N-Glycan Analysis: From High-Throughput

N-Glycan Analysis: From High-Throughput Screening to In-Depth Characterization Aled Jones, Ph.D. Senior Product Manager ProZyme, Inc., Hayward CA The 20th Symposium on the Interface of Regulatory and Analytical Sciences for Biotechnology Health Products (WCBP) Mayflower Hotel, Washington DC Tuesday 26 th January, 2016

Introduction San Francisco Bay Area company Founded in 1990 Supplier of kits, reagents & services for biopharmaceutical, diagnostic and academic customers Over 400 products: glycobiology/glycoanalysis, streptavidin, phycobiliproteins

History Gly-Q system 1990 1996 2003 2010 2012 2013 2015 2016 ProZyme Founded Glycoanalysis Enzymes Commercialized Glyko/OGS (1989-2003) GlykoPrep InstantAB GlykoPrep 2-AB and APTS GlykoPrep -plus 2-AB GlykoPrep -plus InstantAB APTS GlykoPrep with InstantPC

Outline Introduction/background High-throughput N-Glycan sample preparation ProZyme s Solution: GlykoPrep GlykoPrep Automation with the Agilent AssayMAP Bravo Exoglycosidases & Glycosyltransferases Analytical Methods for N-Glycan Screening & Characterization UPLC -FLR CE-LIF: GlykoPrep/APTS CE-LEDIF: Gly-Q, developmental platform for N-glycan screening (P-107-T) LC-MS: InstantPC, novel glycan label for MS (P-211-W)

Introduction Glycosylation attachment of glycan (carbohydrate) structures to proteins Various biological functions (e.g., folding, stability, assembly, signaling, etc.) Antibody and antibody-derived biotherapuetic proteins are glycosylated, > 40 on the market, + other glycosylated biotherapeutics Glycosylation can be a critical quality attribute (CQA) Increasing regulatory pressure to control processes for better safety, efficacy and product quality Patent expirations and the emergence of biosimilars Harmonization/communication between regulatory agencies worldwide

IgG Fc N-Linked Glycosylation at N297 Vidarsson et al., Front Immunol. 2014; 5: 520

Therapeutic Effects of N-Glycans Terminal Sialic Acid -clearance, antiinflammatory High Mannose ++clearance Galactosylation +CDC Core fucose --ADCC Fucose Galactose Mannose N-acetylglucosamine N-acetylneuraminic acid Reviewed in: Liu, J Pharm Sci. 2015; 104: 1866-1884 Abes & Teillaud, Pharmaceuticals 2010; 3:146-57 ADCC: Antibody-Dependent Cell-mediated Cytotoxicity CDC: Complement-Dependent Cytotoxicity

Non-Human/Immunoreactive N-Glycans Galα(1,3)Gal (Galili antigen) Macher & Galili, Biochim Biophys Acta. 2010; 1780 (2): 75-88 Cetuximab - N-glycans with α-gal on Fab region Associated with adverse immunological responses Abatacept - N-glycans with α-gal on Fc region No reported adverse effects Van Bueren et al., Nat Biotechnol. 2011; 29: 574-576 Bosques et al., Biotechnol. 2010; 28: 1153-1156 Chung et al., N Eng J Med. 2008; 358(11): 1109-1117 Fucose Galactose Mannose N-acetylglucosamine N-glycoylneuraminic acid

Non-Human/Immunoreactive N-Glycans Galα(1,3)Gal (Galili antigen) NeuGc NGNA Potentially immunogenic Ghaderi et al., Biotechnol Genet Eng Rev. 2012; 28:147-75 Fucose Galactose Mannose N-acetylglucosamine N-glycoylneuraminic acid

N-Glycan Analysis Material Structure Analytical Methods Intact glycoproteins Electrophoresis, lectin arrays, LC-ESI-MS: HR/AM (+ reduction) Glycopeptides LC-MS, MALDI, CE Released glycans Derivatized Underivatized F Exoglycosidases (linkage assignment) LC-FLR, LC-MS, CE-LIF MALDI-TOF, HPAE-PAD Monosaccharides Derivatized Underivatized F A RP LC-FLR HPAE-PAD

