Nlinked Glycan Characterization and Profiling Combining the Power

N-linked Glycan Characterization and Profiling: Combining the Power of a Novel Labeling Reagent and a Streamlined Analytical Workflow Ying Qing Yu Ph. D Waters Corporation Glycobiology World Congress 2015 © 2015 Waters Corporation 1

Glycosylation is a Key Critical Quality Attribute The International Conference on Harmonization Guideline Q 6 B requires the analysis of carbohydrate content, structural profiles, and characterization of the glycosylation site(s) within the polypeptide chain(s). Trastuzum. Ab, 1 N-linked site 150 KDa © 2015 Waters Corporation figures are from Wikipedia Erythropoietin 3 N-linked sites 1 O-linked site 34 KDa Entanercept 3 N-linked sites 13 O-linked sites 51 Kda 2

Glycoprotein Characterization Multiple Strategies – Complementary Information © 2015 Waters Corporation 3

Carbohydrate Complexity © 2015 Waters Corporation 4

Classes of N-Linked Glycans High Mannose © 2015 Waters Corporation Complex Hybrid 5

Influence on Biopharmaceutical Production Asn Loss of sialylation decreases EPO half-life from 2 h to 10 min Fukuda et al (1989). Blood; 73(1): 84 -89 Desialylation of intravenous immunoglobulin abrogates its anti-inflammatory properties Kaneko et al (2006). Science; 313(5787): 670 -673 Asn Presence of gal- (1, 3)-gal can induce anaphylaxis (shock) and can be present on biotherapeutics Chung et al (2006). N Engl J Med; 358(11): 1109 -1117 Asn © 2015 Waters Corporation Half of all people contain antibodies against (1, 2)xylose and (1, 3)-core fucose Bardor et al (1995). Glycobiology; 13(6): 427 -434 6

Released Glycan Analysis HILIC Profiling Conventional 2 -AB 5 Hours to 2 Days FLR

Conventional Workflow Conventional Glycoprotein Deglycosylation 16 hrs (Pre-Labeling Clean-Up) (Drying) 1 -3 hrs Released N-Glycans + Protein Labeling 3 hrs Labeled Reaction N-Glycans Byproducts + Reagents Clean-up 0. 1 hrs (Drying) Labeled Glycans 2 hrs >24 HOURS © 2015 Waters Corporation Analysis LC-FLR-MS 8

What is new? Novel Rapi. Fluor-MSTM (RFMS) Reagent © 2015 Waters Corporation Patent Pending

Rapi. Fluor-MS Reagent Rapid Reaction Kinetics NHS Carbamate Rapid Tagging Group Tertiary Amine MS-Active Charge Tag Glycan ΔMass Quinoline Fluorophore 312 Da Reaction Time = Seconds Glycan Procedure Time: Highly stable urea linkage © 2015 Waters Corporation 10

Simplified Sample Preparation Conventional Glyco. Works Rapi. Fluor-MS N-Glycan Kit Glycoprotein Deglycosylation 16 hrs (Pre-Labeling Clean-Up) (Drying) 1 -3 hrs <15 min Released N-Glycans + Protein Labeling 3 hrs Labeled Reaction N-Glycans Byproducts + Reagents 5 min Clean-up 0. 1 hrs (Drying) 2 hrs >24 HOURS © 2015 Waters Corporation 10 min Labeled Glycans Analysis LC-FLR-MS 30 min Patent Pending 11

RFMS vs. 2 AB for MS sensitivity comparison Greater than 100 x MS response

Glycan Characterization with RFMS Labeling Comparable FLR and MS response across a broad range of glycans Ig. G FLR Simple bi-antennery structures Fetuin Large, complex structures BPI MS FA 2 A 3 G 3 S 3 A 3 S 1 G 3 S 3 FA 2 G 2 S 1 Xevo G 2 -XS QTof © 2015 Waters Corporation 13

Detailed MS Profiling of a Wide Dynamic Range of Glycans labeled with RFMS Fetuin FLR Zoom In 10. 00 ~20 x 14. 00 16. 00 18. 00 20. 00 22. 00 24. 00 26. 00 28. 00 30. 00 32. 00 34. 00 30. 0016. 00 18. 00 20. 00 22. 00 24. 00 36. 00 38. 00 40. 00 35. 0026. 00 28. 00 30. 00 32. 00 34. 00 36. 00 40. 00 38. 00 Time 40. 00 BPI MS 10. 00 12. 00 15. 00 © 2015 Waters Corporation 20. 00 25. 00 0 10. 00 12. 00 14

