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1 Complementary HILIC Techniques for the Successful Characterization of Therapeutic Protein Glycosylation 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 our NEW GlycoWorks RapiFluor-MS N-Glycan Analysis Kit and our NEW ACQUITY UPLC Glycoprotein BEH Amide 300Å 1.7 µm Columns 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 Product specific information / Reference materials Discount Offers - GlycoWorks RapiFluor-MS N-Glycan Analysis Kits ACQUITY UPLC Glycoprotein BEH Amide 300Å 1.7 µm Columns 2015 Waters Corporation 2

3 Today s Presenter Matthew Lauber, Ph.D. Waters Corporation, Milford, MA USA Matthew Lauber is a Principal Applications Chemist within the Consumables Business Unit at Waters Corporation, where he applies his expertise in protein chemistry and LC-MS based protein characterization methods toward the development and application of state-of-the-art reagents and separation chemistries. Most recently, Matthew has led investigations into the rapid and sensitive analyses of released N- glycans as well as the development of new strategies for characterizing protein glycosylation that are based on the use of a widepore HILIC column technology Waters Corporation 3

4 Complementary HILIC Techniques for the Successful Characterization of Therapeutic Protein Glycosylation Matthew A. Lauber, Ph.D Waters Corporation 4

5 Glycosylation of Biotherapeutics Fucose GlcNAc Mannose Effector Functions (ADCC/CDC) (fucosylation/galactosylation) Low Half Life (high g mannose) Anti-Inflammatory (sialylation) Immunogenic (αgal / N-glycolylneuraminic gy y acid) Overall profile sensitive to manufacturing conditions Galactose N-acetylneuraminic acid N-glycolylneuraminic acid 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 RapiFluor-MS Reagent Rapid Labeling Kinetics 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 Anal Chem 2015, 87 (10), Patent Pending 2015 Waters Corporation 8

9 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 Charge Tag + Δmass (glycosylamine) Glycosylamine = Da (reducing end) = Da +C 17 H 20 N 4 O 2 Δtime = Quinoline Fluorophore Monoisotopic mass shift (from glycosylamine) seconds 1586 Da H 2 O CO 2 Anal Chem 2015, 87 (10), Patent Pending 2015 Waters Corporation 9

10 Revolutionizing N-Glycan Sample Prep Sensitivity Fluorescence MS (BPI) RapiFluor-MS Labeled Instant AB Labeled 2-AB Labeled Procainamide Labeled ve Performan nce (%) Relati * * (*) Comparative result extrapolated from a published comparison of N-glycans, gy 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). Anal Chem 2015, 87 (10), Patent Pending 2015 Waters Corporation 10

11 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 Anal Chem 2015, 87 (10), Patent Pending 2015 Waters Corporation 11

12 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 l Labeling SPE 30 min , E+6 FA2G2Ga1 Anal Chem 2015, 87 (10), Patent Pending Example courtesy of Y. Yu 2015 Waters Corporation 12

13 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 Peak Obs Mass Mass Error Approximate Glycan Name No. (M+H) + (ppm) Relative% 1 A FA A FA A2G FA1G < E+6 6 A2G1(iso) FA2G FA2G1(iso) FA2G1B FA2G1B (iso) FA2G FA2G1Ga F: Fucose 13 FA2G2Ga A: Antennary 14 FA2G2Sg G: Galactose B: Bisecting 15 FA2G2Sg1(iso) Anal Chem 2015, 87 (10), Ga: Alpha-galactose 16 FA2G2Ga Sg: N-glycolylneuraminic acid 17 FA2G2GaSg Patent Pending iso: Structural isomer 18 FA2G2Ga1Sg1(iso) Waters Corporation 13

14 GlycoWorks RapiFluor-MS N-Glycan Kit Preparation of labeled N-glycans (from glycoprotein to analysis ready sample) in 30 minutes Unprecedented sensitivity RapiFluor-MS N-glycan detection facilitated by combining RapiFluor-MS labeling and recent advances in mass spectrometry GlycoWorks RapiFluor-MS N-Glycan Kit Anal Chem 2015, 87 (10), Patent Pending 2015 Waters Corporation 14

15 gy Anal Chem 2015 DOI: /acs.analchem.5b New Application Notes on Released N-Glycan Analysis Genetic Engineering g News Webinars Broadcast: March 25, 2015 June 18, :00 AM EST Currently On Demand 2015 Waters Corporation 15