Traditional N-Glycan Analysis Time to results is long (3-5 days or longer) - Tedious sample preparation - Only some steps automatable - Complex results, difficult to analyze and compare Quality of the results can be improved - Method may bias results - Detection - Labeling - Resolution

High Throughput Glycoanalysis? What the solution should look like Decreased time to results (<4 hours) Automation (from purification to results) High-throughput (1,000 s of samples/day) High-quality results (quantitative, consistent, robust, reproducible, sensitive, precise, etc.) Full characterization (N- & O-linked analysis, specific monosaccharides and occupancy/profiles by site)

ProZyme s Solution: GlykoPrep GlykoPrep N-Glycan Sample Preparation Short time to results High throughput Modular Microplate or microtube format GlykoPrep-plus Automation on the Agilent AssayMAP Bravo Liquid Handling Workstation Reduces errors and complexity Reduces hands-on time Improves reproducibility

GlykoPrep N-Glycan Sample Preparation Platform Purify Antibody (30-60 minutes) Denature & immobilize (30-60 minutes) Digest with N-Glycanase (15-60 minutes) Fluorescent labels (0-1 hour) Cleanup & elute in water (15-30 minutes) Agilent AssayMAP Bravo Liquid Handling Platform Microfuge Format Plate Format Automation Format Up to 24 samples Microfuge tubes Microcentrifuge ~4-5 hours Attended Up to 4 x 96 samples 96-well microplates Centrifuge ~4-5 hours Attended n x 96 samples 96-well microplates AssayMAP Bravo ~4-5 hours per plate Walk-away

GlykoPrep Label Choices Glycoprotein N-Glycanase

GlykoPrep Label Choices Glycoprotein N-Glycanase InstantDyes InstantAB InstantPC InstantDye for LC-FLR Similar HILIC elution profile to 2-AB GPPNG-LB (Bravo), GP24/96NG-LB (manual) InstantDye for LC-MS Beta testing ongoing Bravo method in development H 2 O NH 3 Rapid- Reductive- Amination 2-AB Label makes N-Glycan less polar Improves HILIC separation for UPLC GPPNG-AB (Bravo), GP24/96NG-AB (manual) Label makes N-Glycan more polar Introduces negative charge for CE GPPNG-APTS (Bravo), GP24/96NG-APTS (manual) APTS

GlykoPrep -plus Automated on Agilent Bravo Traditional workflow, optimized conditions Up to 50 µg any glycoprotein Optional reduction protocol for difficult targets 2-AB/InstantAB/APTS workflows automated on Agilent AssayMAP Bravo 3-5 hours per 96-well plate Output: labeled glycans in water

Agilent Automation: AssayMAP Bravo Liquid Handling Workstation

Agilent AssayMAP Bravo: Example 1 4 lots of Rituximab, 24 replicates each, 50ug/rep GlykoPrep-plus 2-AB workflow on Agilent AssayMAP Bravo (GPPNG-AB) Analysis by UHPLC, HILIC (10-Minute Screening Method) ~20 nanograms of N-glycan sample in aqueous buffer 1 µl aqueous injection

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Minutes Rituximab 2-AB N-Glycans 10-Minute UPLC-HILIC Screening Method G0F G1F[6] G1F[3] G2F G0F-N G0 Man 5 G1 Man 6 G2 A1F A2F

Area % Rituximab 2-AB N-Glycans: Lot Comparison 50 45 40 35 30 25 20 15 10 5 0 MabThera_B6055 MabThera_B6069 Rituxan_930814 Rituxan_938802 n = 24 Glycan

