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Introducing GlycoWorks RapiFluor-MS: Label N-Glycans Simply and Rapidly Without Sacrificing Sensitivity Thank you for joining us! Our Webinar will begin shortly.

1 Introducing GlycoWorks RapiFluor-MS: Label N-Glycans Simply and Rapidly Without Sacrificing Sensitivity Thank you for joining us! Our Webinar will begin shortly. While you are waiting, please feel free to browse our library of Webinar content: Click below to learn more about CORTECS, our newest Solid-Core LC Column platform: Waters Corporation 1

2 Friendly Reminders Please use text chat functionality to submit questions during the Webinar. Upon conclusion, follow up information will be available: Recorded version of today s presentation PDF Copies of today s slides Discount Offers on GlycoWorks RapiFluor MS Products Product specific information Reference materials 2015 Waters Corporation 2

Today s Presenters Matthew Lauber, Ph.D - Senior Research Chemist, Waters Corporation Matthew Lauber is a Senior Applications Chemist in the Chemistry Applied Technology group.

Most recently Matt has worked extensively on chemistry products for bioseparations, with a specific focus on the new GlycoWorks RapiFluor MS kit we will discuss today. Paula Magnelli, Ph.

3 Today s Presenters Matthew Lauber, Ph.D - Senior Research Chemist, Waters Corporation Matthew Lauber is a Senior Applications Chemist in the Chemistry Applied Technology group. He has an academic background in protein chemistry and proteomic methods for the structural characterization of biomolecular complexes. Most recently Matt has worked extensively on chemistry products for bioseparations, with a specific focus on the new GlycoWorks RapiFluor MS kit we will discuss today. Paula Magnelli, Ph.D New England Biolabs Dr. Paula Magnelli is a Scientist at NEB Glycobiology Group. Paula is responsible for developing and characterizing novel glycosidases, creating methods for glycomics and glycoproteomics research with an emphasis in therapeutic glycoproteins workflows and glycan sequencing Waters Corporation 3

4 Introducing GlycoWorks RapiFluor-MS: Label N-Glycans Simply and Rapidly Without Sacrificing Sensitivity Matthew A. Lauber, Ph.D. Paula Magnelli, Ph.D Waters Corporation 4

5 Glycosylation of Biotherapeutics Fucose GlcNAc Mannose Effector Functions (ADCC/CDC) (fucosylation/galactosylation) Galactose N-acetylneuraminic acid N-glycolylneuraminic acid Low Half Life (high mannose) Anti-Inflammatory (sialylation) Immunogenic (αgal / N-glycolylneuraminic acid) Overall profile sensitive to manufacturing conditions N-glycosylation is a quality attribute of biotherapeutics N-glycan profiles are frequently characterized in detail and routinely monitored Anal Chem 2013, 85 (2), Waters Corporation 5

6 Glycoprotein Characterization Multiple Strategies Complementary Information 2015 Waters Corporation 6

7 Released Glycan Analysis HILIC Profiling Conventional 5 Hours to 2 Days Conventional 2+ 2-AB 5.3e2 FLR m/z 2015 Waters Corporation 7

8 Released Glycan Analysis Recent Developments Procainamide Klapoetke, S.; Zhang, J.; Becht, S.; Gu, X.; Ding, X., J Pharm Biomed Anal 2010, 53 (3), Rapid Rapid Tagging for MS Gong, B.; Hoyt, E.; Lynaugh, H.; Burnina, I.; Moore, R.; Thompson, A.; Li, H., Anal Bioanal Chem 2013, 405 (17), FLR Rapid Tagging for FLR Cook, K. S.; Bullock, K.; Sullivan, T., Biologicals 2012, 40 (2), MS 2015 Waters Corporation 8

