| Autor: |
Upton R; Manchester Institute of Biotechnology , Michael Barber Centre for Collaborative Mass Spectrometry , University of Manchester , 131 Princess Street , Manchester , M1 7DN , UK . Email: Perdita.barran@manchester.ac.uk., Migas LG; Manchester Institute of Biotechnology , Michael Barber Centre for Collaborative Mass Spectrometry , University of Manchester , 131 Princess Street , Manchester , M1 7DN , UK . Email: Perdita.barran@manchester.ac.uk., Pacholarz KJ; Manchester Institute of Biotechnology , Michael Barber Centre for Collaborative Mass Spectrometry , University of Manchester , 131 Princess Street , Manchester , M1 7DN , UK . Email: Perdita.barran@manchester.ac.uk., Beniston RG; Covance Laboratories Ltd. , Otley Road , Harrogate , HG3 1PY , UK., Estdale S; Covance Laboratories Ltd. , Otley Road , Harrogate , HG3 1PY , UK., Firth D; Covance Laboratories Ltd. , Otley Road , Harrogate , HG3 1PY , UK., Barran PE; Manchester Institute of Biotechnology , Michael Barber Centre for Collaborative Mass Spectrometry , University of Manchester , 131 Princess Street , Manchester , M1 7DN , UK . Email: Perdita.barran@manchester.ac.uk. |
| Abstrakt: |
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. |
