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Material Measurement Laboratory

Mass Spectral Database of Glycans and Glycopeptides in Therapeutic Drugs. Maria Lorna A. De Leoz, Xinjian (Eric) Yan, Xiaoyu (Sara) Yang, Lisa Kilpatrick, Yuxue Liang, Michael Tarlov, Stephen Stein. Material Measurement Laboratory.

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Material Measurement Laboratory

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  1. Mass Spectral Database of Glycans and Glycopeptides in Therapeutic Drugs • Maria Lorna A. De Leoz, Xinjian (Eric) Yan, Xiaoyu (Sara) Yang, • Lisa Kilpatrick, Yuxue Liang, Michael Tarlov, Stephen Stein Material Measurement Laboratory

  2. Therapeutic Monoclonal Antibodies are Widely Used in Medicine • Used as treatment for cancer and several other diseases • $20B market/year • >30 approved mAbs • 6/10 best-selling drugs, 2012 mAbs – monoclonal antibodies

  3. Therapeutic Monoclonal Antibodies Are Glycosylated • Monoclonal Antibody Therapeutic Protein Fab Glycans Fc Glycans Adapted from Oxford Glycobiology Institute • Glycans are attached to the protein molecules • Changes in glycosylation may affect drug safety and efficacy • Glycosylation highly heterogeneous • Glycan analysis remains a formidable challenge

  4. N-GLYCANS ARE RELEASED FROM PROTEINS N-glycan release Protein denaturation Glycoprotein Decantate Ethanol precipitation Solid phase extraction Liquid chromatography Mass spectrometry Precipitate De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics2011; 10, M110.002717.

  5. N-GLYCANS ARE ANALYZED BY MASS SPECTROMETRY ESI-LTQ Velos/Orbitrap MS • Can analyze using ion trap or orbitrap MS • Sample injected through LC or direct infusion • Several MS/MS activation methods • Higher-energy C-trap Dissociation (HCD) • Collisionally-Induced Dissociation (CID by ion trap or orbitrap) • Electron Transfer Dissociation (ETD) • High mass resolution • Max at 125,000 at m/z 400 (i.e. it can distinguish peaks at m/z 400.0000 and 400.0032) • High mass accuracy • < 5 ppm error routinely

  6. N-Glycan Profiling of Serum Glycoproteins * 10% ACN (+) * * Relative Intensity * * 20% ACN (+) * * * Hexose (Hex)N-acetylhexosamine (HexNAc) Fucose (Fuc)Sialic Acid (NeuAc) 40% ACN (-) De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics2011; 10, M110.002717.

  7. STARTERGLYCAN MS/MSLIBRARY 1Purchased; 2Kindly provided by the Consortium for Functional Glycomics through Dr. Jim Paulson

  8. Glycan Consensus Spectrum [M+Na]+ Y4 Y4 Y4/Y4 B3, B4/Y4, B4/Y4 Y3 Y3 B3 B4 B4 B3/Y4, B3/Y4 Y4, Y4 [M+2Na]2+ B3/Y3, B3/Y3 B4/Y3 B4/Y3 HCD, 55eV Y3, Y3 -H2O 0,2A4/Y4 0,2A4/Y4 0,2A4 0,2A5 -(2H2O+CH2O) [M+2Na]2+ 2,4A5 Mannose N-acetylglucosamine

  9. Glycopeptide Consensus Spectrum oxonium -(2H2O+CH2O) (TKPREEQYNSTYR)=p p oxonium 0,2A1 2,4A1 m/z 963.417, z=3 HCD, 62eV p y1 p p [M+2H]2+ -H2O p-NH3 p oxonium p p p b2 -H2O -H2O -H2O p-NH3 p -NH3 b6 y2 y4 b5

  10. Glycan from Rituximab Matches Library Rituximab Head to Tail Comparison Glycan Library

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