Bringing Glycan Analysis to a New Age of Enlightenment

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1 Bringing Glycan Analysis to a New Age of Enlightenment A discussion of disruptive LCMS technologies and workflows for released glycan and glycoprotein analysis Waters Corporation 1

2 RapiFluor-MS: Enhanced Analytical Workflows for Glycan Characterization and Monitoring Ying Qing Yu, Ph.D 2015 Waters Corporation 2

Glycosylation is a Critical Quality Attribute The International Conference

content, structural profiles, and characterization of the glycosylation

TrastuzumAb, 2 N-linked sites 150 KDa figures are from Wikipedia

3 Glycosylation is a Critical Quality Attribute The International Conference on Harmonization Guideline Q6B requires the analysis of carbohydrate content, structural profiles, and characterization of the glycosylation site(s) within the polypeptide chain(s). TrastuzumAb, 2 N-linked sites 150 KDa 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 2015 Waters Corporation 3

4 Glycoprotein Characterization Multiple Strategies Complementary Information 2015 Waters Corporation 4

Labeled N-Glycans + Reagents Reaction Byproducts Clean-up 0.

5 Conventional Workflow Conventional Glycoprotein 16 hrs (Pre-Labeling Clean-Up) (Drying) 1-3 hrs Released N-Glycans + Protein Deglycosylation Labeling 3 hrs Labeled N-Glycans + Reagents Reaction Byproducts Clean-up 0.1 hrs (Drying) 2 hrs >24 HOURS Labeled Glycans LC-FLR Analysis LC-FLR-MS 2015 Waters Corporation 5

6 What is new from Waters? Novel RapiFluor-MS TM (RFMS) Reagent Patent Pending 2015 Waters Corporation 6

ΔMass 312 Da Quinoline Fluorophore Glycan Reaction Time =

7 RapiFluor-MS Reagent Rapid Reaction Kinetics NHS Carbamate Rapid Tagging Group Tertiary Amine MS-Active Charge Tag Glycan ΔMass 312 Da Quinoline Fluorophore Glycan Reaction Time = Seconds Procedure Time: Highly stable urea linkage 2015 Waters Corporation 7

8 Simplified Sample Preparation Conventional Glycoprotein GlycoWorks RapiFluor-MS N-Glycan Kit Deglycosylation 16 hrs (Pre-Labeling Clean-Up) (Drying) 1-3 hrs Released N-Glycans + Protein Labeling <15 min 3 hrs Labeled N-Glycans + Reagents Reaction Byproducts Clean-up 5 min 0.1 hrs (Drying) 2 hrs >24 HOURS Labeled Glycans LC-FLR Analysis LC-FLR-MS 10 min 30 min Patent Pending 2015 Waters Corporation 8

9 How Sensitive is RapiFluor-MS label compare to conventional label? Fluorescence MS (BPI) RapiFluor-MS Labeled Instant AB Labeled 2-AB Labeled Procainamide Labeled Relative Performance (%) * * RFMS vs InstantAB: 2x in FLR and 1000x in MS RFMS vs 2AB: 14x in FLR and 160x in MS RFMS vs PCA: 14x in FLR and 3x in MS 2015 Waters Corporation 9

921, 2+ FA2G2Ga1 1: TOF MS ES+ BPI 6.93e6 RapiFluor-MS Glycan from 0.

10 Direct Sensitivity Comparison using UPLC/FLR/Xevo G2-XS QTOF Conventional 2-AB Glycan from 10 µg of Protein 1.5 Day Sample Prep , 2+ FA2G2Ga1 1: TOF MS ES+ BPI 6.93e6 RapiFluor-MS Glycan from 0.1 µg Protein <1 hr Sample Prep m/z min Xevo G2-XS QTOF 2015 Waters Corporation 10

11 GlycoWorks RapiFluor-MS N-Glycan Kit: Product Information Formats: Large Volume (4 x 24 Samples) & Small Volume (4 x 8 Samples) 2015 Waters Corporation 11

12 Characterizing RFMS labeled N-Glycans 2015 Waters Corporation 12

Glycan Characterization ACQUITY UPLC H-Class

Informatics GlycoWorks RapiFluor-MS N-Glycan

13 Glycan Characterization ACQUITY UPLC H-Class Bio System ACQUITY UPLC Column Manager ACQUITY UPLC FLR Detector Xevo G2-XS QTof MS UNIFI Glycan Application Solution or MassLynx Informatics GlycoWorks RapiFluor-MS N-Glycan Kit ACQUITY UPLC Glycan BEH Amide Column 2015 Waters Corporation 13