Rituximab 2-AB N-Glycans: Lot Comparison Peak ID MabThera _B6055 MabThera_B6059 Rituxan _930814 Rituxan_938802 Average % CV Average % CV Average % CV Average % CV G0F-N 0.8 3.1% 0.6 3.6% 0.6 2.2% 0.7 1.3% G0 1.4 1.5% 1.0 2.2% 1.3 1.1% 1.5 0.8% G0F 43.2 0.5% 35.7 0.3% 40.0 0.3% 41.1 0.4% Man-5 2.1 1.4% 1.4 1.7% 1.6 1.1% 1.7 1.4% G1 1.2 1.1% 1.1 0.9% 1.1 1.6% 1.2 0.7% G1F[3] 10.3 0.5% 11.3 0.3% 10.9 0.4% 10.7 0.5% G1F[6] 28.6 0.5% 32.2 0.4% 30.3 0.4% 29.6 0.3% Man-6 0.9 7.1% 0.3 3.1% 0.5 3.1% 0.5 1.9% G2 0.3 7.4% 0.4 2.5% 0.3 4.3% 0.4 2.6% G2F 7.7 0.5% 11.3 0.4% 9.4 0.4% 8.9 0.5% A1F 1.2 2.1% 1.9 1.0% 1.5 1.1% 1.5 1.7% A2F 0.6 3.8% 1.1 2.3% 0.8 1.5% 0.8 2.9% n = 24 %CV for peaks 1% are 2.3%

Avg % Peak Area (Manual, IAB) Correlation of results between methods & dyes 50 45 40 35 30 25 20 15 10 5 0 2 Dyes, Manual vs. Automation y = 0.9921x - 0.215 R² = 0.999 0 10 20 30 40 50 Avg % Peak Area (Automation, 2-AB)

Agilent AssayMAP Bravo: Example 2 Enbrel ; 15 replicates GlykoPrep-plus 2-AB workflow on Agilent AssayMAP Bravo (GPPNG-AB) UHPLC-HILIC 60-minute gradient (more complex N-glycan profile)

Enbrel 2-AB N-Glycans 60-Minute UPLC-HILIC Method Automation (n = 15) 1 2 4 5 7 8 9 10 13 14 15 18 19 21 24 26 Peak Number Peak ID 1 G0-N 2 G0 4 G0F 5 Man-5 7 G1-6 8 G1-3 9 G1F-6 10 G1F-3 13 Man-6 14 G2 15 G2F 18 G1FS-1 19 A1 21 A1F 24 A2 26 A2F 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 Minutes

Enbrel 2-AB N-Glycans Automation (n = 15) Peak Number Peak ID Average % CV 1 G0-N 0.2 6.8 2 G0 1.1 0.9 4 G0F 18.4 0.8 5 Man-5 4.2 0.8 7 G1-6 0.7 1.5 8 G1-3 0.4 2.3 9 G1F-6 8.3 0.6 10 G1F-3 3.9 0.4 13 Man-6 0.9 2.3 14 G2 2.5 2.1 15 G2F 4.6 0.8 18 G1FS-1 3.5 0.8 19 A1 13.9 0.9 21 A1F 18.4 0.5 24 A2 4.3 1.2 26 A2F 9.3 1.0 %CV for peaks 1% are 2.1%

Screening & Characterization Labeled N-glycans from 50 µg protein (in water) UPLC-FLR Screening 1 µl 10 minute method Exoglycosidase Sequencing Reactions Mass Spectrometry UPLC-FLR Characterization 60 minute method

RFU Exoglycosidase Characterization of Peaks No exoglycosidase + Sialidase A + Sialidase A + β(1-4)-galactosidase + Sialidase A + β(1-4)-galactosidase + β-n-acetylhexoseaminidase + Sialidase A + β(1-4)-galactosidase + β-n-acetylhexoseaminidase + α-mannosidases 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 (MabThera B6069) Minutes

Generation of Glycovariants Treated Rituxan with sialidase A and β(1,4)-galactosidase (GKX-5014) Built different glycoprofile with β(1,4)-galactosyltransferase (GKT-GA14) and α(2,3)-sialyltransferase agalacto + GalT + ST3 agalacto + GalT asialo, agalacto G0F G1F G2F A1F A2F Rituxan 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 (~2 hrs reaction) Minutes

60-Minute HILIC Method 2-AB Partitioned Biantennary N-Glycan Standard Library (GKSB-520)

