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1 Thank you for joining us! Our session will begin shortly Strategies for Successful Characterization of N-Linked Oligosaccharides: Using HILIC Separation Method and the New GlycoWorks Sample Preparation Approach Stephan M. Koza, Ph. D. Waters Corporation June 5, Waters Corporation 1

2 Friendly Reminders Please use text chat functionality to submit your questions today. Jennifer Fournier, Waters Corp. LIVE Technical support during today s event Upon conclusion, follow up information will be available: Recorded version of today s presentation Copies of today s slides Product discount offers Product specific information Reference materials 2013 Waters Corporation 2

3 Agenda: Glycan Analysis Webinar ointroduction to Glycans oprotein Glycosylation oimportance of Monitoring Protein Glycosylation osample Preparation oglycoworks oprocedure osupporting Data osample Analysis ohilic otheory ogst Column Solutions ocharacterization Strategies oglycobase oexoglycosidases oms/ms ohilic for Glycopeptide Analysis oreferences and Summary 2013 Waters Corporation 3

4 Biopharmaceuticals and Glycosylation Glycans Bound to Proteins Monoclonal Antibody e.g. Herceptin EPO Insulin MW 6,000 MW 34,000 MW 147, Waters Corporation 4

5 Oligosaccharide Structures N-linked and 0-Linked O-linked glycosylation to the hydroxy Oxygen of serine or threonine side chains N-linked glycosylation to the amide Nitrogen of asparagine side chains 2013 Waters Corporation 5

6 How Are Proteins Glycosylated? ER Golgi An enzymatically driven process with many enzymes involved 2013 Waters Corporation 6

7 N-linked glycan biosynthesis is a highly complex process 2013 Waters Corporation Struwe WB, Cosgrave EFJ, and Rudd PM. (2011). Glycoproteomics in Health and Disease. Functional and Structural Proteomics of Glycoproteins. 7

8 Glycosylation plays a critical role in biology Nearly 50% of all proteins are Glycosylated. So what do these glycans do? N-linked glycans play a role in Protein folding Cell-cell communication Biological activity Protein half-life Cell attachment IgG-FcγRIIIa Interaction PDB file 3SGJ 2013 Waters Corporation 8

9 What Does This Mean? Heterogeneity of N-Linked Structures High Mannose Complex Hybrid 2013 Waters Corporation 9

10 Glycosylation Functions: Risks and Regulatory Concerns Mediates biological activity Impact safety and efficacy Adverse immunological reaction Modulate PK Consistent glycan distribution indicates process stability 2013 Waters Corporation 10

11 Glycan Separation Technology Sample Preparation Informatics UPLC and HPLC Separations Chemistry Mass Spectrometry 2013 Waters Corporation 11

12 Agenda: Glycan Analysis Webinar ointroduction to Glycans oprotein Glycosylation oimportance of Monitoring Protein Glycosylation osample Preparation oglycoworks oprocedure osupporting Data osample Analysis ohilic otheory ogst Column Solutions ocharacterization Strategies oglycobase oexoglycosidases oms/ms ohilic for Glycopeptide Analysis oreferences and Summary 2013 Waters Corporation 12

13 GlycoWorks TM 2013 Waters Corporation 13

14 Enzymatic Deglycosylation Considerations Reproducible and complete release Conditions maintain glycanase enzyme activity Conditions denature the protein to enhance glycan cleavage Conditions keep the protein in solution N-Linked Glycans of IgG Fc Domain RapiGest SF is an acid-labile surfactant that meets these requirements 2013 Waters Corporation 14

15 The Chemistry of GlycoWorks TM N-Glycan Protein PNGase F Released N-Glycan (Glycosylamine) + Protein HILIC SPE 2013 Waters Corporation 15

16 The Chemistry of GlycoWorks TM Mild Acid Hydrolysis N-Glycan (Glycosylamine) N-Glycan ( Acyclic Free Reducing End) 2013 Waters Corporation 16