UNIFI Glycan Application Workflows Acquire

Scientific Library SimGlycan Workflows support

14 UNIFI Glycan Application Workflows Acquire Glycan FLR+MS Confirmation Glycan DDA Process/search Calibrated RT and Accurate Mass Deconvolute Fragment Ion Report GU Glycan Scientific Library SimGlycan Workflows support conventional and the new RapiFluor-MS labels 2015 Waters Corporation 14

15 The Utility of Glucose Unit (GU) Value What is a GU Value? GU stands for Glucose Unit A GU value is a normalized glycan retention time, 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. GU Values facilitate more routine glycan assignments by enabling the search of a single glycan GU library Waters Corporation 15

16 Glycan FLR +MS Confirmation Workflow Dextran ladder FLR GU 5 GU 10 GU 15 Retention time Calibration Curve Scientific Library Search FLR Analyte XIC CFG and Oxford structure displays are available Waters Corporation 16

17 GlycoWorks RapiFluor-MS Standards RapiFluor-MS Dextran Calibration Ladder RFMS-Dextran Ladder Dextran RapiFluor-MS Label Ethanolamino Urea Linkage FLR GU 3 MS (BPI) min Patent Pending 2015 Waters Corporation 17

18 Waters RFMS Glycan GU Library Exoglycosidase digestion for structure characterization Curated glycan structure library 2015 Waters Corporation 18

19 Glycan DDA Workflow UNIFI Processed Glycan DDA Data Data Export.mzml or.lcs 2015 Waters Corporation 19

20 Enhanced MSMS with RFMS Labeling Information-Rich MS/MS Spectra FA2 Higher glycan precursor ion intensity better MSMS fragmentation Simplified fragmentation pattern for easy data interpretation 2015 Waters Corporation 20

21 Example: NIST mab N-Glycan UNIFI Scientific Library Result: F(6)A2G2Ga1Sg1 (M+2H)2+ (M+3H)3+ MSMS 2015 Waters Corporation 21

MSMS MSMS 528.186 FA2G2Ga1Sg1 673.226 366.137 Tag 1733.671 Tag Tag Fucose GlcNAc Manoses Galactose NeuGc 204.086 Tag Tag Tag Tag 835.276 1368.562 1206.501 882.401 1571.626 1895.747 2260.

22 MSMS MSMS FA2G2Ga1Sg Tag Tag Tag Fucose GlcNAc Manoses Galactose NeuGc Tag Tag Tag Tag (M +H) Tag Tag Tag mass alpha (1,3)-Gal (Ga1) SimGlycan Search Results NeuGc (Sg1) 2015 Waters Corporation 22

23 RFMS Label Enables Routine Glyan Profiling and Monitoring 2015 Waters Corporation 23

The ACQUITY QDa Mass Detector - a pioneering product with mass appeal

analytical chemists everywhere with orthogonal mass detection added

constant use with a wide variety of chromatographic conditions Seamlessly

24 The ACQUITY QDa Mass Detector - a pioneering product with mass appeal Revolutionary innovative design focused on ease of use for analysts Empowering analytical chemists everywhere with orthogonal mass detection added information with every sample Compact, robust and affordable - built for constant use with a wide variety of chromatographic conditions Seamlessly integrates with Empower -based UPLC Systems for ease-of-use and GMP compliance 2015 Waters Corporation 24

integrated with Empower CDS Minimal training required

25 Easily Added to Existing Systems Existing stack upgrade Existing stack +1 Easy to deploy Fully integrated with Empower CDS Minimal training required 110/220V operation Minimal maintenance QDa 2015 Waters Corporation 25

26 A Mass Detector that Starts-Up, Setsup, and is Maintained Like and Optical Detector Disposable Source Aperture 2015 Waters Corporation 27

9e4 500 2000 m/z 500 2000 m/z FA2BG2S2 3+ 2+ 2+ 1.

