biophysical techniques report nuanced results, in which CD detects no change to the secondary structure and little change in the tertiary structure. Yet, DSC measurements show the destabilization of Fab and Fc domains due to Met oxidation. More importantly, our study demonstrates that 1D and 2D NMR and chemometric analysis can provide semi-quantitative analysis of chemical modifications and resolve localized conformational changes with high sensitivity. Furthermore, we leveraged a novel 15N-Met labeling technique of the antibody to directly observe structural perturbations at the oxidation sites. The NMR methods described here to probe HOS changes are highly reliable and practical in biopharmaceutical characterization.https://www.tandfonline.com/doi/10.1080/19420862.2023.2292688Biophysical characterizationCDDSCfluorescence spectroscopyhigher order structureLC-MS |
| spellingShingle | Mingyue Li Victor A. Beaumont Shahajahan Akbar Hannah Duncan Arch Creasy Wenge Wang Kelly Sackett Lisa Marzilli Jason C. Rouse Hai-Young Kim Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches mAbs Biophysical characterization CD DSC fluorescence spectroscopy higher order structure LC-MS |
| title | Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches |
| title_full | Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches |
| title_fullStr | Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches |
| title_full_unstemmed | Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches |
| title_short | Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches |
| title_sort | comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via nmr chemometric analysis and biophysical approaches |
| topic | Biophysical characterization CD DSC fluorescence spectroscopy higher order structure LC-MS |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2023.2292688 |
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