Using LC-Based Technologies for Elucidating Complex Carbohydrate Structures Eoin Cosgrave, PhD Formerly with NIBRT Glycobiology Group Currently Senior Scientist with Waters Biopharmaceutical Life Sciences Group Biopharma LC Meeting Eschborn, Germany 17 October, 2012
Overview An introduction to NIBRT N-linked glycosylation in Biopharma LC-based analysis of N-linked glycans Benefits of LC-FLR in complex carbohydrate analysis
An Introduction to NIBRT Research and Training for the Biopharma Industry
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N-Linked Glycosylation in Biopharma
Glycan Nomenclature Saccharide Abbreviation Structure Symbol CFG N-acetylglucosamine GlcNAc Glucose Glc Galactose Gal N-acetylgalactosamine GalNAc Fucose Fuc Mannose Man N-acetylneuraminic acid NeuNAc (NANA) N-glycolylneuraminic acid NeuNGc (NGNA) Xylose Xyl Harvey DJ et al. Proteomics, 2009, 9(15), 3796-3801
Biological Impact of N-linked Glycosylation Struwe WB, Cosgrave EFJ, and Rudd PM. (2011). Glycoproteomics in Health and Disease. Functional and Structural Proteomics of Glycoproteins.
Categorization of N-linked Structures High Mannose Complex Hybrid
No Longer an Emerging Market? Blood Proteins Blood Proteins Hormones Hormones Growth Factors Growth Factors Cytokines Cytokines Vaccines Monoclonal Antibodies Vaccines Bone Proteins Other mabs Bone Proteins 190 EMA/FDA Approved Walsh (2010). Nature Biotech; 28(9):917-924
No Longer an Emerging Market? Blood Proteins Blood Proteins Hormones Hormones Growth Factors Growth Factors Cytokines Cytokines Vaccines Monoclonal Antibodies Vaccines Bone Proteins Other mabs Bone Proteins 127 are Glycoproteins (>66%) Walsh (2010). Nature Biotech; 28(9):917-924
LC-based Analysis of N-linked Glycans Other Amgen Genentech J & J Novo Nordisk Biogen Idec Sanofi Abbott Serono BMS $48.9 Billion USD Aggarwal (2010). Nature Biotech; 28(11):1165-1171
Glycoprotein R&D is Gaining Momentum
Non-Human Glycosylation Introduces Risk Asn Asn 50% of non-allergic blood donors contain antibodies against (1,2)-xylose and (1,3)-core fucose Bardor et al (1995). Glycobiology; 13(6): 427-434 Asn N-glycolylneuraminic acid is an oncofetal antigen in humans Muchmore et al (1989). J Biol Chem; 264(34): 20216-20223 Asn Presence of gal- (1,3)-gal can induce anaphylaxis Chung et al (2006). N Engl J Med; 358(11): 1109-1117
Changes in Bioprocessing Can Introduce Variability in Glycans Mack G. Nat Biotech, 2008, 26(6): 592
Regulatory Agencies Have Responded with Tighter Demands
LC-Based Analysis of N-linked Glycans
Glycan Analytical Instrumentation in NIBRT HPLCs 2x Agilent 1200 with Jasco 2020 FLR 1x Agilent 1200 with DAD 1x Agilent 1200 with VWD 4x Waters Alliance 2695 with 2475 FLR 2x Waters Alliance 2795 with 2475 FLR UHPLCs 5x Waters H-Class with FLR 4x Waters Acquity with FLR 2x Waters Acquity I-Class 1x Waters Nano 2D Acquity Mass Spectrometers 1x Waters Global Micromass Q-ToF 1x Waters Micromass MALDI-ToF 1x Agilent LC-MS ChipCube Iontrap 6430 1x Agilent LC-MS SQ 6130 3x Waters Xevo G2 Q-ToF 1x Waters Synapt G2 Q-ToF Capillary Electrophoresis-LIF 1x Beckman Coulter PA800 1x Agilent 7100
Why Use HILIC-FLR? 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?