27 RFMS Shifts Glycans to Higher Charge States RapiFluor-MS 2-AB FA e e m/z m/z FA2BG2S e5 5.3e m/z m/z 2015 Waters Corporation 28

28 Routine N-Glycan Detection with Matching FLR and MS response Detection across a broad range of glycoforms: IgG Simple bi-antennary structures Minutes RNase B High mannose structures Fetuin Large, complex and highly sialylated structures Minutes 2015 Waters Corporation 29

29 Routine RFMS Labeled N-Glycan Detection with Matching FLR and MS Response 60 FLR EU Near Equivalent FLR and MS. 0 6x10 6 5x Minutes TIC The added benefit of mass confirmation. Intensity 4x10 6 3x10 6 2x10 6 1x Minutes 2015 Waters Corporation 30

30 IgG Glycan Profile and Structure Confirmation Using the QDa Intensity FA2 20.5% RPA EU m/z Intensity 3.2x x Intensity A2G1b 0.5% RPA Minutes Key take away: Large dynamic range can clearly see most abundant and least abundant glycoforms Waters Corporation m/z

31 Routine Mass Data for Every Peak confidence in results.ease of method development quick diagnosis of problems EU A FA FA2B A2G1a A2G1b FA2G1a FA2G1b FA2BG1a FA2BG1b A2G FA2G FA2BG FA2G1S FA2G2S FA2BG2S FA2G2S FA2BG2S Minutes 2015 Waters Corporation 32

32 Enabling Accelerated Bioprocess Development Trastuzumab N-Glycan Analysis RapiFluor-MS labeled glycans - 10 minute method Intensity SIR M m/z 40 FLR Intensity A m/z Intensity EU x10 6 1x10 6 SIR Overlay Intensity Intensity Intensity 2x10 6 1x x F(6)A m/z A2G(4) m/z F(6)A2G(4) m/z Minutes Intensity F(6)A2G(4) m/z Intensity F(6)A2G(4)2S m/z Minutes 2015 Waters Corporation 33

33 Monitoring Key Glycan Ratios Keeping Tabs on Mannose Intensity M5 SIR: m/z Intensity F(6)A2G(4)1 SIR: m/z Minutes Minutes Man5: F(6)A2G(4)1 Ratio Time 1 Time 2 Time 3 Low Medium High Mean StDev % RSD Waters Corporation 34

34 Summary RFMS labeling technology shortens and simplifies the released glycan sample preparation, while enhancing Fluorescence, MS and MS/MS response. RFMS improves released glycan characterization, particularity for low abundant glycan assignment. The workflows developed with UNIFI streamlined and automated the process of glycan characterization. RFMS enables routine glycan monitoring with Mass Detection for confidence in profile and rapid resolution of unexpected results. ACQUITY QDa enables higher throughput targeted quantitation using the selectivity of mass detection Waters Corporation 35

35 Innovations in HILIC for Improved Characterization of Intact Glycoproteins, Glycosylated Protein Fragments, and Glycopeptides Matthew A. Lauber, Ph.D Waters Corporation 36

36 Glycoprotein Characterization Multiple Strategies Complementary Information HILIC Hydrophilic Interaction Chromatography 2015 Waters Corporation 37

37 Proof of Concept on Large Molecule HILIC RNase B High Mannose Man5 to Man9 PDB: 1RBB Glycan structure shown to scale with protein Modified from Structure 1999, 7 (7), R Waters Corporation 38

38 HILIC Stationary Phases A Unbonded BEH 130Å Time (min) 2015 Waters Corporation 39

39 HILIC Stationary Phases BEH Particle 0.4 A Amide Bonded BEH 130Å Unbonded BEH 130Å Increased Retentivity (>10% H 2 O) Improved Resolution Time (min) 2015 Waters Corporation 40

Highly Branched Glycans 0.4 Intact mabs A 214 0.

40 HILIC Stationary Phases Amide Bonded BEH 300Å Improved Resolution for Large Analytes Highly Branched Glycans 0.4 Intact mabs A Amide Bonded BEH 130Å Unbonded BEH 130Å Time (min) Patent pending 2015 Waters Corporation 41

41 Method Development Mobile Phase Additive RNase B % FA TFA A mm Ammonium Formate ph 4.4 F 3 C-COO - - OOC-CF % TFA F 3 C-COO - + H 3 N Time (min) - OOC-CF 3 Protonation and Strong Ion Pairing with a Hydrophobic Acid Hydrophilicity Reduced Minimize protein hydrophilicity Improve selectivity of the separation for the glycan Patent pending 2015 Waters Corporation 42

