Current Glycoprotein Analysis. Glycan Characterization: Oligosaccharides. Glycan Analysis: Sample Preparation. Glycan Analysis: Chromatography

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1 Bio Day DENMARK MARCH 2013 Analysis of N-linked Glycans of GlycoProteins

2 Agenda Importance of Glycan Analysis Current Glycoprotein Analysis Glycan Characterization: Oligosaccharides Glycan Analysis: Sample Preparation Glycan Analysis: Chromatography Empower: GU units NIBRT Glycobase 3+ Summary 2013 Waters Corporation 2

3 Biopharmaceuticals Glycan Mixtures Monoclonal Antibody e.g. Herceptin EPO Insulin MW 6,000 MW 34,000 MW 147, Waters Corporation 3

4 Glycan Separation Technology 2013 Waters Corporation 4

5 Glycosylation Functions Risks and Regulatory Concerns Mediates biological activity Alteration may eliminate or alter activity Immune response triggered by unrecognized glycan Glycan distribution indicates process stability Glycans impact safety and efficacy Correct and consistent structure of the glycans Obtaining the desired medical effect Avoid adverse immunological reaction 2013 Waters Corporation 5

6 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 6

7 Isomeric Structures The basic carbohydrate units are called monosaccharides Hexose HexNAc D-(+)-Glucose D-(+)-Galactose D-(+)-Mannose D-(+)-Fucose DeoxHex Sialic Acid Sialic acid N-Acetyl-D-glucosamine N-Acetyl-D-galactosamine 2013 Waters Corporation 7

8 Glycan Analysis Biopharmaceutical Development Cycle Target ID Clone Selection Formulations & Discovery Characterization Stability Development Comparability Production PAT- Process Analytical Technologies Glycan Analysis QC/QA Post-Approval ID Test? Comparability 2013 Waters Corporation 8

9 Glycoprotein Characterization Requirements Selectivity for Key Glycans Sensitivity for Minor Glycoforms Reproducibility of Separation Quantitative Results Robust Complete Solution 2013 Waters Corporation 9

10 Agenda Importance of Glycan Analysis Current Glycoprotein Analysis Glycan Characterization: Oligosaccharides Glycan Analysis: Sample Preparation Glycan Analysis: Chromatography Empower: GU units NIBRT Glycobase 3+ Summary 2013 Waters Corporation 10

11 Glycoprotein Characterization Work Flows Glycoprotein MW Profile ESI MS Glycopeptides & Peptides N- Oligosachharides Peptide Mapping UPLC-UV UPLC-MS Label MALDI HILIC / Fluorescence Detection 2013 Waters Corporation 11

12 Glycoprotein Characterization Glycoprotein Galactose N-Acetyl glucosamine Mannose Fucose Glycoprotein 2013 Waters Corporation 12

13 Glycoprotein Characterization LC/MS analysis of Intact IgG G0F/G1F G1F/G1F G0F/G2F 0.5 µg IgG1 GOF/G0F G0/GOF G1F/G2F G2F/G2F Desalting Cartridge Deconvoluted Mass Spectrum IgG Waters Corporation 13

14 Glycoprotein Characterization Glycopeptide Analysis Glycoprotein Protease Digestion and Separation 6 Glycopeptides Galactose N-Acetyl glucosamine Mannose Fucose 2013 Waters Corporation 14

15 Antibody Glycopeptide Microheterogeneity IgG Glycopeptides 1.0e-2 8.0e-3 AU 6.0e-3 4.0e-3 UV 2.0e % TIC Time The UPLC chromatograms of tryptic digest of mouse IgG show resolution of the glycoforms Waters Corporation 15

16 IgG Glycopeptides UPLC MS and UV % G2F (M+H) +3 m/z = % G1F (M+H) +3 m/z = % G0F (M+H) +3 m/z = Time 1522 UV Waters Corporation 16

17 IgG Complex N-Glycans - Glycopeptides Galactose N-Acetyl glucosamine Mannose Fucose 2013 Waters Corporation 17

18 Glycopeptides with HILIC en 2013 Waters Corporation 18

19 Agenda Importance of Glycan Analysis Current Glycoprotein Analysis Glycan Characterization: Oligosaccharides Glycan Analysis: Sample Preparation Glycan Analysis: Chromatography Empower: GU units NIBRT Glycobase 3+ Summary 2013 Waters Corporation 19