17 The Chemistry of GlycoWorks TM + N-Glycan (Free Reducing End) HOAc DMSO 2-AB HILIC SPE NaBH 3 CN Reduction N-Glycan (2-AB Labeled) 2013 Waters Corporation 17

18 SPE Optimization 2013 Waters Corporation 18

19 HILIC Hydrophilic-Interaction Chromatography Reversed-Phase Mode Hydrophilic compounds Hydrophobic stationary phase Polar Solvents Aqueous to organic gradient HILIC mode Hydrophilic compounds Hydrophilic stationary phase Polar Solvents Organic to aqueous gradient 2013 Waters Corporation 19

20 HILIC Retention Mechanisms Stationary Phase Combination of partitioning and hydrogen bonding Polar analyte partitions between bulk organic mobile phase and the immobilized water layer Hydrogen bonding between the analyte and hydrophilic surface 2013 Waters Corporation 20

21 Optimizing Recovery for a Diverse Set of N-Linked Glycans Glycan Performance Test Standard 1x +A3 (Trisialylated) Glycans 1 2 Peak 2-AB Labeled Glycan 1 G0-GN 2 G0 3 G0F 4 Man5 5 G0FN 6 G1F 7 G1F 8 G1FN 9 Man6 10 G2 11 G2F 12 G2FN 13 G1FS1 14 G2FS1 15 A3 16 A x , ,16 Neutral Glycans Acidic Glycans Nature 446, (26 April 2007) 2013 Waters Corporation 21

22 Elution Optimization for Quantitative Recovery of 2-AB Labeled N-Glycans 20% ACN Varying [NH 4 HCO 3 ] 25 mm NH 4 HCO 3 Varying %ACN 5% ACN Varying [NH 4 OAc] Lyophilization Control 100 mm NH 4 HCO Lyophilization Control 5% ACN Lyophilization Control 25 mm NH 4 HCO mm NH 4 OAc % Reco overy Neutral Monosialylated 25 mm NH 4 HCO 3 % Reco overy 10 mm NH 4 HCO % ACN 20% ACN % Reco overy mm NH 4 OAc 10 mm NH 4 OAc 20 0 mm NH 4 HCO Trisialylated Experimental GU Experimental GU Experimental GU 2013 Waters Corporation 22

23 GlycoWorks HILIC SPE with the optimized elution conditions A Control Before SPE C 20 Control Before SPE Control Lyophilization Dried Control HILIC SPE Processed 5% ACN, 100 Elution mm with 100 mm NH4OAc 4 OAc, 5% ACN B GlycoWorks HILIC SPE Processed Elution with 100 mm NH 4 OAc, 5% ACN % Abundance Peak 3 G0F Peak 16 A Waters Corporation 23

24 Assessing the Quantitative Recovery of Unlabeled Glycans on HILIC SPE 8000 Intensity Intensity Man5 A3 Unlabeled A Unlabeled Control Pos Lyophilization Control Dried Control HILIC SPE Processed HILIC Elution with 100 mm Unlabeled NH 4 OAc, 5%ACN MS Inten nsity Unlabeled Man5 m/z % Abund dance min 0 Man5 A3 (XIC, m/z) 2013 Waters Corporation 24

25 Robustness Testing of the SPE Elution Conditions Protocol Elution (100 mm NH4OAC, 5.0% ACN) Elution with 110 mm NH4OAC, 4.5% ACN Elution with 90 mm NH4OAc, 5.5% ACN % Abun ndance Peak 1 G0-GN Peak 2 G0 Peak 3 G0F Peak 4 Man5 Peak 5 G0FN Peak 6 G1F Peak 7 G1F Peak 8 G1FN Peak 9 Man6 Peak 10 G2 Peak 11 G2F Peak 12 G2FN Peak 13 G1FS1 Peak 14 G2FS1 Peak 15 A3 Peak 16 A Waters Corporation 25