42 Orthogonality and LC-MS Man5 +Man6 +Man7 +Man8 +Man9 Reversed Phase BEH C4 300Å A RNase B aglycosylated Waters Corporation 43

43 Orthogonality and LC-MS Man5 +Man6 +Man7 +Man8 +Man9 Reversed Phase BEH C4 300Å 0.06 A RNase B aglycosylated Man5 +Man6 HILIC BEH Amide 300Å A 214 RNase B aglycosylated +Man Man7 +Man Time (min) Patent pending 2015 Waters Corporation 44

44 Orthogonality and LC-MS Man5 +Man6 +Man7 +Man8 +Man9 Reversed Phase BEH C4 300Å 0.06 A RNase B aglycosylated 0 Man9 Glycoform Man5 +Man6 HILIC BEH Amide 300Å A 214 RNase B aglycosylated +Man Man7 +Man Time (min) m/z Patent pending 2015 Waters Corporation 45

45 Glycoprotein Characterization Multiple Strategies Complementary Information HILIC Hydrophilic Interaction Chromatography 2015 Waters Corporation 46

46 Orthogonal Peptide Mapping Techniques Trastuzumab Lys-C Digest 1.0 Peptide BEH C18 300Å 1.7 µm A214 Reversed Phase Non-glycosylated Peptides Peptide Glycoprotein BEH Amide 300Å 1.7 µm HILIC A214 Glycopeptides Non-glycosylated Peptides Waters Corporation Time (min)

Orthogonal Peptide Mapping Techniques Glycopeptides 0.2 Peptide A214 TKPREEQYNSTYRVVSVLTVLHQDWLNGK Reversed Phase 0.1 0.0 55 57 59 61 63 65 0.

47 Orthogonal Peptide Mapping Techniques Glycopeptides 0.2 Peptide A214 TKPREEQYNSTYRVVSVLTVLHQDWLNGK Reversed Phase Same gradient slope A214 HILIC Waters Corporation Time (min) Patent pending 48

48 MS-Based Identification of Glycoforms as simple as peptide mapping UV FA2 FA2G1 HILIC HC:K16 TKPREEQYNSTYRVVSVLTVLHQDWLNGK Glycoforms Peptide A2 FA1 M5 FA1G1 A2G1 FA2G1 FA2G2 A1 FA Da FA2G Da 2E+6 TIC Intensity 1E+6 HILIC 0E Time (min) Patent pending 2015 Waters Corporation 49

49 MS-Based Identification of Glycoforms as simple as peptide mapping UV FA2 FA2G1 HILIC HC:K16 TKPREEQYNSTYRVVSVLTVLHQDWLNGK Glycoforms Peptide A2 FA1 M5 FA1G1 A2G1 FA2G1 FA2G2 FA2G2S , ppm A1 FA Da FA2G Da m/z 2E+6 TIC FA2G2S , ppm Intensity 1E+6 HILIC m/z 0E Time (min) Patent pending 2015 Waters Corporation 50

50 A 214 HILIC Glycopeptide Mapping to Investigate Multi-Domain Glycosylation Cetuximab Lys-C / Tryptic Digest Peptide HILIC Time (min) Patent pending 2015 Waters Corporation 51

51 HILIC Glycopeptide Mapping to Investigate Multi-Domain Glycosylation Cetuximab Lys-C / Tryptic Digest 9 Fc glycoforms 16 Fab >2% Peptide T22 +FA2 HILIC T22 +FA2G1 HC:T22 EEQYNSTYR Glycoforms T8 +FA2G2Ga2 A 214 T8 +FA2G1 T22 +FA1 T8 +FA2G2 T22 +M5 T22 +FA1G1 T22 +FA2G1 T8 +FA2G2Sg1 T8 +FA2G2Ga1 T22 +M6 T22 +FA2G2 T8 +FA2G2Ga1Sg1 T22 +FA2G2Ga1 T22 +Hex6HexNAc3DHex1 HC:T8 MNSLQSNDTAIYYC(am)AR Glycoforms T8 T8 +Hex9HexNAc5DHex1 +Hex7HexNAc5DHex1NGNA1 T22 +Hex5HexNAc3DHex Time (min) T8 (deamidated) +FA2G2Ga2 T8 +FA2G2Sg2 T8 +Hex8HexNAc5DHex1 T8 +Hex8HexNAc5DHex1NGNA1 Patent pending 2015 Waters Corporation 52