9 Part I Development of the GlycoWorks RapiFluor-MS N-Glycan Kit 2015 Waters Corporation 9

10 Glycan Labeling Techniques Rapid Tagging N-Glycan PNGase F Protein Reductive Amination (conventional) Glycosylamine labeling with rapid tagging reactive groups circumvents these issues Released N-Glycan (Glycosylamine) N-Glycan (Glycosylamine) + Mild Acid Hydrolysis Protein Relies on numerous chemical conversions Laborious Time consuming Often produces heterogenous reaction products N-Glycan ( Acyclic Free Reducing End) + N-Glycan (Free Reducing End) HOAc DMSO 2-AB SPE NaBH 3 CN Reduction N-Glycan (2-AB Labeled) 2015 Waters Corporation 10

RapiFluor-MS TM Reagent Built Upon Our Expertise From Waters expertise in rapid, fluorescence labeling of amino acids Enhanced chemical properties for glycan analysis:

11 RapiFluor-MS TM Reagent Built Upon Our Expertise From Waters expertise in rapid, fluorescence labeling of amino acids Enhanced chemical properties for glycan analysis: Rapid Tagging Efficient Fluorescence Enhanced Ionization Efficiency RapiFluor-MS AccQ Fluor Rapid Fluorescence Patent Pending Exclusive Licensing 2015 Waters Corporation 11

12 RapiFluor-MS Reagent Rapid Labeling Kinetics Mechanism for Rapid Tagging of Glycosylamines with RapiFluor-MS NHS Carbamate Rapid Tagging Group rapifluor-ms Tertiary Amine MS-Active Charge Tag + mass (glycosylamine) Glycosylamine = Da (reducing end) = Da +C 17 H 20 N 4 O 2 time = Quinoline Fluorophore Monoisotopic mass shift (from glycosylamine) secondsda H 2 O CO Waters Corporation 12

13 Revolutionizing N-Glycan Sample Prep Sensitivity Comparison Instant AB TM 2.6E+6 RapiFluor-MS Labeled N-Glycans FA2 FLR 400 FLR MS (BPI) 0.0E+0 1.7E+6 2.6E+6 0.0E+0 1.7E+6 FA2 FA2 300x zoom MS (BPI) 0.0E min Instant AB Labeled N-Glycans FA2 FLR MS (BPI) actors ple of N-Glycans ss Check Std/ 1000) Response Fa (FA2 Peak Area per Samp from 1 µg of Intact mab Mas RapiFluor-MS Labeled Instant AB Labeled 0.0E min 2015 Waters Corporation 13

14 Revolutionizing N-Glycan Sample Prep Quantitative Analysis to Compare to Convention Rapid Tagging RapiFluor-MS rapifluor-ms Different Mechanisms Reductive Amination 2-AB H O 3.3E+6 FLR 0.0E+0 4.0E+6 MS (BPI) 0.0E+0 FA2 (2.2 pmol) (Area = x 10 3 ) RapiFluor-MS Labeled Human IgG N-Glycans FA2 (Area = x 10 3 ) higg FA2 Peak Area (Fluorescence - Ex 265 nm / Em 425 nm) y = x Quantity (pmol) E+6 8E+5 6E+5 4E+5 2E+5 0E+0 y = x R² = RapiFluor-MS Label (pmol) 2015 Waters Corporation 14

15 Revolutionizing N-Glycan Sample Prep Quantitative Analysis to Compare to Convention Rapid Tagging RapiFluor-MS rapifluor-ms Different Mechanisms Reductive Amination 2-AB H O 3.0E+5 FLR 0.0E+0 3.0E+4 MS (BPI) 0.0E+0 FA2 (2.6 pmol) (Area = 19.3 x 10 3 ) 2-AB Labeled Human IgG N-Glycans FA2 (Area = 2.0 x 10 3 ) higg FA2 Peak Area (Fluorescence - Ex 330 nm / Em 420 nm) y = x Quantity (pmol) E+4 6E+4 5E+4 4E+4 3E+4 2E+4 1E+4 0E+0 y = x R² = AB Label (pmol) 2015 Waters Corporation 15