16 Glycoprotein Characterization Multiple Strategies Complementary Information HILIC Hydrophilic Interaction Chromatography 2015 Waters Corporation 16

17 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 17

18 HILIC Stationary Phases A Unbonded BEH 130Å Time (min) 2015 Waters Corporation 18

19 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 19

20 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 20

21 HILIC Stationary Phases Amide Bonded BEH 300Å Improved Resolution for Large Analytes Highly Branched Glycans 0.4 Intact mabs A Amide Bonded BEH 130Å 130Å Unbonded BEH 130Å 300Å Time (min) Patent pending 2015 Waters Corporation 21

22 Method Development Mobile Phase Additive RNase B %FA 0.5% TFA A mm Ammonium Formate ph H 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 22

23 Orthogonality and LC-MS Man5 +Man6 +Man7 +Man8 +Man9 Reversed Phase BEH C4 300Å A RNase B aglycosylated Waters Corporation 23

24 Orthogonality and LC-MS Man5 +Man6 +Man7 +Man8 +Man9 Reversed Phase BEH C4 300Å 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 24

25 Orthogonality and LC-MS Man5 +Man6 +Man7 +Man8 +Man9 Reversed Phase BEH C4 300Å 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 25

26 Glycoprotein Characterization Multiple Strategies Complementary Information HILIC Hydrophilic Interaction Chromatography 2015 Waters Corporation 26

27 Orthogonal Peptide Mapping Techniques Trastuzumab Lys-C Digest 1.0 Reversed Phase Peptide BEH C18 300Å 1.7 µm Non-glycosylated Peptides Peptide A HILIC A Glycoprotein BEH Amide 300Å 1.7 µm Non-glycosylated Peptides Glycopeptides Time (min) 2015 Waters Corporation 27

28 Orthogonal Peptide Mapping Techniques 0.2 Glycopeptides TKPREEQYNSTYRVVSVLTVLHQDWLNGK Peptide A Reversed Phase Same gradient slope HILIC A Time (min) Patent pending 2015 Waters Corporation 28

29 MS-Based Identification of Glycoforms as simple as peptide p mapping 0.07 FA2 UV FA2G1 HILIC HC:K16 TKPREEQYNSTYRVVSVLTVLHQDWLNGK Glycoforms Peptide A A2 FA1 M5 FA1G1 A2G1 FA2G1 FA2G2 A1 FA2 FA2G Da Da E+6 TIC Intensity 1E+6 HILIC 0E Time (min) Patent pending 2015 Waters Corporation 29

30 MS-Based Identification of Glycoforms as simple as peptide p mapping 0.07 FA2 UV FA2G1 HILIC HC:K16 TKPREEQYNSTYRVVSVLTVLHQDWLNGK Glycoforms Peptide A A2 FA1 M5 FA1G1 A2G1 FA2G1 FA2G2 FA2G2S , ppm A1 FA2 FA2G Da Da E+6 TIC m/z FA2G2S , ppm Intensity 1E+6 HILIC m/z 0E Time (min) Patent pending 2015 Waters Corporation 30

31 HILIC Glycopeptide Mapping to Investigate Multi-Domain Glycosylation g y y Cetuximab Lys-C / Tryptic Digest Peptide HILIC A Time (min) Patent pending 2015 Waters Corporation 31

32 HILIC Glycopeptide Mapping to Investigate Multi-Domain Glycosylation g y y Cetuximab Lys-C / Tryptic Digest 9 Fc glycoforms 16 Fab >2% Peptide T22 HILIC +FA2 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) T8 +FA2G2Ga2 +FA2G2Sg2 T8 +Hex8HexNAc5DHex1 T8 +Hex8HexNAc5DHex1NGNA1 Patent pending 2015 Waters Corporation 32

33 Glycoprotein Characterization Multiple Strategies Complementary Information HILIC Hydrophilic Interaction Chromatography 2015 Waters Corporation 33

34 Middle-Up/Down mab Analysis Middle-Up (Fab )2 2x Light Chain 2x Fd Cleavage Site -G G- IdeS Digestion 2x Fc/2 Denaturation ti Reduction 2x Fc/ Waters Corporation 34