52 Glycoprotein Characterization Multiple Strategies Complementary Information HILIC Hydrophilic Interaction Chromatography 2015 Waters Corporation 53

53 Middle-Up/Down mab Analysis Middle-Up (Fab )2 2x Light Chain 2x Fd Cleavage Site -G G- IdeS Digestion 2x Fc/2 Denaturation Reduction 2x Fc/ Waters Corporation 54

54 HILIC Middle-Up Analysis IdeS-Digested Trastuzumab UV Fd LC Fc/2 Middle-Up TIC 5 10 Patent pending 2015 Waters Corporation 55

55 HILIC Middle-Up Analysis IdeS-Digested Trastuzumab UV Fd LC Fc/2 Middle-Up TIC 5 10 Fc/2 +FA Fc/2 +FA2G Da Da Da Fc/2 +FA2G1 Fc/2 +A Waters Corporation Fc/2 +A2G1 Fc/2 +FA2G Da Da Da Molecular Weight Da Patent pending 56

56 Glycoprotein Characterization Multiple Strategies Complementary Information HILIC Hydrophilic Interaction Chromatography 2015 Waters Corporation 57

57 Assaying Glycan Occupancy and Deglycosylation on Intact mabs Intact 2 N-Glycans 1 N-Glycans 0 N-Glycans 2015 Waters Corporation 58

Assaying Deglycosylation and Glycan Occupancy

58 Assaying Deglycosylation and Glycan Occupancy on Intact mabs Intrinsic Fluorescence Detection Trastuzumab Partially Deglycosylated 2 N-Glycans 1 N-Glycan 21.5% 0 N-Glycans 3.1% min 2 N-Glycans 1 N-Glycans Intact 0 N-Glycans Patent pending 2015 Waters Corporation 59

59 Assaying Deglycosylation and Glycan Occupancy on Intact mabs Trastuzumab (2 N-Glycans) Intrinsic Fluorescence Detection Trastuzumab Partially Deglycosylated min Trastuzumab Native 2 N-Glycans 1 N-Glycan 21.5% 0 N-Glycans 3.1% 2 N-Glycans 1,000 5,000 m/z FA2/FA2G Da FA2/FA2 148,057 Da kda Trastuzumab (1 N-Glycan) FA2 146,614 Da FA2G1 146,773 Da Intact 1 N-Glycan 1.0% min 1,000 5,000 m/z kda Patent pending 2015 Waters Corporation 60

60 Assaying Deglycosylation and Glycan Occupancy on Intact mabs Intrinsic Fluorescence Detection Intact Bevacizumab 1 N-Glycan 4.9% Trastuzumab 1 N-Glycan 1.0% IgG1κ Reference Material 1 N-Glycan 1.3% Murine IgG1 (Intact mab Mass Check Std) 1 N-Glycan 0.7% min Patent pending 2015 Waters Corporation 61

61 Glycoprotein Characterization Multiple Strategies Complementary Information Combining Approaches for Comprehensive Characterization 2015 Waters Corporation 62

62 Cetuximab Complementary Analyses RapiFluor-MS Labeled N-Glycans Glycan BEH Amide 2.1 x 50 mm 4E+6 FA2 FA2G1 FA2G2Ga2 High Resolution High Sensitivity Released N-Glycan Profile EU M5 FA2G2Sg1 FA2G2 FA2G2Ga1 FA2G2Ga1Sg1 Hex9HexNAc5DHex1 Released Glycan 0E Time (min) Patent pending 2015 Waters Corporation 63

63 Cetuximab Complementary Analyses RapiFluor-MS Labeled N-Glycans Glycan BEH Amide 2.1 x 50 mm 4E+6 FA2 FA2G1 FA2G2Ga2 High Resolution High Sensitivity Released N-Glycan Profile EU M5 FA2G2Sg1 FA2G2 FA2G2Ga1 FA2G2Ga1Sg1 Hex9HexNAc5DHex1 Released Glycan 0E Fc/2 +FA2 Fc/2 Glycosylated Time (min) Domain-Specific Glycan Information Fab Reduced, IdeS-Digested Cetuximab (Carboxypeptidase B treated) Glycoprotein BEH Amide 2.1 x 150 mm A Fc/2 +M5 Fc/2 +FA2G1 Fd pe + (FA2G2Sg1) Fd pe + (FA2G2Ga2) Fd pe + (FA2G2Ga1Sg1) Fd Glycosylated N-term pe N-Linked Glycans 0.02 Fc/2 +FA2G2 Fd pe + (Hex9HexNAc5DHex1) Fc 0.01 Fd pe + (FA2G2Ga1) Time (min) Subunit Patent pending 2015 Waters Corporation 64