16 Revolutionizing N-Glycan Sample Prep Sensitivity Comparison 2-AB 3.3E+6 0.0E+0 4.0E+6 0.0E+0 3.3E+6 0.0E+0 4.0E+6 0.0E+0 RapiFluor-MS Labeled N-Glycans from Pooled Human IgG FA2 (2.2 pmol) FA2 FLR MS (BPI) AB Labeled N-Glycans from Pooled Human IgG FA2 (2.6 pmol) 100x zoom FLR actors d FA2 Glycan / 1000) FA2 7.4 MS (BPI) Response Fa (Peak Area per pmol of Labeled RapiFluor-MS Labeled FLR MS (BPI) AB Labeled 2015 Waters Corporation 16

17 Revolutionizing N-Glycan Sample Prep Sensitivity Comparison Fluorescence MS (BPI) RapiFluor-MS Labeled Instant AB Labeled 2-AB Labeled Procainamide Labeled Relative Per rformance (%) * * (*) Comparative result extrapolated from a published comparison of N-glycans, wherein it was found that procainamide provided comparable fluorescence and up to 50 fold greater ESI-MS sensitivity when compared to 2-AB(Klapoetke et al. 2010) Waters Corporation 17

18 Simplified Workflow Conventional 5 Hours to 2 Days Direct Analysis (Organic Solvent Dilution) 10 min 5 min 10 min GlycoWorks RapiFluor-MS N-Glycan Kit Total Sample Prep Time 30 min Patent Pending 2015 Waters Corporation 18

19 Powered by New England Biolabs :: Global leader in the production and supply of reagents for the life sciences :: Innovator of technologies from basic molecular biology through to next generation sequencing and high throughput genomics :: Provider of best-in-class products with unparalleled technical support :: Manufacturer of reagents for glycan analysis for over 20 years 1974 NEB founded 1992 Maltose Binding Protein Expression Kit Released 1994 Chitin research begins 2007 K. lactis glycoengineered strains 2011 Heparinases released 2014 Rapid PNGase F released Dwek coins term glycobiology 1992 PNGase F, Endo H & first exos released 1990 NEB glycobiology program formed 2000 α-n-acetyl- Galactosaminidase released 2005 Cellulase research begins 2008 Novel O-glycosidase released 2012 Endo S released 2014 Human GPI glycoproteome published 2015 Waters Corporation 19

Rapid Buffer GlycoWorks Rapid PNGase F Enzymatic

20 Rapid Deglycosylation RapiGest SF and Glycoworks Rapid PNGase F RapiGest SF Surfactant GlycoWorks Rapid Buffer GlycoWorks Rapid PNGase F Enzymatic Deglycosylation 2 min 80 C 5 min 50 C 2015 Waters Corporation 20

21 Revolutionizing N-Glycan Sample Prep Rapid Deglycosylation (-) Negative Control (no enzyme) (+) Positive Control (SDS/DTT + PNGase F) (R) Rapid Deglycosylation (RapiGest, Rapid PNGase F, 10 min). Positive control: denaturation with DTT-SDS (incompatible with MS applications), followed by PNGase F treatment is a well established method for complete N-glycan removal. Rapid deglycosylation: complete unbiased N-glycan removal in 10 minutes, compatible with rapid labeling and mass spectrometry analysis Waters Corporation 21

22 Revolutionizing N-Glycan Sample Prep Speed: Fast Deglycosylation (-) Negative Control (no enzyme) (R) Rapid Deglycosylation (RapiGest, Rapid PNGase F, 10 min) (RT) Rapid Deglycosylation with 4mM TCEP (+) Positive Control (SDS/DTT + PNGase F) 2015 Waters Corporation 22

23 Revolutionizing N-Glycan Sample Prep Speed: Fast Deglycosylation Glycoprotein + Rapid Buffer + Rapigest SF + Rapid PNGase F 2 min 80 C 5 min 50 C 2015 Waters Corporation 23

24 Revolutionizing N-Glycan Sample Prep Robustness: Quantitative Extraction Labeled Glycan Reaction Byproducts Labeled Deglycosylated Protein Collect Byproducts Labeled Glycan 2015 Waters Corporation 24