20 Glycoprotein Characterization Glycan Analysis 6 3 Glycan Release Glycoprotein 6 3 Oligosaccharides Galactose N-Acetyl glucosamine Mannose Fucose 2013 Waters Corporation 20

21 Studying Glycans: The Workflow 1) Release glycans from the protein 2) Label the free glycans for detection 3) Separate a complex pool of glycans 4) Assign glycan structures to peak 2013 Waters Corporation 21

22 What does this look like at the bench? Glycoprotein Sample Identified Glycan Structure(s) Sample Preparation Glycan Separation Data Processing Data Interpretation Enzymatic Deglycosylation System equilibration Adjust Dextran Amounts GlycoBase 3.1 Free Glycan Capture Dextran Run Process Sample Set Tentative Assignments Glycan Dry Down Standard Run Calibrate Dextran MS or Enzyme Analysis? Formic Acid Treat Sample Set Run Sample Integration Glycan Dry Down Data Export 2AB Labeling Excess label removal Glycan Dry Down Reconstitution Where does Waters contribute? Waters Corporation 22

23 What Waters Offers for Glycan Analysis` Solid Phase Extraction Offerings Standards and Reagents Offerings Informatics Offerings Instrument Offerings Sample Preparation Glycan Separation Data Processing Data Interpretation Enzymatic Deglycosylation System equilibration Adjust Dextran Amounts GlycoBase 3.1 Free Glycan Capture Dextran Run Process Sample Set Analyst Defined General Lab Supplies Standard Run Calibrate Dextran Analyst Defined General Lab Supplies Sample Set Run Sample Integration General Lab Supplies Data Export 2AB Labeling Excess label removal General Lab Supplies General Lab Supplies 2013 Waters Corporation 23

24 Agenda Importance of Glycan Analysis Current Glycoprotein Analysis Glycan Characterization: Oligosaccharides Glycan Analysis: Sample Preparation Glycan Analysis: Chromatography Empower: GU units NIBRT Glycobase 3+ Summary 2013 Waters Corporation 24

25 Glycoanalysis Workflow Glycoprotein Denaturation Deglycosylation Free Glycans And Proteins RapiGest TM SF IAM DTT Isolation of Glycans GlycoWorks HILIC µelution Plate UPLC - FLR Isolation of Labeled Glycans Derivatize with 2-AB 2013 Waters Corporation 25

26 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 RapiGest SF is an acid-labile surfactant that meets these requirements 2013 Waters Corporation 26

27 The Journey of Complex Glycans (1.5 day sample prep. Process Recommended) Reduction/alkylation using DTT/IAM -Denature and unfold the protein Enzymatic removal of N-linked glycans - using PNGase F *In order to ensure complete deglycosylation, it is recommended to leave your sample overnight 1 hr Overnight* Adding 1% formic acid to the lyophilized glycans and set for 30 minutes - to convert glycans to free reducing form 40 min Lyophilization of the free glycans 1 hr Extraction of free glycans - using HILIC SPE device 30 min. Lyophilize the sample again FLR derivatization e.g., using 2AB (heating at 65 C for 2-3 hrs) 1 hr 30 min Extraction of the 2ABglycans - using HILIC SPE device 30 min HILIC-UPLC/FLR/MS analysis Lyophilize the sample 1 hr 2013 Waters Corporation 27

28 The Journey of Simplier Glycans (1 day sample prep. Process, 10 hrs) Reduction/alkylation using DTT/IAM -Denature and unfold the protein 1 hr Enzymatic removal of N-linked glycans - using PNGase F 2-4 hrs* * This step needs to be optimized to your protein and can be less or more Adding 1% formic acid to the lyophilized glycans and set for 30 minutes - to convert glycans to free reducing form 40 min Lyophilization of the free glycans 1 hr Extraction of free glycans - using HILIC SPE device 30 min. Lyophilize the sample again FLR derivatization e.g., using 2AB (heating at 65 C for 2-3 hrs) 1 hr 30 min Extraction of the 2ABglycans - using HILIC SPE device 30 min HILIC-UPLC/FLR/MS analysis Lyophilize the sample 1 hr 2013 Waters Corporation 28

