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Database of Glycans and Glycopeptides in Therapeutic Monoclonal Antibodies

The Mass Spectral Database of Glycans and Glycopeptides provides critical insights into the glycosylation patterns of therapeutic monoclonal antibodies (mAbs). With a global market exceeding $20 billion, and over 30 approved mAbs, their efficacy and safety heavily rely on glycan structures. This resource facilitates in-depth glycan analysis through advanced mass spectrometry techniques, addressing the challenges posed by heterogeneous glycosylation. The database includes profiling of N-glycans from serum glycoproteins, aiding drug development and ensuring therapeutic effectiveness.

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Database of Glycans and Glycopeptides in Therapeutic Monoclonal Antibodies

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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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