The Road to Glycan Analysis Without Compromise WCBP 2015 Waters Technical Seminar Jan 27, 2015 Washington, DC 2015 Waters Corporation 1
Today s Agenda Overview and Introduction RapiFluor-MS N-Glycan Labeling: A breakthrough technology for released glycan LC and MS analysis Matthew A. Lauber (Consumables Business Unit, Waters) RapiFluor-MS Technology & Glycan Characterization Ying Qing Yu (Biopharmaceutical Sciences, Waters) Impact of RapiFluor-MS Technology on Released Glycan Profile Monitoring Sean M. McCarthy (Biopharmaceutical Sciences, Waters) Scientific Panel Questions & Discussion 2015 Waters Corporation 2
Glycosylation of Biotherapeutics Fucose GlcNAc Mannose Effector Functions (ADCC/CDC) (fucosylation/galactosylation) Low Half Life (high mannose) Anti-Inflammatory (sialylation) Immunogenic (αgal / N-glycolylneuraminic acid) Overall profile sensitive to manufacturing conditions Galactose N-acetylneuraminic acid N-glycolylneuraminic acid N-glycosylation is a quality attribute of biotherapeutics Glycosylation profiles are characterized and routinely monitored Anal Chem 2013, 85 (2), 715-36. 2015 Waters Corporation 3
Glycoprotein Characterization Multiple Strategies Complementary Information 2015 Waters Corporation 4
Customer voice brought us focus More Important Less Important Just compatible with UV detection Just compatible with Fluorescence Just compatible with MS detection Lower Interest Reagents are less toxic versus many existing offerings Compatible with both MS and optical detection Labeling reagent and protocol is automation friendly. Lower cost per sample Provides rapid labeling versus many existing offerings Higher Interest 2015 Waters Corporation 5
The Road to Glycan Analysis Without Compromise RELEASED GLYCAN ANALYSIS LC or LCMS Rapid or Traditional Quan or Qual Cost/ROI Training Requirements 2015 Waters Corporation 6
RapiFluor-MS N-Glycan Labeling: A breakthrough technology for released glycan LC and MS analysis 2015 Waters Corporation 7
Released Glycan Analysis HILIC Profiling Conventional 2-AB 5 Hours to 2 Days FLR MS 2+ 5.3e2 500 2000 m/z 2015 Waters Corporation 8
Out with Conventions Reductive Amination is Laborious Rapid Tagging Glycosylamine labeling circumvents these issues N-Glycan Released N-Glycan (Glycosylamine) N-Glycan (Glycosylamine) + PNGase F Mild Acid Hydrolysis Protein Protein Reductive Amination (conventional) Anhydrous sample Numerous chemical conversions Laborious Heterogenous reaction products N-Glycan ( Acyclic Free Reducing End) + N-Glycan (Free Reducing End) HOAc DMSO 2-AB SPE NaBH 3 CN Reduction N-Glycan (2-AB Labeled) 2015 Waters Corporation 9
RapiFluor-MS TM Reagent Built Upon Our Expertise From Waters expertise in rapid, fluorescence labeling of amino acids AccQ Fluor Enhanced chemical properties for glycan analysis: Rapid Tagging Efficient Fluorescence Enhanced Ionization Efficiency Rapid Fluorescence RapiFluor-MS Patent Pending 2015 Waters Corporation 10
RapiFluor-MS Reagent Rapid Reaction Kinetics NHS Carbamate Rapid Tagging Group Tertiary Amine MS-Active Charge Tag Glycan ΔMass 312 Da Quinoline Fluorophore Glycan Highly stable urea linkage Reaction Time = Seconds Procedure Time: 2015 Waters Corporation 11
RapiFluor-MS Reagent Sensitivity Comparison Instant AB 2.6E+6 RapiFluor-MS Labeled N-Glycans FA2 FLR RapiFluor-MS TM Labeled N-Glycans 0.0E+0 1.7E+6 FA2 MS (BPI) 0.0E+0 4.5 5 5.5 6 6.5 7 min 2.6E+6 Instant AB Labeled N-Glycans FLR Instant AB TM Labeled N-Glycans FA2 0.0E+0 1.7E+6 FA2 MS (BPI) 300X Zoom 0.0E+0 4.5 5 5.5 6 6.5 7 min Instant AB is a trademark of Prozyme Inc. 2015 Waters Corporation 12