25 Revolutionizing N-Glycan Sample Prep Robustness: Quantitative Extraction Mixture of higg and bovine fetuin N-glycans 2.3E+6 FLR No SPE Crude Reaction Mixture A3G3S3 FA2 FA2G2S1 A3S1G3S3 RapiFluor-MS FA2 Fluorescence Peak Area 0.0E+0 6E+8 1.9E+6 After SPE (1x SPE) SPE Recovery 5E+8 ~74% 4E+8 3E+8 2E+8 Specified elution volume 0.0E E+8 0E SPE Elution Volume (µl) 2015 Waters Corporation 25

26 Revolutionizing N-Glycan Sample Prep Robustness: Quantitative Extraction 1.2E+6 1x SPE A3G3S3 30 FLR 25 1x SPE Positive Control FA2 A3S1G3S3 FA2G2S1 0.0E E+6 2x SPE Relative Ab bundance (%) x SPE SPE Processed Positive Control 5 5.7% 6.1% 0 0.0E+0 min GlycoWorks HILIC SPE of RapiFluor-MS N-Glycans is quantitative No significant deviation in the glycan profile upon SPE processing 2015 Waters Corporation 26

27 Revolutionizing N-Glycan Sample Prep Robustness: Optimized Labeling Labeling optimized for high yield and minimization of artifacts Glycoprotein Concentration 0.36 mg/ml RapiFluor-MS Concentration 18 mm 36 mm 54 mm 72 mm 108 mm 1.6E+5 FA2 G0F Fluorescence Peak Area Maximized yield 4 9 min 0.0E RapiFluor-MS Reagent Concentration (mm) Patent Pending 2015 Waters Corporation 27

28 Revolutionizing N-Glycan Sample Prep Robustness: Optimized Labeling Labeling optimized for high yield and minimization of artifacts Not Optimized High Ionic Strength ph mm Reagent Overlabeled BPI FA2 +2 Labels 10x zoom FA2G1 ~1% +2 Labels 0.3 Optimized 50 mm HEPES ph mm Reagent Overlabeled 0.1% BPI A2 10x zoom (FA2 +1 Label / FA2 +2 Labels) % Overlabeled Minimization of artifacts + Maximized yield RapiFluor-MS Reagent Concentration (mm) Patent Pending 2015 Waters Corporation 28

Revolutionizing N-Glycan Sample Prep Robustness: High Yield N-Glycan Yield (entire workflow) ~73% 2E+6 0E+0 FA2 Rep #1 1.6 pmol Rep #2 1.7 pmol 100% Theoretical Yield = 2.3 pmol 1.

29 Revolutionizing N-Glycan Sample Prep Robustness: High Yield N-Glycan Yield (entire workflow) ~73% 2E+6 0E+0 FA2 Rep #1 1.6 pmol Rep #2 1.7 pmol 100% Theoretical Yield = 2.3 pmol 1.5x pmol 2 pmol glycan 0.45 pmol FA2 10 µl injection pg IgG X X X X 150,000 pg 1 pmol IgG 1 pmol total 400 µl = 2.3 pmol glycan pool sample prepared Peak Area (Fluorescence - Ex 265 nm / Em 425 nm) 1E+6 8E+5 6E+5 4E+5 2E+5 0E+0 y = x R² = RapiFluor-MS Label (pmol) 2015 Waters Corporation 29

30 Revolutionizing N-Glycan Sample Prep Robustness: High Yield with Minimal Bias Step Yield Testing to Confirm Minimal Bias Deglycosylation FA2 Rep #1 1.6 pmol Rep #2 1.7 pmol Complete Labeling >95% SPE ~74% GlycoWorks RapiFluor-MS N-Glycan Kit GlykoPrep Rapid N-Glycan Preparation with 2-AB 100% Theoretical Yield = 2.3 pmol Intact mass analysis / Subunit LC-MS Gel shift assays Released glycan profile vs subunit derived glycan information Recovery measurements Glycan profile before vs after SPE ~73% Yield ~35% Yield GlykoPrep is a copyright of Prozyme. These values may not be representative of all applications Waters Corporation 30