The Analytical Workflow DMB NeuAc Speciation Glycans 2AB Total Analysis Charged Glycans Glycoprotein PNGase F Unlabelled Mass Spec Protein Currently (but this is changing)
The Analytical Workflow Glycans 2AB Total Analysis Glycoprotein PNGase F HILIC-FLR RPLC-FLR
EU HILIC-FLR Separates Neutral and Charged Structures Waters Acquity H-Class Bio 1.7 m Waters BEH Glycan 2.1 mm x 150 mm 30 min gradient HILIC-FLR Waters IgG 2AB Glycan Standard 50.0 40.0 30.0 20.0 10.0 0.0 2 4 6 8 10 12 14 Retention Time (min)
EU CQAs Can Be Determined From a Single Chromatogram Waters Acquity H-Class Bio 1.7 m Waters BEH Glycan 2.1 mm x 150 mm 30 min gradient HILIC-FLR Waters IgG 2AB Glycan Standard 50.0 40.0 Fucosylated Sialylated High Mannose Structures Terminal Galactose 30.0 size 20.0 10.0 0.0 2 4 6 8 10 12 14 Retention Time (min)
EU HILIC-FLR Separates Neutral and Charged Structures F(6)A1 A2 FA2 M5 F(6)A2B A2G1 A2BG1 FA2G1 FA2G1 FA2BG1 M6 A2G2 A2BG2 FA2G2 FA2BG2 F(6)A2G1S1 A2G2S1 F(6)A2G2S1 F(6)A2BG2S1 Waters Acquity H-Class Bio 1.7 m Waters BEH Glycan 2.1 mm x 150 mm 30 min gradient HILIC-FLR Waters IgG 2AB Glycan Standard 50.0 40.0 30.0 20.0 10.0 0.0 2 4 6 8 10 12 14 Retention Time (min)
<2 m Particles Maximize Speed and Resolution 5 m Tosoh TSKgel Amide-80 4.6 mm x 250 mm 3 h gradient 3 m Tosoh TSKgel Amide-80 4.6 mm x 150 mm 1 h gradient 1.7 m Waters BEH Glycan 2.1 mm x 150 mm 30 min gradient 1.7 m Waters BEH Glycan 2.1 mm x 150 mm 30 min gradient Retention Time (min) 15 30 60 Retention Time (min) 180
Glycans Can Be Analyzed in Reversed Phase Glycans 2AB Total Analysis Glycoprotein PNGase F RPLC-FLR
RP-LC Separates N-Glycans on CQAs RPLC-FLR Chen X et al. Anal Biochem, 2007, 370: 147-161
Sialic Acid Species Are Separated in RPLC-FLR DMB NeuAc Speciation Glycans Glycoprotein PNGase F RPLC-FLR
Sialic Acid Species Are Separated in RP-LC RPLC-FLR Sialic Acids Neu5Ac Neu5Gc,9Ac Neu5,7Ac2 Neu5,9Gc2 Waters 3.5 μm Xbridge C18 (4.6 mm x 100 mm) Isocratic 30 min Separation Ex : 578 nm Em : 450 nm Neu5Gc Neu5,7(8),9Ac3 Neu5Ac Neu5Gc
Mapping Retention Times with Standards Identifies Neu5Gc RPLC-FLR Waters 3.5 μm Xbridge C18 (4.6 mm x 100 mm) Isocratic 30 min Separation Ex : 578 nm Em : 450 nm Sialic Acid Neu5Ac Neu5Gc IgG Sample Neu5Gc = 7.2% Neu5Ac = 86.8%
The Charged State of Glycans is Determined Using LC-FLR Glycans 2AB Charged Glycans Glycoprotein PNGase F WAXLC-FLR
WAX-Analysis Identifies Charged Glycan Structures Sialylation R R R R R R R R R R Sialic acid Phosphates/Sulphates Fetuin Standard Prozyme 10 m DEAE (4.6 mm x 50 mm) 50 min Gradient Method Ex : 330 nm Em : 420 nm Sample X Undigested Sample X ABS-digested
2D-Analysis Aids in Elucidating Complex Glycan Pools Initial Profile * Fc RIIIb 1 2 3 4 5 6 7 8 9 10 Fc RIIIb First Dimension (WAX-HPLC) 30 Neutral * 1 2 Mono- 3 4 * Second Dimension (HILIC) Di- 5 6 Tri- 7 Tetra- 8 9 * WAXLC-FLR 10 60 70 80 90 100 110 120 130 140 Retention Time (min) HILIC-FLR
GlycoBase 3.1
Complex Carbohydrate Characterization
EU EU Glycosylation Presents Extensive Complexity GU Value 3 4 5 6 7 8 9 10 11 12 13 14 8.00 6.00 4.00 Human IgG1 Waters Acquity H-Class 1.7 μm BEH Glycan (2.1 mm x 250 mm) FLR detection ( ex 330 nm, em 420 nm) 53% to 70% Linear gradient (30 min) Solvent A: 50 mm NH4 + HCOO - Solvent B: Acetonitrile 2.00 0.00 5.00 Glycoprotein Therapeutic 4.00 3.00 2.00 1.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes
Digestion to Core Mannose Identifies Unexpected Modifications EU EU GU Value 3 4 5 6 7 8 9 10 11 12 13 14 30.00 20.00 10.00 Human IgG1 Waters Acquity H-Class 1.7 μm BEH Glycan (2.1 mm x 250 mm) FLR detection ( ex 330 nm, em 420 nm) 53% to 70% Linear gradient (30 min) Solvent A: 50 mm NH4 + HCOO - Solvent B: Acetonitrile 0.00 15.00 10.00 Glycoprotein Therapeutic 5.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes
Identifying Phosphorylation on N-linked Glycans A B B C lysosome M6P receptor A: GlcNAc phosphotransferase B: Golgi -mannosidase C: Phosphodiester glycosidase
Phosphorylation Generates Several N-linked Possibilities Mono-PO 4 Bis-PO4
WAX-HPLC Identifies Sialylated and Phosphorylated Glycans EU EU EU EU 40.00 20.00 0.00 60.00 Undigested Waters Alliance 2795 10 μm DEAE (4.6 mm x 50 mm) FLR detection ( ex 330 nm, em 420 nm) 0% to 100% Linear gradient (50 min) Solvent A: 20% Acetonitrile Solvent B: 20 mm NH4 + CH 2 HCOO - 40.00 ABS 20.00 0.00 80.00 * * Sialylated glycans 60.00 40.00 20.00 0.00 * * CIAP Phosphorylated glycans 60.00 40.00 20.00 * * ABS CIAP Sulphated glycans (?) 0.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Minutes
EU EU EU Enzymatic Digests Aid in Pinpointing Unique Pathway Structures M1 M3 M3 M4 M4 M3P-GlcNAc1 M3P-GlcNAc1 M3P-GlcNAc1 M5 M5 M4P-GlcNAc1 M4P-GlcNAc1 M4P-GlcNAc1 M6 M6 M5P M7 M7 M6P iso M6P iso M8 M8 M7P GU Value 3 4 5 6 7 8 9 10 11 60.00 50.00 40.00 30.00 20.00 A GU = 1.13 GU = 1.10 GU = 1.08 Waters Acquity H-Class 1.7 μm BEH Glycan (2.1 mm x 150 mm) FLR detection ( ex 330 nm, em 420 nm) 30% to 47% Linear gradient (30 min) Solvent A: 50 mm Ammonium formate, ph 4.4 Solvent B: Acetonitrile 10.00 0.00 30.00 B 20.00 10.00 0.00 100.00 80.00 C 60.00 40.00 20.00 0.00 * 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 Minutes
Techniques Combine to Characterize Complex Samples EU GU Value 4 5 6 7 8 9 10 11 4.00 11 3.50 3.00 2.50 23 2.00 2 1.50 1.00 0.50 1 3 5 9 10 7 4 6 8 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 0.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 Minutes
UPLC-FLR with Enzymatic Arrays Identifies All Structures
Combining LC Quantitation with MS Accuracy FLR UPLC QToF Separation resolution of UPLC technology Quantitation from FLR detection Compositional confirmation from QToF MS
Fluorescence Quantitation with MS Verification Protein Sample X FLR BPI Inlet: Waters Acquity H-Class 1.7 m BEH Glycan (2.1 mm x 150 mm) FLR detection ( ex 330 nm, em 420 nm) MS: Waters Xevo G2 ESI-Q-ToF Negative mode MS e configuration XIC [M-H] 1- MS [M-2H] 2-
Summary N-linked glycans play an important functional role in many biotherapeutics Detailed analysis of glycan structures is performed using LC methods RP, HILIC, and WAX are used to report on varying glycan characteristics Enzyme technology allows for elucidation of complex glycan pools UPLC-FLR-MS technology combines two powerful analytical techniques
Acknowledgements Principal Investigator Amgen Oxford U Waters Prof Pauline M Rudd O-link Analytical Group Tharmala Tharmalingam (RBSC) Rebecca Duke (O-glycanase) Weston Struwe (AGRC) Contract Glycan Analysis Niaobh McLoughlin Patrick Jennings Rebecca Duke Eoin Cosgrave Weston Struwe Jo Withers Bioinformatics Giorgio Carta John O Rourke Agilent Industrial Collaborations Eoin Cosgrave Jerrard Hayes Mark Hilliard Oscar Potter Glycobiology in Health & Disease Radka Saldova (ovarian/breast cancer) Mohan Kumar Muniyappa (Asthma) Simone Albrecht (Gut-Microbe interactions) Barbara Adamczyk (prostate/colon cancer) Margaret Doherty (Biomarker discovery) Louise Unwin (Biomarker discovery) Technology Development Group Oscar Potter (LC-MS, CE-LIF) Weston Struwe (MS, LC-MS) Eoin Cosgrave (NP-HPLC, LC-MS) Henning Stockmann (Robotics) Mark Hilliard (UPLC, MS) Tharmala Tharmalingam (LC-MS, CAD)