RapiFluor-MS Reagent Sensitivity Comparison Instant AB 2.6E+6 RapiFluor-MS Labeled N-Glycans FA2 FLR 400 342.8 FLR MS (BPI) ~2x nearly 1000x 0.0E+0 1.7E+6 0.0E+0 4.5 5 5.5 6 6.5 7 min 2.6E+6 FA2 Instant AB Labeled N-Glycans FA2 MS (BPI) FLR Response Factors 300 200 100 233.7 179.9 0.0E+0 1.7E+6 FA2 MS (BPI) 300x Zoom 0 RapiFluor-MS Labeled 0.3 Instant AB Labeled 0.0E+0 4.5 5 5.5 6 6.5 7 min 2015 Waters Corporation 13
RapiFluor-MS Reagent Sensitivity Comparison Fluorescence MS (BPI) RapiFluor-MS Labeled 100 90 100 90 Instant AB Labeled 2-AB Labeled Procainamide Labeled Relative Performance (%) 80 70 60 50 40 30 20 10 0 52.5 7.0 7.0* 80 70 60 50 40 30 20 10 0 0.1 0.6 30.0* (*) Comparative result extrapolated from a published comparison of N-glycans, 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). 2015 Waters Corporation 14
Simplified Workflow Conventional 5 Hours to 2 Days Direct Analysis (Organic Solvent Dilution) 10 min 5 min 10 min Total Sample Prep Time GlycoWorks RapiFluor-MS N-Glycan Kit 30 min Patent Pending 2015 Waters Corporation 15
Rapid Deglycosylation RapiGest SF Assisted 1% RapiGest SF Surfactant 2 min 80 C GlycoWorks Rapid Buffer GlycoWorks Rapid PNGase F Enzymatic Deglycosylation 5 min 50 C 2015 Waters Corporation 16
Rapid Deglycosylation RapiGest SF Assisted 148560 148.4 kda No PNGase F (control) 148240 145000 150000 146786 146.8 kda 146952 5 min 50 C 145000 150000 145345 145.3 kda 2 Step 2 min Heat Denaturation 5 min 50 C 145000 150000 2015 Waters Corporation 17
Robust HILIC SPE Labeled Glycan Reaction Byproducts Labeled Deglycosylated Protein Collect Byproducts Labeled Glycan 2015 Waters Corporation 18
Robustness Quantitative Extraction higg and fetuin N-glycans 1.2E+6 1x SPE A3G3S3 30 FLR 0.0E+0 1.2E+6 FA2 A3S1G3S3 FA2G2S1 10 15 20 25 30 35 2x SPE Relative Abundance (%) 25 20 15 10 1x SPE Positive Control 2x SPE SPE Processed FLR Positive Control 5 5.7% 6.1% 0 0.0E+0 min 10 15 20 25 30 35 GlycoWorks HILIC SPE of RapiFluor-MS N-glycans is quantitative No significant deviation in the glycan profile upon SPE processing 2015 Waters Corporation 19
Robustness High Yield and Minimal Bias Step Yield Testing to confirm minimal bias Deglycosylation FA2 Rep #1 1.6 pmol Rep #2 Complete 1.7 pmol Recovery measurements Glycan profile before vs after SPE 100% Theoretical Yield = 2.3 pmol 1.5x10 7 pg IgG 1 pmol 2 pmol glycan 0.45 pmol FA2 10 μl injection X X X X 150,000 pg 1 pmol IgG 1 pmol total 400 μl = 2.3 pmol glycan pool sample prepared Intact mass analysis Gel shift assays Subunit LC-MS Labeling >95% Released glycan profile vs subunit derived glycan information SPE ~74% Entire Workflow (experimentally determined) ~73% Yield 2015 Waters Corporation 22
Summary GlycoWorks RapiFluor-MS N-Glycan Kit Simple, streamlined protocol Fast and complete deglycosylation RapiFluor-MS Rapid and efficient labeling Unbiased and robust SPE for neutral to tetrasialylated N-glycans Unprecedented FLR and MS sensitivity Glycoprotein GlycoWorks RapiFluor-MS N-Glycan Kit Analysis-Ready N-glycans 30 min 2015 Waters Corporation 23
GlycoWorks RapiFluor-MS Kit Smart Workflow with No Compromise Available February 2015 2015 Waters Corporation 24
Poster WCBP 2015 Poster P-216-W Rapid Preparation of N-Glycans Using a Novel Fluorescence and MS Active Labeling Reagent Download pdf 2015 Waters Corporation 25