26 Adaptability of HILIC SPE for Processing Varied Quantities of Glycoprotein 96 Well µelution Plate 25 µg IgG processed 125 µl reconstitution 4 µl injection 96 Well µelution Plate 2.5 µg IgG processed 12.5 µl reconstitution 4 µl injection 2013 Waters Corporation 26

27 Sample Prep Flexibility: Comparison of 96-well µelution Plate and a Single Use Cartridge 96 Well µelution Plate 5 mg sorbent 25 µg IgG processed 125 µl reconstitution 4 µl injection 1 2 3, Cartridge 96 Well µelution Plate Single-Use 1cc Cartridge 96 Well Plate Manose Galactose Sialic Acid Fucose N-Acetylglucosamine Single-Use 1cc Cartridge 10 mg sorbent 50 µg IgG processed 250 µl reconstitution 4 µl injection % Abundan nce Peak 1 G0F Peak 2 G0FN Peak 3 G1F Peak 4 G1F Peak 5 G1FN Peak 6 G2F Peak 7 G2FN Peak 8 Peak 9 G1FS1 G2FS Waters Corporation 27

28 GlycoWorks Digestion/Labeling Protocol 2013 Waters Corporation 28

29 Agenda: Glycan Analysis Webinar ointroduction to Glycans oprotein Glycosylation oimportance of Monitoring Protein Glycosylation osample Preparation oglycoworks oprocedure osupporting Data osample Analysis ohilic otheory ogst Column Solutions ocharacterization Strategies oglycobase oexoglycosidases oms/ms ohilic for Glycopeptide Analysis oreferences and Summary 2013 Waters Corporation 29

30 Developed, Optimized, and Tested For Glycan Analysis ACQUITY UPLC BEH Glycan Column HILIC mode separation of carbohydrates Amide bonded phase Stable BEH Particles 1.7 µm Diameter Particles Optimized for use on ACQUITY UPLC System with fluorescence detection Quality Control tested with 2-AB human IgG glycan standards 2013 Waters Corporation 30

31 UPLC of Labeled Glycans Improved resolution and quantitative assays For typical IgG glycans o G0, G0F, G1, G1F, G1F+SA, G2, G2F, G2F+SA, Man5 For neutral glycans and mono-sialylated to penta-sialyated glycans Faster Analyses Column-to-column reproducibility No detectable carryover 2013 Waters Corporation 31

32 UPLC Technology for Glycan Solution Improved Analysis of Labeled N-Glycans Improved resolution Improved analysis time Solution Components ACQUITY UPLC System ACQUITY UPLC FLR Detector ACQUITY UPLC BEH Glycan Column 2013 Waters Corporation 32

33 Ethylene Bridged Hybrid (BEH) Particles Technology Bridged Ethanes within a silica matrix U.S. Patent No. 6,686,035 B Waters Corporation Anal. Chem. 2003, 75,

34 ACQUITY UPLC BEH Glycan Column Chemistry BEH Particle Ligand type: Trifunctional Amide BEH Particle size: 1.7 µm Endcap style: None Recommended ph range: 2 to Waters Corporation 34

35 HILIC Retention Mechanisms 2013 Waters Corporation 35

36 Waters UPLC Glycan Separation Technology Provide Outstanding Glycan Profile Resolutions 5 µm Tosoh TSKgel Amide mm x 250 mm 3 h method 3 µm Tosoh TSKgel Amide mm x 150 mm 1 h method 1.7 µm Waters BEH Glycan 2.1 mm x 150 mm 30 min method Zoomed View Retention Time (min) Retention Time (min) Waters Corporation 36

37 Comparison to Mixed-Mode Mode Glycan Column ACQUITY BEH Glycan 1.7 µm (2.1 X 150 mm, 0.50 ml/min ) , , min Mixed-Mode Glycan Column, 1.9 µm (2.1 X 150 mm, 0.40 ml/min) 2013 Waters Corporation 37

