Hybrid mass spectrometry methods reveal lot-to-lot differences and delineate the effects of glycosylation on the tertiary structure of Herceptin®† †Electronic supplementary information (ESI) available: Supporting results (Fig. S1–S23 and Tables S1–S3) as mentioned in the text. See DOI: 10.1039/c8sc05029e
| Autor: | Upton, Rosie, Migas, Lukasz G., Pacholarz, Kamila J, Beniston, Richard G., Peters, Shirley, Davis, Rachel, Estdale, Sian, Firth, David, Barran, Perdita |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Glycan
Glycosylation biology 010405 organic chemistry Ion-mobility spectrometry Allosteric regulation General Chemistry 010402 general chemistry Mass spectrometry 01 natural sciences Protein tertiary structure 0104 chemical sciences chemistry.chemical_compound Chemistry Biopharmaceutical chemistry Biochemistry biology.protein Hydrogen–deuterium exchange skin and connective tissue diseases |
| Zdroj: | Chemical Science Upton, R, Migas, L G, Pacholarz, K J, Beniston, R G, Peters, S, Davis, R, Estdale, S, Firth, D & Barran, P 2019, ' Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and Delineate the Effects of Glycosylation on the Tertiary Structure of Herceptin ', Chemical Science . https://doi.org/10.1039/C8SC05029E |
| ISSN: | 2041-6539 2041-6520 |
| DOI: | 10.1039/C8SC05029E |
| Popis: | To quantify the measurable structural heterogeneity of a biopharmaceutical product and the effect of glycosylation on this we systematically evaluate three lots of Herceptin®, two mAb standards and an intact 5 Fc-hinge fragment. To quantify the measurable variations in the structure of a biopharmaceutical product we systematically evaluate three lots of Herceptin®, two mAb standards and an intact Fc-hinge fragment. Each mAb is examined in three states; glycan intact, truncated (following endoS2 treatment) and fully deglycosylated. Despite equivalence at the intact protein level, each lot of Herceptin® gives a distinctive signature in three different mass spectrometry approaches. Ion mobility mass spectrometry (IM-MS) shows that in the API, the attached N-glycans reduce the conformational spread of each mAb by 10.5–25%. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) data support this, with lower global deuterium uptake in solution when comparing intact to the fully deglycosylated protein. HDX-MS and activated IM-MS map the influence of glycans on the mAb and reveal allosteric effects which extend far beyond the Fc domains into the Fab region. Taken together, these findings and the supplied interactive data sets establish acceptance criteria with application for MS based characterisation of biosimilars and novel therapeutic mAbs. |
| Databáze: | OpenAIRE |
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