31 Revolutionizing N-Glycan Sample Prep Robustness: Stable Glycan Derivatives Fluorescence 10x zoom Intial 0 Hours 3 Days 72 Hours Initial min 10x zoom Fluorescen nce Peak Areas After 3 days 10 C min No significant change in the glycan FLR signal even after 3 days at 10 C Stable glycan derivatives 0 A2 FA2 FA2G1 FA2G1(iso) FA2G2 FA2G2Sg1 FA2G2Ga Waters Corporation 31

Summary Part I Preparation of labeled N-glycans (from glycoprotein to analysis ready sample) in 30 minutes Unprecedented sensitivity for labeled N- glycans Complete deglycosylation producing

32 Summary Part I Preparation of labeled N-glycans (from glycoprotein to analysis ready sample) in 30 minutes Unprecedented sensitivity for labeled N- glycans Complete deglycosylation producing unbiased results Simple, streamlined protocol Accurate profiling based on robust SPE for neutral to tetrasialylated N-glycans RapiFluor-MS GlycoWorks RapiFluor-MS N-Glycan Kit 2015 Waters Corporation 32

33 Part II GlycoWorks RapiFluor-MS N-Glycan Work 2015 Waters Corporation 33

34 Glycan Characterization Using UPLC/FLR/Xevo G2-XS QTof Conventional 2-AB Glycan from 10 µg of Protein 1.5 Day Sample Prep 1: TOF MS ES+ BPI 6.93e6 RapiFluor-MS Glycan from 0.1 µg Protein <1 hr Sample Prep 100x Sensitivity Characterization min High Resolution LC-MS(/MS) Example courtesy of Y. Yu 2015 Waters Corporation 34

Glycan Characterization Using UPLC/FLR/Xevo G2-XS Conventional 2-AB Glycan from 10 µg of Protein 1.5 Day Sample Prep 1130.921, 2+ FA2G2Ga1 RapiFluor-MS Glycan from 0.

35 Glycan Characterization Using UPLC/FLR/Xevo G2-XS Conventional 2-AB Glycan from 10 µg of Protein 1.5 Day Sample Prep , 2+ FA2G2Ga1 RapiFluor-MS Glycan from 0.1 µg Protein <1 hr Sample Prep 100x Sensitivity min 1: TOF MS ES+ BPI 6.93e m/z Characterization High Resolution LC-MS(/MS) Example courtesy of Y. Yu 2015 Waters Corporation 35

Glycan Characterization Enhanced Electrospray Ionization RapiFluor-MS 2-AB FA2 2+ 1+ 3.5e6 2.9e4 2.

36 Glycan Characterization Enhanced Electrospray Ionization RapiFluor-MS 2-AB FA e6 2.9e m/z m/z FA2BG2S e5 1.15e e m/z m/z 2015 Waters Corporation 36

37 Glycan Characterization Information-Rich MS/MS Spectra QTof Collision Induced Dissociation FA2 Contiguous series of glycosydic bond fragments (Near exclusively containing label) MS/MS spectra additionally contain some cross ring fragmentation 2015 Waters Corporation 37

38 Glycan Characterization Detailing the N-Glycan Profile of a mab 2.1E+6 FLR Intact mab Mass Check Standard anti-citrinin murine monoclonal IgG1 0.0E+6 8.4E+6 MS Rapid Preparation Deglycosylation Labeling SPE 30 min 0.0E Waters Corporation 38

39 Glycan Characterization Detailing the N-Glycan Profile of a mab FLR Intact mab Mass Check Standard anti-citrinin murine monoclonal IgG1 MS Peak No. Glycan Name Obs Mass Mass Error Approximate (M+H) + (ppm) Relative% F: Fucose A: Antennary G: Galactose B: Bisecting Ga: Alpha-galactose Sg: N-glycolylneuraminic acid iso: Structural isomer 1 A FA A FA A2G FA1G < A2G1(iso) FA2G FA2G1(iso) FA2G1B FA2G1B (iso) FA2G FA2G1Ga FA2G2Ga FA2G2Sg FA2G2Sg1(iso) FA2G2Ga FA2G2GaSg FA2G2Ga1Sg1(iso) Waters Corporation 39