Characterization of EPO N and O-Linked Glycosylation APPRLICDSR VLERYLLEAK EAENITTGCA EHCSLNENIT VPDTKVNFYA N-Linked Glycans WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVNSSQPWEP LQLHVDKAVS

64 Characterization of EPO N and O-Linked Glycosylation APPRLICDSR VLERYLLEAK EAENITTGCA EHCSLNENIT VPDTKVNFYA N-Linked Glycans WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVNSSQPWEP LQLHVDKAVS O-Linked Glycan GLRSLTTLLR ALGAQKEAIS PPDAASAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGD O-Linked Glycan EPO Recombinant Human Epoetin Alpha N-Linked Glycans 2015 Waters Corporation 65

65 Released Glycan Analysis of EPO RapiFluor-MS Labeling 3E+6 Fluorescence N-Linked Glycans Released Glycan 0E+0 2E+5 BPI 0E Patent pending 2015 Waters Corporation 66

66 Released Glycan Analysis of EPO RapiFluor-MS Labeling 3E+6 Fluorescence FA4G4Lac1S4 FA4G4S4 FA4G4Lac2S4 N-Linked Glycans Released Glycan FA4G4Lac1S3 FA4G4S3 FA4G4Lac1S4+Ac FA4G4Lac2S3 FA4G4Lac3S3 FA2G2S , ppm FA4G4S4+Ac , ppm FA2G2S1 FA2G2S2 FA4G4S4+Ac FA3G3S3 FA4G4Lac3S4 FA4G4Lac4S4 0E+0 2E+5 BPI 890 m/z m/z 1349 FA4G4Lac2S4 FA4G4Lac4S , ppm , ppm 0E m/z m/z Patent pending 2015 Waters Corporation 67

67 HILIC of Intact N-Deglycosylated EPO O-Linked Glycan Characterization 1E+7 Intrinsic Fluorescence Detection O-Linked Glycan O-Linked Glycan N-Linked Glycans Rapid Deglycosylation Intact 0E min Patent pending 2015 Waters Corporation 68

HILIC of Intact N-Deglycosylated EPO O-Linked Glycan Characterization Intrinsic Fluorescence Detection 1E+7 0E+0 Aglycosylated * O-Linked Glycan * * O-Linked Glycan

68 HILIC of Intact N-Deglycosylated EPO O-Linked Glycan Characterization Intrinsic Fluorescence Detection 1E+7 0E+0 Aglycosylated * O-Linked Glycan * * O-Linked Glycan PNGase F Da +TFA Da Da min kda +TFA +TFA Intact *Truncated C-terminus Patent pending 2015 Waters Corporation 69

glycosylation Middle-Up Middle-Down Rapid domain specific glycan profiling using IdeS digestion Higher quality MS of

69 Novel Widepore HILIC Column for Supporting Multiple Analysis Workflows Large Molecule HILIC ACQUITY UPLC Glycoprotein BEH Amide Column Intact Glycoprotein Glycan occupancy on intact mabs Profiling occupancy and heterogeneity of EPO O- glycosylation Middle-Up Middle-Down Rapid domain specific glycan profiling using IdeS digestion Higher quality MS of glycoforms Glycopeptides Traditional proteolytic digestion to yield site-specific glycan profiling Patent pending 2015 Waters Corporation 70

70 Innovative Glycan Analyses Released Glycan Analysis RapiFluor-MS Xevo G2-XS QTof ACQUITY QDa Detector ACQUITY UPLC Glycoprotein BEH Amide Column Intact Glycoprotein Glycopeptides Middle-Up Middle-Down 2015 Waters Corporation 71

71 Anal Chem 2015 DOI: /acs.analchem.5b New Application Notes on Glycan Analysis Genetic Engineering News Webinars Broadcast: March 25, 2015 Currently Streaming June 18, :00 AM EST 2015 Waters Corporation 72

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