35 HILIC Middle-Up Analysis IdeS-Digested Trastuzumab g 0.24 Protein BEH C4 300Å 1.7μm Fc/2 Glycoforms 0.18 LC Fd Middle-Up A Time (min) 2015 Waters Corporation 35

36 HILIC Middle-Up Analysis IdeS-Digested Trastuzumab g UV Fd LC Fc/2 Middle-Up TIC 5 10 Patent pending 2015 Waters Corporation 36

37 HILIC Middle-Up Analysis IdeS-Digested Trastuzumab g UV Fd LC Fc/2 Middle-Up TIC Fc/2 +FA2 Fc/2 +FA2G Da Da Da A Fc/2 +A2 Fc/2 +A2G1 Fc/2 +FA2G1 Fc/2 +FA2G Time (min) Da Da Da Molecular Weight Da Patent pending 2015 Waters Corporation 37

38 Middle-Up Analysis Orthogonality 0.24 Protein BEH C4 300Å 1.7μm Fc/2 Glycoforms 0.18 LC Fd Middle-Up A Time (min) Glycoprotein BEH Amide 300Å 1.7μm LC 0.12 Fd A Fc/2 Glycoforms Time (min) Patent pending 2015 Waters Corporation 38

39 Glycoprotein Characterization Multiple Strategies Complementary Information HILIC Hydrophilic Interaction Chromatography 2015 Waters Corporation 39

40 Assaying Deglycosylation and Glycan Occupancy on Intact mabs Intact 2 N-Glycans 1 N-Glycans 0N-Glycans 2015 Waters Corporation 40

41 Assaying Deglycosylation and Glycan Occupancy on Intact mabs Detection Intr rinsic Fluo orescence Trastuzumab Partially Deglycosylated 2 N-Glycans 1 N-Glycan 21.5% 0 N-Glycans 3.1% min 2 N-Glycans 1 N-Glycans Intact 0N-Glycans Patent pending 2015 Waters Corporation 41

42 Assaying Deglycosylation and Glycan Occupancy on Intact mabs Trastuzumab (2 N-Glycans) Detection Intr rinsic Fluo orescence Trastuzumab Partially Deglycosylated 2 N-Glycans 1 N-Glycan 21.5% 0 N-Glycans 3.1% min Trastuzumab Native 2 N-Glycans FA2/FA2G Da FA2/FA2 148,057 Da 1,000 5,000 m/z 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 42

43 Assaying Deglycosylation and Glycan Occupancy on Intact mabs Intrinsic Fluorescence Detection Intact Bevacizumab 1N-Glycan 4.9% Trastuzumab 1N-Glycan 1.0% IgG1κ Reference Material 1 N-Glycan 13% 1.3% Murine IgG1 (Intact mab Mass Check Std) 1 N-Glycan 0.7% min Patent pending 2015 Waters Corporation 43

44 Glycoprotein Characterization Multiple Strategies Complementary Information Increasing Peak Capacity for High Antennarity Released N-Glycans 2015 Waters Corporation 44

45 Increasing Peak Capacity for High Antennarity Released N-Glycans 3E+6 Glycan BEH Amide 130Å 1.7 µm 1 A2 2 P * c = 104 ΔRT 1,4 = min W h,avg = 7.43 sec 6 P * c = 53 ΔRT 5,8 = 6.45 min W h,avg = 7.47 sec W h 2 FA2G1 3 FA2G2S E Retention Time (min) 4 FA2BG2S2 3E+6 Glycoprotein BEH Amide 300Å 1.7 µm 5 - FA3G3S3 2 P c * = 101 ΔRT 1,4 = min W h,avg = 6.89 sec 6 P c * = 62 ΔRT 5,8 = 6.70 min W h,avg = 6.60 sec 6 - FA4G4S % Increase in Peak Capacity 7 - FA4G4Lac1S4 5 1x 0E FA4G4Lac2S Retention Time (min) 2x 2015 Waters Corporation 45

46 Increasing Peak Capacity for High Antennarity Released N-Glycans 3E+5 Glycan BEH Amide 130Å 1.7 µm FA3G3S3 6 - FA4G4S4 0E Glycoprotein BEH Amide 300Å 1.7 µm 3E x 7 - FA4G4Lac1S4 2x 8 - FA4G4Lac2S4 FA4G4S3 FA4G4S2 FA4G4Lac1S3 0E Retention Time (min) 2015 Waters Corporation 46