40 Glycan Characterization Detailing the N-Glycan Profile of a mab FLR Intact mab Mass Check Standard anti-citrinin murine monoclonal IgG1 MS FA2G2? Zoom 10x 2015 Waters Corporation 40

41 Glycan Characterization Detailing the N-Glycan Profile of a mab Relative Abundance 8.0% Relative Abundance 0.7% Prominent 528 m/z ion and loss of GlcNAc 2015 Waters Corporation 41

42 Glycan Characterization Detailing the N-Glycan Profile of a mab FLR Intact mab Mass Check Standard anti-citrinin murine monoclonal IgG1 MS FA2G2? FA2G1Ga1 Zoom 10x 2015 Waters Corporation 42

43 Glycan Monitoring Transferability UPLC / HPLC H-Class BIO UPLC ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7µm 2.1 X 150 mm Flow Rate: 0.40 ml/minute Fluorescence (relative) Total Run Time = 55 minutes Alliance HPLC XBridge Glycan BEH Amide, 130Å, 2.5µm 3.0 X 225 mm total length (150 mm + 75 mm) Flow Rate: 0.56 ml/minute Total Run Time = minutes Time (minutes) Comparable sensitivity and resolution 3x sample load / 2x increase in time Transfer between labs with different LC equipment capabilities 2015 Waters Corporation 43

44 Glycan Monitoring UPLC/FLR/ACQUITY QDa Mass Detector FLR Rountine Analysis with Mass Detection Detection across a broad range of glycan species: IgG Simple bi-antennery structures RNase B High mannose structures QDa Fetuin Large, complex structures 3x 3x Monitoring Minutes Intermediate Technology UPLC-FLR UPLC-Mass Detection Example courtesy of S. McCarthy 2015 Waters Corporation 44

45 Glycan Monitoring UPLC/FLR/ACQUITY QDa Mass Detector FLR QDa 2015 Waters Corporation 45

46 GlycoWorks RapiFluor-MS Standards RapiFluor-MS Glycan Performance Test Standard FLR Labeled Glycans of IgG Pooled from Human Serum 400 pmol FA FA2BG2S2 Peak Glycan Approx RT (min) Approx Rel Abund (%) 1 A FA FA2B A2G A2G FA2G FA2G FA2BG FA2BG A2G FA2G FA2BG FA2G1S FA2G2S FA2BG2S FA2G2S FA2BG2S MS (BPI) System suitability Method familiarization Benchmarking Troubleshooting Waters Corporation 46

47 LC Calibration with Labeled Dextran 10 Calibration Curve EU n GU Value Log Mol Wt Cubic Spline Fitting Retention Time (min) Retention Time (min) Retention Time (min) 2015 Waters Corporation 47

48 LC Calibration with Labeled Dextran 10 Calibration Curve min GU Value g Mol Wt Log Cubic Spline Fitting EU Retention Time (min) Retention Time (min) GU 5.85 GU 5.95 GU Retention Time (min) 2015 Waters Corporation 48

49 LC Calibration with Labeled Dextran 35 Multiple sites reporting on one glycan profile Cubic Spline Fit Type Retentio on Time (min) Significant deviations in RTs GU Value RSD max = 8.50 RSD max = Glycan Profile Component Glycan Profile Component GU values improve the robustness of data reporting 2015 Waters Corporation 49