29 2-AB (2-aminobenzamide aminobenzamide) Glycan Labeling 2013 Waters Corporation 29

30 What is in the GlycoWorks Kits? Sample clean-up devices - High-throughput: GlycoWorks HILIC micro-elution SPE plate for free glycan and labeled glycan extraction - Single Use: GlycoWorks HILIC 1cc Cartridge (10 pack) Protein denaturation - RapiGest SF (denaturation) - DTT and IAM (reductive/alkylation) FLR labeling chemistry - Acetic acid - DMSO - Reductive reagent, Sodium cyanoborohydride Standard - GlycoWorks Control Standard Additional Standards - Dextran Calibration Ladder (2AB-labeled) - Glycan Performance Test Standard (2AB-labeled) Care and Use with Protocol 2013 Waters Corporation 30

31 Conventional Dextran Ladder (supplied by Sigma or Prozyme) Dextran n Waters Corporation 31

32 Waters GlycoWorks Dextran Ladder Dextran n Minutes 2013 Waters Corporation 32

33 Waters GlycoWorks Glycan Performance Test Standard Based on pooled human serum IgG Spiked with M5 and M6 Certificate of Analysis using the ACQUITY BEH Glycan Column 3 (1) ACQUITY FLR ChA Ex330,Em420 nm Range: FLR TOF MS ES+ BPI 4.95e3 MS Time (minutes) 2013 Waters Corporation 33

34 New GlycoWorks Product Guide EN 2013 Waters Corporation 34

35 Agenda Importance of Glycan Analysis Current Glycoprotein Analysis Glycan Characterization: Oligosaccharides Glycan Analysis: Sample Preparation Glycan Analysis: Chromatography Empower: GU units NIBRT Glycobase 3+ Summary 2013 Waters Corporation 35

36 HILIC Hydrophilic-Interaction Chromatography Reversed-Phase Mode Mid range hydrophilic to hydrophobic compounds Hydrophobic stationary phase Polar Solvents Aqueous to organic gradient Normal Phase Mode Hydrophobic compounds Hydrophilic stationary phase Non-polar solvents Organic to organic gradient HILIC mode Hydrophilic compounds Hydrophilic stationary phase Polar Solvents Organic to aqueous gradient 2013 Waters Corporation 36

37 HILIC Retention Mechanisms Combination of partitioning and hydrogen bonding Polar analyte partitions between bulk mobile phase and the immobilized water layer Hydrogen bonding between the analyte and amide hydrophilic surface 2013 Waters Corporation 37

38 HILIC - HPLC 2-AB Labeled Murine IgG N-Glycans Vicam IgG Gly_tos _12a G0F G1F Amide Column ACQUITY FLR ChA Ex330,Em420 nm 2 x 100 Range: mm 5 µm FL EU EU x 10e G2F Time Time (minutes) Waters Corporation 38

39 ACQUITY UPLC BEH Glycan Column Chemistry BEH Particle Ligand type: Trifunctional Amide BEH Particle size: 1.7 µm Endcap style: None Recommended ph range from 2 to Waters Corporation 39

40 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 40

41 ACQUITY UPLC BEH Glycan Column Certificate of Analysis Chemical Tests Chromatographic Test with Glycan Performance Test Standard Individual Column Tests 2013 Waters Corporation 41

42 Waters Glycan Separation Technology 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 1.7 µm Waters BEH Glycan 2.1 mm x 150 mm 30 min method Retention Time (min) Retention Time (min) 2013 Waters Corporation

43 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 43

44 ACQUITY UPLC Conditions Column: ACQUITY UPLC BEH Glycan 1.7µm, 2.1 x 150 mm Eluent A: 100 mm Ammonium Formate, ph 4.5 Eluent B: Acetonitrile Temperature: 60 C Fluorescence: λex = 330 nm, λem = 420 nm Sample amount: 15 pmol UPLC Gradient: Time Flow rate (min) (ml/min) % A % B Init * * *Flow rate lowered during aqueous regeneration 2013 Waters Corporation 44