38 Effect of Temperature on Retention and Selectivity (2-AB IgG Glycans) EU x 10e EU C EU x 10e4 EU C EU x 10e4 EU C EU x 10e4 EU C Time 2013 Waters Corporation 38

39 Effect of Temperature Backpressure ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm C psi C Time 2013 Waters Corporation 39

40 Effect of Ionic Strength (Ammonium Formate) on Retention and Selectivity Acidic Glycans Ammonium Formate 250 mm mm EU x 10e mm 25 mm mm Time Waters Corporation 40

41 ph Effects Ammonium Formate vs. Formic Acid Acidic Glycans EU x 10e mm Ammonium Formate EU x 10e % Formic Acid Time Waters Corporation 41

42 Glycan Separation Effect of Column Length G1F Isomers Greater Resolution (QC) Higher Throughput (DEVELOPMENT) 2013 Waters Corporation 42

43 UPLC Separation of Human IgG N-linked 2-AB Labeled Glycans Using BEH Glycan Column EU x 10 0e size 13 1 G0 2 G0F 3 Man5 4 G0FGN 5 G1 6 G1Fa 7 G1Fb 8 G1FGN 9 Man6 10 G2 11 G2F 12 G1F+SA 13 G2F+SA 0 Time ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm A: 100mM ammonium formate ph 4.5 B: Acetonitrile 75% B to 60% B over 46.5 mins, 0.5mL/min, 60 C Fluorescence: λex = 330 nm, λem = 420 nm 2013 Waters Corporation 43

44 Disialo-Biantennary Glycans on ACQUITY UPLC BEH Glycan Column (2.1 x 150 mm) Bovine AGP G,N G,G Prozyme N-linked IgG Rapid AB std 1pmol/uL 2 ulb 20 min 16Jan12_Glycan_MS_3_4 (1) ACQUITY FLR ChA Ex278,Em344 nm Range: N,N EU x 10e N= NeuAc G= NeuGc Time NeuAc: (N-acetyl-neuraminic acid), NeuGc: (N-glycolylneuraminic acid) 2013 Waters Corporation 44

45 ACQUITY UPLC BEH Glycan Column Certificate of Analysis Chemical Tests Chromatographic Test with Glycan Performance Test Standard Individual Column Tests 2013 Waters Corporation 45

46 Batch-to-Batch Reproducibility of ACQUITY UPLC BEH Glycan Material Using 2-AB Labeled Human IgG N-Linked Glycans Batch 1 Batch 2 Batch 3 Batch 4 2AB Labeled Glycan Standard - Ref Waters Corporation 46

47 BEH Glycan 2-AB Labeled N-Glycans Method Scaling 1.7, 2.5, and 3.5 µm Particle Sizes (2.1 mm X 150 mm) Peak 8= ACQUITY BEH Glycan 1.7 µm P c * half-height = ml/min RT 1,16 = 20.2 min psi (Column, Max) 16 Peak 9= 5.0 XBRIDGE BEH AMIDE 2.5 µm XP P c * half-height = ml/min RT 1,16 = 30.2 min 40.0 min 4600 psi (Column, Max) 7.4 XBRIDGE BEH AMIDE 3.5 µm P c * half-height = ml/min RT 1,16 = 42.1 min 58.8 min 1800 psi (Column, Max) min o Scaling method for fraction collection of low abundance glycans o LC System Limitations o Longer Run Times and Lower Resolution 2013 Waters Corporation 47

48 Relative Abundance Determinations , , um, 0.50 ml/min) um XP, 0.34 ml/min % Abun ndance um, 0.24 ml/min Peak 1 G0-GN Peak 2 G0 Peak 3 G0F Peak 4 Man5 Peak 5 G0FN Peak 6 G1F Peak 7 G1F Peak 8 G1FN Peak 9 Man6 Peak 10 G2 Peak 11 G2F Peak 12 G2FN Peak 13 G1FS1 Peak 14 G2FS1 n=3 Peak 15 A3 Peak 16 A3 *Peaks 8 and µm resolution insufficient, accuracy of the integration is poor 2013 Waters Corporation 48

