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

Bio Day DENMARK MARCH 2013 Analysis of N-linked Glycans of GlycoProteins marleen_van_wingerden@waters.com

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

Biopharmaceuticals Glycan Mixtures Monoclonal Antibody e.g. Herceptin EPO Insulin MW 6,000 MW 34,000 MW 147,000 2013 Waters Corporation 3

Glycan Separation Technology 2013 Waters Corporation 4

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

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

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

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

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

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

Glycoprotein Characterization Glycoprotein 6 3 6 3 Galactose N-Acetyl glucosamine Mannose Fucose Glycoprotein 2013 Waters Corporation 12

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 IgG1 2013 Waters Corporation 13

Glycoprotein Characterization Glycopeptide Analysis 6 3 6 3 Glycoprotein Protease Digestion and Separation 6 Glycopeptides 6 3 3 Galactose N-Acetyl glucosamine Mannose Fucose 2013 Waters Corporation 14

Antibody Glycopeptide Microheterogeneity IgG Glycopeptides 1.0e-2 8.0e-3 AU 6.0e-3 4.0e-3 UV 2.0e-3 0.0 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 93 % TIC -7 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 Time The UPLC chromatograms of tryptic digest of mouse IgG show resolution of the glycoforms. 2013 Waters Corporation 15

IgG Glycopeptides UPLC MS and UV % G2F (M+H) +3 m/z =976.02 2 47.40 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00 % G1F (M+H) +3 m/z =922.00 1 47.40 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00 % G0F (M+H) +3 m/z =867.99 1 47.40 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.00 Time 1522 UV 214 918 825 575 47.60 47.80 48.00 48.20 48.40 48.60 48.80 49.0 2013 Waters Corporation 16

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

Glycopeptides with HILIC 720003363en 2013 Waters Corporation 18

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

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

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?... 2013 Waters Corporation 22

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

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

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

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

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

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

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

Conventional Dextran Ladder (supplied by Sigma or Prozyme) 10000 Dextran n 50000 60000 70000 80000 90000 100000 110000 120000 130000 140000 150000 40000 30000 20000 2013 Waters Corporation 31

Waters GlycoWorks Dextran Ladder Dextran 8 9 10 11 12 13 14 15 16 17 18 19 7 20 n 6 5 4 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 Minutes 2013 Waters Corporation 32

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: 184647 8 13 FLR 4 1 2 5 6 7 9 10 11 12 14 15 16 17 18 19 TOF MS ES+ BPI 4.95e3 MS 10 15 20 25 30 Time (minutes) 2013 Waters Corporation 33

New GlycoWorks Product Guide 720004584EN 2013 Waters Corporation 34

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

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

HILIC - HPLC 2-AB Labeled Murine IgG N-Glycans Vicam IgG Gly_tos 012308_12a 12000.001 11000.001 G0F G1F Amide Column ACQUITY FLR ChA Ex330,Em420 nm 2 x 100 Range: 12686 mm 5 µm 10000.001 9000.001 8000.000 FL EU EU x 10e4 7000.000 6000.000 5000.000 G2F 4000.000 3000.000 2000.000 1000.000 0.000 Time 48.00 49.00 50.00 51.00 52.00 53.00 54.00 55.00 56.00 57.00 58.00 59.00 60.00 61.00 62.00 63.00 64.00 65.00 Time (minutes) 65.0 2013 Waters Corporation 38

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 11 2013 Waters Corporation 39

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

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

Waters Glycan Separation Technology 5 µm Tosoh TSKgel Amide-80 4.6 mm x 250 mm 3 h method 3 µm Tosoh TSKgel Amide-80 4.6 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) 15 30 60 Retention Time (min) 2013 Waters Corporation 42 180

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 186006349 2013 Waters Corporation 43

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 0.5 25 75 46.5 0.5 40 60 48 0.25* 100 0 49 0.25* 100 0 50 0.5 25 75 63 0.5 25 75 *Flow rate lowered during aqueous regeneration 2013 Waters Corporation 44

Neutrals and Charged Structures are Separated in a Single Injection 50.0 40.0 Afucosylated Fucosylated Sialylated High Mannose Structures Terminal Galactose EU 30.0 size 20.0 10.0 0.0 2 4 6 8 10 12 14 Retention Time (min) 2013 Waters Corporation 45

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

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

Why Empower and GU Values? GU Value 3 4 5 6 7 8 9 10 11 12 13 14 8.0 6.0 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 4.0 2.0 0.0? 5.0 Glycoprotein X EU 4.0 3.0 2.0 1.0?????????????????????????????? 0.0 2 4 6 8 10 12 14 16 18 20 RetentionTime (min) 2013 Waters Corporation 48

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 2733.9729 2733.9729 2733.9729 GU ~10.2 ~11.1 ~10.6 Risk? None Immunogenic anaphylaxis? 2013 Waters Corporation 49

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

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

GU Values Aid Dramatically in Identifying Glycan Structures GU Value 4 5 6 7 8 9 10 11 4.0 11 3.5 3.0 2.5 23 EU 2.0 2 1.5 1.0 0.5 1 3 5 9 10 7 4 68 12 13 16 14 15 17 18 19 20 21 22 25 24 26 29 34 27 28 30 33 38 3536 31 37 39 42 43 32 40 41 44 45 0.0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Retention Time (min) 2013 Waters Corporation 52

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

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

Integrate the Dextran Peaks 2013 Waters Corporation 55

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

For Samples, Integrate Peaks of Interest EU 5.584 8.832 12.215 15.384 18.235 20.776 23.027 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 9.904 10.370 13.220 16.237 12.933 15.032 15.271 15.557 17.253 18.075 18.958 EU 19.892 12.498 14.484 11.733 14.132 16.665 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes 2013 Waters Corporation 57

Select Process Quantitate Quantitate: Retention Times Shift to GU Values EU 40000 50000 60000 70000 80000 90000 100000 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 3.5 53538 54904 57183 58917 63387 65565 62514 68005 69069 69845 70781 73053 76567 78715 78203 79529 82842 87367 89266 98516 EU 86500 61199 67317 66208 74512 3.0 2.5 2.0 1.5 1.0 0.5 0.0 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Minutes 2013 Waters Corporation 58

www.waters.com/glycans 2013 Waters Corporation 60

Where to next? Database interrogation 2013 Waters Corporation 61

Search Database Using GU Value 2013 Waters Corporation 62

Interesting Application Notes/posters on Glycan Analysis A new column for improved resolution for glycan analysis App Note 720003112en Batch to Batch Profiling using UPLC/FLR/MS App Note 720003576en Method Development of 2AB labeled Glycans 720003238en Analysis of Procaïnamide labeled N-Glycans 720004212en Analysis of N-linked Glycans from Coag Factor IX 720004019en Usage of UPLC of 2AB labeled Fetuin Glycans Removed by Exoglycosidase 720003865EN A Holistic Workflow for Acquiring, Processing, and Reporting Fluorescent-Labeled Glycans w Unify 720004619en Type green reference in search box on www.waters.com 2013 Waters Corporation 63

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

2013 Waters Corporation 65