RapiFluor-MS: Enhanced Workflows for Glycan Characterization 2015 Waters Corporation 26
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 27
UNIFI Glycan Workflows HILIC FLR GU +Accurate Mass HILIC FLR GU + DDA MS/MS Automated Data Acquisition Processing Review and Reporting Workflows support conventional glycan labels and new RapiFluor-MS label technology 2015 Waters Corporation 28
RapiFluor-MS Labeling for Characterization Greater than 100x MS response over 2AB labeling BPI MS Sample: NIST RM 8670 mab lot #3F1b 2015 Waters Corporation 29
HILIC FLR GU + Accurate Mass Method Robustness and Transferability, Confident Assignments RapiFluor-Dextran Ladder Retention Time Calibration Curve GU Assign GU values to N-glycans BPI MS for accurate mass confirmation RapiFluor-MS Labeled Dextran and System Performance Standard (higg) are now available to support this GU workflow 2015 Waters Corporation 30
UNIFI Scientific Library for Automated GU or GU+Mass Glycan Confirmation FLR label mass Waters Glycan GU Library: Experimentally derived GU Retention (>10 injections/protein) Data from proteins representing spectrum of glycan diversity All entries confirmed with exoglycosidase digestion Experimental GU value 2015 Waters Corporation 31
UNIFI Scientific Library for Confident Glycan Assignments 2015 Waters Corporation 32
Powerful UNIFI Reporting Architecture Simplifies Communication of Results Example 2015 Waters Corporation 33
UNIFI Glycan DDA Workflow Glycan DDA data acquired Processing and file format conversion SimGlycan Database Search 2015 Waters Corporation 34
Optional UNIFI Export to SimGlycan for MS/MS Database Search A2G2S1 2015 Waters Corporation 35
Enhanced MS/MS with RapiFluor-MS Labeling MS performance extends to fragmentation data FLR BPI MS MSMS 2015 Waters Corporation 36
Enhanced MSMS with RapiFluor-MS Labeling MS performance extends to MS/MS fragmentation data 2015 Waters Corporation 37
Poster WCBP 2015 Poster P-115-T Developing a Scientific Library for UPLC/FLR/MS Analysis of Released N-glycans Labeled with a Novel Labeling Reagent Download pdf 2015 Waters Corporation 38
RapiFluor-MS: Enhanced Workflows for Glycan Monitoring 2015 Waters Corporation 39
Transferability between UPLC and HPLC RapiFluor-MS Labeled Glycans H-Class BIO UPLC ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7µm 2.1 X 150 mm Flow Rate: 0.40 ml/minute Fluorescence (relative) Total Run Time = 55 minutes 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 Alliance HPLC XBridge Glycan BEH Amide, 130Å, 2.5µm 3.0 X 225 mm total length (150 mm + 75 mm) Flow Rate: 0.56 ml/minute 30.00 Total Run Time = 121.3 minutes 24.00 28.00 32.00 36.00 40.00 44.00 48.00 52.00 56.00 60.00 64.00 Time (minutes) Comparable sensitivity and resolution 3x sample load / 2x increase in time Transfer between labs with different LC equipment capabilities 2015 Waters Corporation 40
Glycan Monitoring ACQUITY UPLC H-Class Bio System ACQUITY UPLC Column Manager ACQUITY UPLC FLR Detector ACQUITY QDa Mass Detector Empower or MassLynx Informatics GlycoWorks RapiFluor-MS N-Glycan Kit ACQUITY UPLC Glycan BEH Amide Column 2015 Waters Corporation 41
ACQUITY QDa Mass Detector A breakthrough 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 HPLC & UPLC 2015 Waters Corporation 42
