Site-specific structural changes in long-term-stressed monoclonal antibody revealed with DEPC covalent-labeling and quantitative mass spectrometry
Studies of structural changes in mAbs under forced stress and storage conditions are essential for the recognition of degradation hotspots, which can be further remodeled to improve the stability of the respective protein. Herein, we used diethyl pyrocarbonate (DEPC)-based covalent labeling mass spe...
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| Hauptverfasser: | , , , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
5 October 2023
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| In: |
Pharmaceuticals
Year: 2023, Jahrgang: 16, Heft: 10, Pages: 1-20 |
| ISSN: | 1424-8247 |
| DOI: | 10.3390/ph16101418 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.3390/ph16101418 Verlag, lizenzpflichtig, Volltext: https://www.mdpi.com/1424-8247/16/10/1418 
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| Verfasserangaben: | Manasi Gaikwad, Florian Richter, Rabea Götz, Aline Dörrbaum, Lena Schumacher, Jason Tonillo, Christian Frech, Roland Kellner and Carsten Hopf |
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| 520 | |a Studies of structural changes in mAbs under forced stress and storage conditions are essential for the recognition of degradation hotspots, which can be further remodeled to improve the stability of the respective protein. Herein, we used diethyl pyrocarbonate (DEPC)-based covalent labeling mass spectrometry (CL-MS) to assess structural changes in a model mAb (SILuMAb). Structural changes in the heat-stressed mAb samples were confirmed at specific amino acid positions from the DEPC label mass seen in the fragment ion mass spectrum. The degree of structural change was also quantified by increased or decreased DEPC labeling at specific sites; an increase or decrease indicated an unfolded or aggregated state of the mAb, respectively. Strikingly, for heat-stressed SILuMAb samples, an aggregation-prone area was identified in the CDR region. In the case of longterm stress, the structural consequences for SILuMAb samples stored for up to two years at 2-8 °C were studied with SEC-UV and DEPC-based CL-MS. While SEC-UV analysis only indicated fragmentation of SILuMAb, DEPC-based CL-MS analysis further pinpointed the finding to structural disturbances of disulfide bonds at specific cysteines. This emphasized the utility of DEPC CL-MS for studying disulfide rearrangement. Taken together, our data suggests that DEPC CL-MS can complement more technically challenging methods in the evaluation of the structural stability of mAbs. | ||
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