Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and the Effects of Glycosylation on the Structure of Herceptin®
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Figure 4: Deuterium uptake difference plots for heavy chain (HC) intact vs. endoS2 treated Herceptin®; endoS2 - intact. Each bar represents a different peptide (HC sequence coverage = 70.2 %, 55 peptides). Blue bars represent the deuterium uptake differences for Herceptin® lot C; orange bars represent deuterium uptake differences for Herceptin® lot A.
Figure S2: Mass spectra for Herceptin® (a) fully glycosylated (intact), (b) following endoS2 treatment (truncated glycans) and (c) following PNGase F treatment (fully deglycosylated).
Figure S3: Mass spectra for the NIST mAb standard (a) fully glycosylated (intact), (b) following endoS2 treatment (truncated glycans) and (c) following PNGase F treatment (fully deglycosylated).
Figure S4: Mass spectra for the IgG1 Fc-hinge fragment (a) fully glycosylated (intact), (b) following endoS2 treatment (truncated glycans) and (c) following PNGase F treatment (fully deglycosylated).
Figure S7: DTCCSDHe plots for fully glycosylated (intact), partially deglycosylated (endo S2 treated) and fully deglycosylated (PNGase F treated) IgG1 mAbs (charge state range 22+ to 25+). Data fitted with Gaussians (cumulative fit plotted) and normalised against (IMS area fraction) x (MS peak height fraction). Width of shading around each peak denotes the standard deviation calculated, n=3.
Figure S9-12: Collision energy activated IM-MS heat map of the IgG1 NIST mAb and mAb lot A-C with (a) glycans, (b) truncated (endoS2 treated) glycans and (c) no glycans. Mass selected 24+ charge state with collision voltage range 4-200 V, n=3.
All interactive figures were generated using ORIGAMIANALYSE available here.
Hybrid Mass Spectrometry Methods Reveal Lot-to-Lot Differences and the Effects of Glycosylation on the Structure of Herceptin®
Rosie Upton1, Lukasz G. Migas1, Kamila J. Pacholarz1, Richard G. Beniston2, vSian Estdale2, David Firth2 and Perdita E.Barran*1
1 Manchester Institute of Biotechnology, Michael Barber Centre for Collaborative Mass Spectrometry, University of Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom
2 Covance Laboratories Ltd., Otley Road, Harrogate, HG3 1PY, United Kingdom
- E-mail: perdita.barran@manchester.ac.uk
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 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 supports 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.