49 Agenda: Glycan Analysis Webinar ointroduction to Glycans oprotein Glycosylation oimportance of Monitoring Protein Glycosylation osample Preparation oglycoworks oprocedure osupporting Data osample Analysis ohilic otheory ogst Column Solutions ocharacterization Strategies oglycobase oexoglycosidases oms/ms ohilic for Glycopeptide Analysis o References and Summary 2013 Waters Corporation 49

50 The Glucose Unit Concept An abstract idea used for the expression of HILIC retention times: Corrects for instrumental variance, Depends upon the interaction of the glycan with the stationary phase, Allows for the prediction of monosaccharide shifts. Fluorescence GU = A2G(4)S Minutes 2013 Waters Corporation 50

51 UPLC-FLR Released analysis complements mass data for glycan characterization 2AB Dextran Standard (GU) HILIC UPLC-FLR Fluorescence Released 2AB Glycans from Biotherapeutic GU HPLC UPLC Empower 3.0 UNIFI 1.6 GU Search 2013 Waters Corporation 51

52 Exoglycosidase Arrays Aid in Determining Glycan Sequence JBM BTG SPG ABS BKF GUH NAN1 AMF CBG 2013 Waters Corporation 52

53 Herceptin (Trastuzumab) N-linked Glycan Structural Assignment using GlycoBase 3.0+ Source: Dr. Mark Hilliard, NIBRT, WCBP Waters Corporation 53

54 Acquire Both FLR and MS Chromatograms From the Same Injection HILIC-UPLC/FLR/QTof MS (2AB-labeling) G0F (1) ACQUITY FLR ChA Ex330,Em420 nm Range: EU x 10e G1Fa FLR FLR G0 - GN G0F - GN G0 Man n5 G1a G1b Man6 G1 1Fb G2 G2F Man7 G2FS1 Man8 G2FS : TOF MS ES+ BPI 2.15e3 G1F - GN BPI MS MS % 0 Time (min.) Waters Application Note: en Trastuzumab Glycan Batch-to-Batch Profiling Using a UPLC/FLR/Mass Spectrometry Platform 2013 Waters Corporation 54

55 MS/MS of 2AB-labeled FIX Glycans 4A/1F/4S x rfix 4: TOF MSMS ES+ 2.15e3 Tag (M+2H) x pd-fix 7: TOF MSMS ES m/z Ying Qing Yu, Analysis of N-Linked Glycans from Coagulation Factor IX, Recombinant and Plasma Derived, Using HILIC UPLC/FLR/QTof MS ( en ) 2013 Waters Corporation 55

56 Separation of Isobaric Oligosaccharide Isomers by IMS Using MALDI SYNAPT G2 HDMS Extracted Mobilograms of Isomers Difucosyl-lacto-Nhexaose 1:1 Mixture Difucosyl-para-lacto- N-hexaose Experimental Conditions Glycans - stock solutions at 0.2 mg/ml DHB (2,5- Dihydroxybenzoic acid) at 20mg/mL in ethanol was used as the matrix Experiments were run on an MALDI Synapt G2, equipped with a 200 Hz laser 2013 Waters Corporation 56

57 MS/MS Spectra From the Front and Tailing Side of the IMS Peak Using MALDI SYNAPT G2 HDMS Mass Spectra of Selected Regions from DriftScope plot can be displayed Difucosyl-lacto-Nhexaose Difucosyl-para-lacto-Nhexaose 2013 Waters Corporation 57

