Unambiguous Identification of Glucose-Induced Glycation in mAbs and other Proteins by NMR Spectroscopy

Jennifer Moises, Christof Regl, Arthur Hinterholzer, Christian G. Huber, Mario Schubert*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Objective
Glycation is a non-enzymatic and spontaneous post-translational modification (PTM) generated by the reaction between reducing sugars and primary amine groups within proteins. Because glycation can alter the properties of proteins, it is a critical quality attribute of therapeutic monoclonal antibodies (mAbs) and should therefore be carefully monitored. The most abundant product of glycation is formed by glucose and lysine side chains resulting in fructoselysine after Amadori rearrangement. In proteomics, which routinely uses a combination of chromatography and mass spectrometry to analyze PTMs, there is no straight-forward way to distinguish between glycation products of a reducing monosaccharide and an additional hexose within a glycan, since both lead to a mass difference of 162 Da.
Methods
To verify that the observed mass change is indeed a glycation product, we developed an approach based on 2D NMR spectroscopy spectroscopy and full-length protein samples denatured using high concentrations of deuterated urea.
Results
The dominating β-pyranose form of the Amadori product shows a characteristic chemical shift correlation pattern in 1H-13C HSQC spectra suited to identify glucose-induced glycation. The same pattern was observed in spectra of a variety of artificially glycated proteins, including two mAbs, as well as natural proteins.
Conclusion
Based on this unique correlation pattern, 2D NMR spectroscopy can be used to unambiguously identify glucose-induced glycation in any protein of interest. We provide a robust method that is orthogonal to MS-based methods and can also be used for cross-validation.
Original languageEnglish
JournalPharmaceutical Research
Early online date13 Dec 2022
DOIs
Publication statusPublished - Jun 2023

Keywords

  • Amadori product
  • Glycation
  • NMR spectroscopy
  • monoclonal antibodies

Fields of Science and Technology Classification 2012

  • 304 Medical Biotechnology
  • 104 Chemistry

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