RFMS Labeling Enables MS Detection of Very Low Abundance Glycans Human Ig. G FLR

RFMS extends MS enhancement throughout the glycan fragmentation pattern © 2015 Waters Corporation 16

RFMS enables easy assignment of two isobaric glycans RFMS labeled N-glycans from a murine Ig. G 1 m. Ab FLR MS BPI ? • The two isobaric glycans, FA 2 G 2 and a minor shoulder peak, partially resolved by HILIC • The minor peak represents only 0. 7% of total FLR signal © 2015 Waters Corporation 17

RFMS enables easy assignment of a minor shoulder peak of FA 2 G 2 as FA 2 G 1 Ga 1 RFMS labeled N-glycans from a murine Ig. G 1 m. Ab FLR MS BPI FA 2 G 1 Ga 1 0. Loss of Glc. NAc 0 4 • The two isobaric glycans, FA 2 G 2 and a minor shoulder peak, partially resolved by HILIC • The minor peak represents only 0. 7% of total FLR signal • Structurally diagnostic ions: 1) predominant 528 m/z ion and 2) prominent Glc. NAc loss • High sensitivity and information rich fragmentation data support the identification of the isobaric, lower abundance species as an α-Gal containing FA 2 G 1 Ga 1. © 2015 Waters Corporation 18

UNIFI Glycan Application Workflows HILIC FLR GU +Accurate Mass HILIC FLR DDA MS/MS Automated Data Acquisition Glycan FLR+MS Confirmation Glycan DDA Processing Calibrated RT and Accurate Mass Deconvolute Fragment Ion Review and Reporting Scientific Library Sim. Glycan © 2015 Waters Corporation 19

The Utility of GU Values What is a GU Value? • GU stands for Glucose Unit • A GU value is a normalized glycan structure retention time observed in HILIC for glycan peaks, obtained using a dextran ladder calibration Why is the GU approach useful? • GU Values assist in normalizing glycan retention time across days, instruments and laboratories, so data can be compared and shared easily. • GU Values facilitate more routine glycan assignments by enabling the creation of a single glycan GU retention library. © 2015 Waters Corporation 20

Generating Waters RFMS GU Glycan Libraries FLR label mass Waters Glycan GU Library: Experimental GU value • Experimentally derived GU Retention (>10 injections/protein) • Data from proteins representing spectrum of glycan diversity • All entries confirmed with exoglycosidase digestion © 2015 Waters Corporation 21

HILIC FLR GU + Accurate Mass Workflow Method Robustness and Transferability, Confident Assignments Retention Time Calibration Curve GU Labeled Dextran Ladder Assign GU values to N-glycans BPI MS for accurate mass confirmation © 2015 Waters Corporation 22

Glycan GU Scientific Library Search for Confident Glycan Assignments Both 2 -AB and RFMS labeled glycan performance test standards are now available to support this workflow © 2015 Waters Corporation 23

UNIFI Glycan DDA Workflow © 2015 Waters Corporation 24

UNIFI Acquire and Process Glycan DDA Data RFMS labeled N-glycans from mouse Ig. G MSMS fragmentation of a highlighted minor glycoform was displayed FLR Raw MSMS Spectrum ~ 0. 1% total FLR BPI MS Deconvolute & deeisotope Export as. LCS (or mzml) format © 2015 Waters Corporation 25

Automated Glycan Identification using. LCS Files with Sim. Glycan The minor glycoform was correctly

Work in progress ¡Developing a scientific library for Rapi. Fluor-MS labeled glycans for automated glycan assignment ¡Applying Rapi. Fluor-MS label for more complex glycosylated proteins © 2015 Waters Corporation 27

Acknowledgements Waters Matt Lauber Ph. D New England Biolabs Paula Magnelli Ph. D ¡ Professor Pauline Rudd (COI) – Mark Hilliard (UNIFI Glycan Scientific Library) – Giogio Carta (UNIFI Glycan Scientific Library) © 2015 Waters Corporation 28