47 Glycoprotein Characterization Multiple Strategies Complementary Information Combining Approaches for Comprehensive Characterization 2015 Waters Corporation 47

48 Cetuximab Complementary Analyses p y y 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 FA2G2 FA2G2Sg1 FA2G2Ga1 FA2G2Ga1Sg1 Hex9HexNAc5DHex1 Released Glycan 0E Time (min) Patent pending 2015 Waters Corporation 48

49 Cetuximab Complementary Analyses p y y 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 FA2G2 FA2G2Sg1 FA2G2Ga1 FA2G2Ga1Sg1 Hex9HexNAc5DHex1 Released Glycan 0E Fc/2 +FA2 Time (min) Fc/2 Glycosylated 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 Fd pe + (FA2G2Ga1) Middle-Up Time (min) Patent pending 2015 Waters Corporation 49

50 Characterization of EPO N and O-Linked Glycosylation y 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 50

51 Released Glycan Analysis of EPO RapiFluor-MS Labeling 3E+6 Fluorescence Glycoprotein BEH Amide 2.1 x 150 mm N-Linked Glycans Released Glycan 0E+0 2E+5 BPI 0E Patent pending 2015 Waters Corporation 51

52 Released Glycan Analysis of EPO RapiFluor-MS Labeling 3E+6 Fluorescence Glycoprotein BEH Amide 2.1 x 150 mm FA4G4S4 FA4G4Lac1S4 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 0.9 ppm 0E m/z m/z Patent pending 2015 Waters Corporation 52

53 HILIC of Intact N-Deglycosylated EPO O-Linked Glycan Characterization 1E+7 Intr rinsic Fluo orescence Detection O-Linked Glycan O-Linked Glycan N-Linked Glycans Rapid Deglycosylation Intact 0E min Patent pending 2015 Waters Corporation 53

54 HILIC of Intact N-Deglycosylated EPO O-Linked Glycan Characterization Intr rinsic Fluo orescence 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 54

55 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 Subunits Rapid domain specific glycan profiling using IdeS digestion Higher quality MS of glycoforms Glycopeptides Glycopeptides Traditional proteolytic digestion to yield site-specific glycan profiling Patent pending 2015 Waters Corporation 55

56 Glycoprotein Performance Test Standard FLR RNase A Man5 Man7 RNase B Glycoforms System Suitability Troubleshooting Benchmarking Man6 Man8 Man9 N-Linked Glycans KETAA AKFER QHMDS STSAA SSSNY CNQMM KSRNL TIC TKDRC KPVNT FVHES LADVQ AVCSQ KNVAC KNGQT Disulfides NCYQS YSTMS ITDCR ETGSS KYPNC AYKTT QANKH IIVAC EGNPY VPVHF DASV Species M Average (Da) Aglycosylated (4 disulfides) RNase B (4 disulfides), Man Rnase B (4 disulfides), Man Rnase B (4 disulfides), Man Rnase B (4 disulfides), Man Rnase B (4 disulfides), Man Waters Corporation 56

57 gy 5+ New Application Notes on Glycoprotein BEH Amide 300Å Separations 2015 Waters Corporation 57

58 Thank You Released Glycan Analysis RapiFluor-MS Xevo G2-XS QTof ACQUITY QDa Detector Innovative Glycan Analyses ACQUITY UPLC Glycoprotein BEH Amide Column Intact Glycoprotein Glycopeptides Middle-Up Middle-Down Acknowledgements S. McCall S. Koza Y. Yu D. Brousmiche P. Iraneta B. Alden E. Chambers J. Fournier G. Mantha B. Warren W. Gu K. Wyndham 2015 Waters Corporation 58

59 Thank You for Attending! Post-Event Home Page: 20% Introductory Offer GlycoWorks RapiFluor-MS N-Glycan Analysis Kits 30% Introductory Offer All Glycan Columns and our NEW ACQUITY UPLC Glycoprotein BEH Amide 300Å 1.7 µm Columns Full Webinar Recording w/pdf Slide Deck Compilation of KEY Literature, Brochures etc For Questions and to Submit your Ideas for our Next Topic Please - mychemrep@waters.com 2015 Waters Corporation 59