58 HILIC Separation of Glycopeptides HILIC mode (Hydrophilic-Interaction Chromatography) For polar compounds Hydrophilic stationary phase Organic to aqueous gradient Example column: ACQUITY UPLC BEH Glycan Column Improves resolution of glycopeptides from non-glycosylated peptides Improves resolution of microheterogeneity (same peptide with different glycans attached) 2013 Waters Corporation 58

59 mab Tryptic Digest UPLC Separation in HILIC Mode Using BEH Glycan Column ACQUITY UPLC BEH Glycan Column, 1.7um, 2.1x150mm 40 C, 0.2 ml/min A: 10mM ammonium formate ph 4.5 B: 10mM ammonium formate ph 4.5 in 90:10 ACN:water 90% to 55% B in 45 mins Glycopeptides (4x magnified) completely separation from non-glycosylated peptides Glycopeptides, both N-linked and O-linked, are resolved from non-glycosylated peptides using the BEH Glycan Column 2013 Waters Corporation 59

60 Combined MS XIC of mab Tryptic Digest in HILIC Mode (BEH Glycan Column) Improved resolution of different glycoforms of EEQYNSTYR 2013 Waters Corporation 60

61 Using NTS to determine which amino acid is glycosylated NxS/T motif Protein Sci October; 10(10): Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1, Bernhard A. Hofmann, 1 Sabine Sydow, 1,2 Olaf Jahn, 1 Lars Van Werven, 1 Thomas Liepold, 1 Klaus Eckart, 1 and Joachim Spiess Waters Corporation 61

62 Using MS to find which amino acid is glycosylated N-linked glycans CID doesn t reveal which amino acid is glycosylated, but... Well known NxS/T motif! MS identification of glycopeptide may be sufficient O-linked glycans May be multiple S or T residues in a glycopeptide in the peptide map ETD best technique for identifying site occupancy ETD of T14 peptide from etanercept Spectra confirms sites of glycosylation 2013 Waters Corporation 62

63 Agenda: Glycan Analysis Webinar ointroduction to Glycans oprotein Glycosylation oimportance of Monitoring Protein Glycosylation osample Preparation oglycoworks oprocedure osupporting Data osample Analysis ohilic otheory ogst Column Solutions ocharacterization Strategies oglycobase oexoglycosidases oms/ms ohilic for Glycopeptide Analysis o References and Summary 2013 Waters Corporation 63

64 Interesting Application Notes/posters on Glycan Analysis Optimization of GlycoWorks HILIC SPE for the Quantitative and Robust Recovery of N-Linked Glycans from mab-type Samples eN Single-Use and High-Throughput HILIC SPE Device Formats and an IgG Control Standard for Facilitating N-Glycan Analyses eN A new column for improved resolution for glycan analysis App Note en Batch to Batch Profiling using UPLC/FLR/MS App Note en Method Development of 2AB labeled Glycans en Analysis of N-linked Glycans from Coag Factor IX en Usage of UPLC of 2AB labeled Fetuin Glycans Removed by Exoglycosidase eN A Holistic Workflow for Acquiring, Processing, and Reporting Fluorescent- Labeled Glycans w Unifi en Type green reference in search box on Waters Corporation 64

65 Summary Waters provides you with a total solution from sample handling, UPLC separation, data workflow and interpretation GlycoWorks provides you easy to use and reproducible sample handling workflows (Plate and Single-Use) in complete kits from one single vendor (offering flexibility by allowing the customer to chose enzyme and fluorescent label) Glycan Separation Technology Columns provide you high resolution separation, with consistent batch to batch performance Waters mass spectrometers and informatics solutions provide the necessary tools for both efficient and thorough glycan characterization and reliable quality control analyses 2013 Waters Corporation 65

66 Thank You! Questions? Landing Page Promotional Offers on Glycan Columns and GlycoWorks Sample Prep Kit Full Webinar Recording of Today s Session PDF Slide Deck Compilation of KEY Application Notes, Literature, White Papers, Brochures etc Questions and Submit your Ideas for our Next Topic mychemrep@waters.com 2013 Waters Corporation 66