Automated Start Up Provides Robust, Reproducible Performance Automated resolution and calibration occurs with each start-up ensuring mass information is accurate and precise ESI interface optimized for UPLC performance to ensure chromatographic resolution, sensitivity and throughput is preserved Disposable sample aperature and capillary for easy maintenance Graphic QDa monitor display enables easy viewing and adjustment of system parameters 2015 Waters Corporation 43
Routine N-Glycan Detection with Comparable FLR and MS response Detection across a broad range of glycoforms: IgG Simple bi-antennery structures 5 10 15 20 25 30 35 40 Minutes RNase B High mannose structures Fetuin Large, complex structures 5 10 15 20 25 30 35 40 Minutes 2015 Waters Corporation 44
IgG Glycan Profile and Structure Confirmation Using ACQUITY QDa 480.00 Intensity 600000 888.2 FA2 20.5% RPA EU 240.00 300000 0.00 0 600 1200 m/z Intensity 3.2x10 6 1.6x10 6 24000.0 Intensity 12000.0 896.0 A2G1b 0.5% RPA 0.0 10.00 15.00 20.00 25.00 30.00 Minutes Key take away: Large dynamic range can clearly see most abundant and least abundant glycoforms. 2015 Waters Corporation 45 0.0 600 1200 m/z
Intuitive GMP compliant Reporting Empower integration enables annotation of peaks with names and m/z EU 480.00 240.00 A2-815.1 FA2-888.2 FA2B - 989.6 A2G1a - 896.2 A2G1b - 896.0 FA2G1a - 969.3 FA2G1b - 969.2 FA2BG1a - 1070.7 FA2BG1b - 1070.6 A2G2-977.1 FA2G2-1050.3 FA2BG2-1151.8 FA2G1S1-1114.6 FA2G2S1-1196.0 FA2BG2S1-865.3 FA2G2S2-894.7 FA2BG2S2-962.2 0.00 10.00 15.00 20.00 25.00 30.00 Minutes 2015 Waters Corporation 46
Developing a Rapid Method for Glycan Analysis 20 55 minute Method EU 10 0 5 10 15 20 Minutes 40 30 10 minute Method EU 20 10 0 2 3 4 5 Minutes 2015 Waters Corporation 47
Rapid Screening Process for Development Samples Trastuzumab N-Glycan Analysis RapiFluor-MS labeled glycans: 10 minute method 40 FLR Intensity Intensity 50000 0 200000 0 M5 774.1 m/z A2 814.8 m/z EU 20 Intensity 2x10 6 1x10 6 0 F(6)A2 887.9 m/z Intensity 0 2x10 6 1x10 6 0 SIR Overlay 1 2 3 4 5 6 Minutes Intensity Intensity Intensity Intensity 100000 50000 0 1x10 6 0 400000 200000 0 20000 0 A2G(4)1 895.9 m/z F(6)A2G(4)1 968.9 m/z F(6)A2G(4)2 1049.9 m/z F(6)A2G(4)2S1 1195.5 m/z 1 2 3 4 5 6 Minutes 2015 Waters Corporation 48
Monitoring Glycan Ratios Keeping Tabs on Mannose 5 240000 140000.0 180000 105000.0 Intensity 120000 M5 SIR: 774.1 m/z Intensity 70000.0 F(6)A2G(4)1 SIR: 968.9 m/z 60000 35000.0 0 2.00 2.50 3.00 3.50 4.00 Minutes Mannose 5 spiked in Low Medium High Inj 1 0.61 0.96 1.20 Inj 2 0.57 0.90 1.11 Inj 3 0.55 0.86 1.18 Mean 0.58 0.91 1.16 StDev 0.03 0.05 0.04 % RSD 5.44 5.47 3.85 2015 Waters Corporation 49 0.0 Man5: F(6)A2G(4)1 Ratio 2.00 2.50 3.00 3.50 4.00 Minutes
Released N-Glycan UPLC Analysis Workflows SAMPLE PREP SEPARATION DETECTION & INFORMATICS FLR GlycoWorks Kits RapiFluor-MS N-Glycan Kit ACQUITY UPLC Glycan BEH Amide Column TIC ACQUITY FLR/QDa and Empower 3 Software FLR Quantification GU Retention MS Confirmation MS/MS Deglycosylation, Labeling and Clean-up in 30 min Unmatched sensitivity for FLR and MS detection FLR/Xevo G2-XS QTof MS and UNIFI Scientific Information System FLR Quantification GU Retention Accurate Mass Confirmation MS/MS Fragmentation 2015 Waters Corporation 50
Poster WCBP 2015 Poster P-206-W Routine Monitoring of N-Glycans Using a Novel Labeling Reagent with Fluorescence and Mass Detection Download pdf 2015 Waters Corporation 51
RapiFluor-MS N-Glycan Labeling: A breakthrough technology for released glycan LC and MS analysis 2015 Waters Corporation 52