Comprehensive characterization of higher order structure changes in methionine oxidized monoclonal antibodies via NMR chemometric analysis and biophysical approaches

ABSTRACT

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.

KEYWORDS:

• Biophysical characterization
• CD
• DSC
• fluorescence spectroscopy
• higher order structure
• LC-MS
• methionine oxidation
• monoclonal antibody
• NMR chemometric analysis

Acknowledgments

The authors acknowledge the support from Beth McCoy, Mingzhang Wang, Leon Shi, Samuel Perakis, and Margaret Ruesch of Pfizer, Inc. We also thank the Summer Growth Experience Program at Pfizer for the support of Hannah Duncan.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available on request from the corresponding author, M. Li.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2292688.

Additional information

Funding

This work was funded by Pfizer, Inc.