45 Neutrals and Charged Structures are Separated in a Single Injection Afucosylated Fucosylated Sialylated High Mannose Structures Terminal Galactose EU 30.0 size Retention Time (min) 2013 Waters Corporation 45

46 Why choose GlycoWorks and UPLC? Looked at the FLR/MS chromatogram. 2AB-labeled Etanercept N-Glycan profile (released from 4 µg of protein) FLR MS ACQUITY UPLC H-class/FLR/Xevo G2-S QTOF MS 2013 Waters Corporation 46

47 Agenda Importance of Glycan Analysis Current Glycoprotein Analysis Glycan Characterization: Oligosaccharides Glycan Analysis: Sample Preparation Glycan Analysis: Chromatography Empower & GU units NIBRT Glycobase 3+ Summary 2013 Waters Corporation 47

48 Why Empower and GU Values? GU Value Human IgG1 Waters H-Class 1.7 µm BEH Glycan (2.1 mm x 250 mm) FLR detection (λ ex 330 nm, λ em 420 nm) 53% to 70% Linear gradient (30 min) Solvent A: 50 mm NH4 + HCOO - Solvent B: Acetonitrile EU ? 5.0 Glycoprotein X EU ?????????????????????????????? RetentionTime (min) 2013 Waters Corporation 48

49 GU Values Provide and Orthogonal Approach to Characterization Prevalent structural isomers makes MS of glycans challenging Structure Comp Fuc 1 Hex 6 HexNAc 5 NeuAc 2 Fuc 1 Hex 6 HexNAc 5 NeuAc 2 Fuc 1 Hex 6 HexNAc 5 NeuAc 2 m/z GU ~10.2 ~11.1 ~10.6 Risk? None Immunogenic anaphylaxis? 2013 Waters Corporation 49

50 Overall Workflow Glycoprotein Sample Known Glycans Unknown Glycans Glycan Profiling Structural Characterization Empower Automation GU Value Generation GlycoBase Interrogation Structure Confirmation (MS and Enzyme Arrays) 2013 Waters Corporation 50

51 Glycoprotein Sample Known Glycans Unknown Glycans Glycan Profiling Structural Characterization Empower Automation GU Value Generation Report Generation GlycoBase Interrogation Structure Confirmation (MS and Enzyme Arrays) 2013 Waters Corporation 51

52 GU Values Aid Dramatically in Identifying Glycan Structures GU Value EU Retention Time (min) 2013 Waters Corporation 52

53 GU Values Aid Dramatically in Identifying Glycan Structures Sialylated Neutral Phosphorylated! No Mass Spec Used for Glycan Characterization! 2013 Waters Corporation 53

54 Brief Summary Empower and GU values 1) Ensure GPC option is installed in Empower 2) GU custom calculation needs be added to your project custom fields 3) Dextran ladder standard is required to calibrate GU calculation 2013 Waters Corporation 54

55 Integrate the Dextran Peaks 2013 Waters Corporation 55

56 Select Process Calibrate: Retention Times Shift to GU Values 2013 Waters Corporation 56

57 For Samples, Integrate Peaks of Interest EU EU Minutes 2013 Waters Corporation 57

58 Select Process Quantitate Quantitate: Retention Times Shift to GU Values EU EU Minutes 2013 Waters Corporation 58

59 Agenda Importance of Glycan Analysis Current Glycoprotein Analysis Glycan Characterization: Oligosaccharides Glycan Analysis: Sample Preparation Glycan Analysis: Chromatography Empower: GU units NIBRT Glycobase 3+ Summary 2013 Waters Corporation 59

60 Waters Corporation 60

61 Where to next? Database interrogation 2013 Waters Corporation 61

62 Search Database Using GU Value 2013 Waters Corporation 62

63 Interesting Application Notes/posters on Glycan Analysis 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 Procaïnamide labeled N-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 Unify en Type green reference in search box on Waters Corporation 63

64 Summary Waters provides you with a total solution from sample handling, UPLC separation, data workflow and interpretation GlycoWorks provides you a easy to use sample handling workflow in a complete kit 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 Empower provides the necessary tools for calculations and calibrations Waters cooperation with NIBRT : Glycobase database for glycan interpretation and trainings 2013 Waters Corporation 64

65 2013 Waters Corporation 65

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