2. 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

3. 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 2 mAbs – monoclonal antibodies

4. Therapeutic Monoclonal Antibodies Are Glycosylated 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 Adapted from Oxford Glycobiology Institute Glycans Fab Fc Monoclonal Antibody Therapeutic Protein 3

5. Protein denaturation Glycoprotein N-glycan release Decantate Precipitate Ethanol precipitation Solid phase extraction Liquid chromatography Mass spectrometry N-GLYCANS ARE RELEASED FROM PROTEINS De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717.

6. N-GLYCANS ARE ANALYZED BY MASS SPECTROMETRY 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 ESI-LTQ Velos/Orbitrap MS 5

7. * * * * * * * * 10% ACN (+) 20% ACN (+) 40% ACN (-) Relative Intensity Hexose (Hex) N-acetylhexosamine (HexNAc)  Fucose (Fuc)  Sialic Acid (NeuAc) De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717. N-Glycan Profiling of Serum Glycoproteins

8. STARTER GLYCAN MS/MS LIBRARY NGA4 2 G0 NGA2F 1 G0F NGA3B 1,2 G0B NA2G1F 1 G1F A1F 1 G2FS NA2F 1 G2F A2F 1 G2FS2 NGA2B 2 G0B NGA2 1 G0 NGA4B 2 G0B 1 Purchased; 2 Kindly provided by the Consortium for Functional Glycomics through Dr. Jim Paulson

9. Glycan Consensus Spectrum Y4,Y4Y4,Y4 B4B4 [M+Na] + Y3,Y3Y3,Y3 Y 4 /Y 4  B 3, B 4 /Y 4 , B 4 /Y 4 0,2 A 5 0,2 A 4 0,2 A 4 /Y 4 0,2 A 4 /Y 4  B 4 /Y 3  B 4 /Y 3 B 3 /Y 4 , B 3 /Y 4 B 3 /Y 3 , B 3 /Y 3 -H 2 O 2,4 A 5 [M+2Na] 2+ -(2H 2 O+CH 2 O) B4B4 Y4Y4 Y4Y4 Y3Y3 Y3Y3 B3B3 Mannose N-acetylglucosamine [M+2Na] 2+ HCD, 55eV

10. Glycopeptide Consensus Spectrum b2 y2y4b5 b6 [M+2H] 2+ -(2H 2 O+CH 2 O) (TKPREEQYNSTYR)= p y1 0,2 A 1 oxonium p-NH 3 -H 2 O p-NH 3 -NH 3 2,4 A 1 m/z 963.417, z=3 HCD, 62eV p p p p p p p p p

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