50 LC Calibration with Labeled Dextran Dextran no amine to modified with rapid tagging reagents Reductive amination with analogs is disadvantageous - different physicochemical properties due to different linkage - FLR wavelengths / basicity / pkas / polarity / HILIC retention RapiFluor-MS Labeled N-Glycans Reductive Amination With an Analog Novel Dextran Ladder Dextran Glycan Dextran x λ Excitation,Max =265 nm 235 nm 240 nm Ex 265 nm 245 Em 425 nm λ Emission,Max =425 nm 250 nm 255 nm 260 nm 265 nm Ex 370 nm Em 480 nm 325 nm 370 nm x x Ex 265 nm 265 nm Em 425 nm 315 nm 265 nm 270 nm 275 nm 365 nm 280 nm 285 nm 290 nm 295 nm nm nm nm 2015 Waters Corporation 50

51 GlycoWorks RapiFluor-MS Standards RapiFluor-MS Dextran Calibration Ladder 50 µg of a Novel Dextran Ladder FLR properties match RapiFluor-MS labeled glycans Tuned for HILIC Retention Dextran RapiFluor-MS Label Ethanolamino Urea Linkage FLR GU 3 MS (BPI) min Patent Pending 2015 Waters Corporation 51

52 GlycoWorks Mobile Phase Concentrate High purity mobile phases are critical to successful glycan LC-MS LC-MS Grade LC-MS grade mobile phase concentrate Eliminate concerns on NH 4 HCOOH purity and glass-ware cleanliness 10 ml concentrate simply poured into a 1L bottle of LC-MS water Not LC-MS Grade Adduct Formation Na K KCl / NaCl 2015 Waters Corporation 52

Summary Part II N-glycan characterization facilitated by combining RapiFluor-MS labeling and high sensitivity QTof MS RapiFluor-MS enhances ESI of

the GlycoWorks RapiFluor- MS N-glycan kit, new glycan standards and reagents have been developed to support analytical workflows, such as GU value

53 Summary Part II N-glycan characterization facilitated by combining RapiFluor-MS labeling and high sensitivity QTof MS RapiFluor-MS enhances ESI of glycans, producing high charge state, protonated ions RapiFluor-MS enables the possibility of QDa mass detection during routine analyses In addition to the GlycoWorks RapiFluor- MS N-glycan kit, new glycan standards and reagents have been developed to support analytical workflows, such as GU value assignment RapiFluor-MS GlycoWorks RapiFluor-MS N-Glycan Kit Calibration Ladder Dextran RapiFluor-MS Label Ethanolamino Urea Linkage 2015 Waters Corporation 53

54 Useful Literature Application Notes / Publications Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent Waters Application Note EN. January Robustness of RapiFluor-MS N-Glycan Sample Preparation and HILIC Analysis Waters Application Note in preparation. March Manuscript in preparation Posters WCBP 2015 / P-206-W Routine Monitoring of N-Glycans Using a Novel Labeling Reagent with Fluorescence and Mass Detection WCBP 2015 / P-216-W Rapid Preparation of N-Glycans Using a Novel Fluorescence and MS Active Labeling Reagent WCBP 2015 / P-115-T Developing a Scientific Library for UPLC/FLR/MS Analysis of Released N-glycans Labeled with a Novel Waters Corporation 54

55 Thank You for Attending! Post-Event Home Page: 30% Introductory Offer On GlycoWorks RapiFluor-MS Full Webinar Recording of Today s Session w/pdf Slide Deck Compilation of TODAY S KEY Literature, Brochures etc For Questions and to Submit your Ideas for our Next Topic Please - mychemrep@waters.com 2015 Waters Corporation 55

Eoin F.J. Cosgrave, Matthew A. Lauber, Robert Birdsall, and Sean M. McCarthy Waters Corporation, Milford, MA, USA APPLICATION BENEFITS INTRODUCTION

Eoin F.J. Cosgrave, Matthew A. Lauber, Robert Birdsall, and Sean M. McCarthy Waters Corporation, Milford, MA, USA APPLICATION BENEFITS INTRODUCTION New Capabilities for Monitoring Released N-Glycans through the Combined Use of RapiFluor-MS Labeling, ACQUITY UPLC H-Class Bio System, and Serial Fluorescence/ACQUITY QDa Mass Detection Eoin F.J. Cosgrave,