1 2 3 4 5 6 7 8 9 10 10-Minute HILIC Method 2-AB Partitioned Biantennary N-Glycan Standard Library (GKSB-520) High mannose 2 3 4 Fucosyl biantennary Afucosyl biantennary

Mixed-Mode Separation of N-Glycans N-Glycans from fetuin, InstantAB AXR-1 (Thermo) Weak anion exchange/rp 3 Sialic Acids 2 Sialic Acids 4 Sialic Acids AXH-1 (Thermo) Weak anion exchange/hilic 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 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35.00 36.00 37.00 38.00 39.00 40.00 41.00 42.00 43.00 44.00 45.00 46.00 47.00 48.00 49.00 50.00 Minutes

N-Glycan Order by Platform HILIC- UPLC Neutral Sialylated -1-2 Small neutral α-gal Glycan size Negative charge Sialylated Neutral Capillary Electrophoresis (CE) -2-1 Hydrodynamic radius Small neutral α-gal

CE-LIF Methods GlykoPrep sample prep, N-glycans labeled with APTS (8-aminopyrene-1,3,6- trisulfonic acid), product codes GP24NG-APTS, GP96NG-APTS SCIEX Separations (Beckman) PA 800 plus with LIF detector SCIEX Separations N-CHO capillary, 60 cm length (effective 50 cm), 50um i.d. N-linked Carbohydrate Separation Gel Buffer (#477623) Samples loaded at 2 psi for 10 seconds Reversed polarity, 30 kv for 20 minutes, 20C Detection by laser-induced fluorescence (LIF), excitation 360nm/emission 428nm Reusch et al. mabs 2015; 7: 167-179 Szekrényes et al. Multi-Site N-glycan mapping study 1: Capillary electrophoresis - laser induced fluorescence. mabs 2016; 7: 167-179

APTS-Labeled N-Glycan Standards for CE APTS Partitioned Biantennary N-Glycan Standard Library (GKSP-520)

APTS-Labeled N-Glycans from Enbrel No Cleanup GlykoPrep C2 Cleanup

CE-LIF Case Study: Cetuximab CE profiles not normalized APTS-labeled N-glycans prepared with GlykoPrep CE-LIF Intact molecule Molecule --RX 9.5% 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 Minutes α-galactosylated N-glycan structure (9.5% relative peak area) confirmed by analysis of the corresponding 2-AA-labeled glycan peak: exoglycosidase digestion with α(1-3,4,6)- Galactosidase (ProZyme GKX-5007), LC-MS Methods for the identification of α-galactosylated N-glycan structures by exoglycosidase digestion and glucose unit (GU)-based assignment using CE are described in: Szabo et al. Ultrasensitive Capillary Electrophoretic Analysis of Potentially Immunogenic Carbohydrate Residues in Biologics: Galactose-α-1,3-Galactose Containing Oligosaccharides. Mol. Pharmaceutics 9(6): 1612-1619 (2012).

FabRICATOR Digestion of Cetuximab CE profiles not normalized Digest to Fc/2 + F(ab ) 2 Protein A to separate domains F(ab ) 2 FabRICATOR: Ides (Genovis) Fc http://www.genovis.com/fabricator-enzyme

Cetuximab Fc vs. Fab N-Glycans CE profiles not normalized Intact molecule 9.5% Fc region Fab region (PA flowthough after FabRICATOR) 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 CE profiles not normalized Note: denaturing conditions needed to remove Fab glycans

Process Development 500 1000 clonal cell lines 100 200 10 20 production cell line Early Screening Emphasizes capacity and speed over accuracy Later screening Increases in complexity and emphasizes fit to manufacturing process Aggregation N-Glycan Profiling Target Binding Growth (titer) Glycan Screen Charge Heterogeneity 6 Months Fc Functional Assays Courtesy of Mark Melville and Joe Siemiatkoski of Epirus Biopharmaceuticals, Inc.

Gly-Q: Platform for Rapid Sample Preparation, Separation & Analysis of N-Glycans 96 cell culture samples in a single workday Rapid, automatable sample prep Simple CE instrument, 2 minute run time Gly-Q instrument Replaceable cartridge with gel matrix & capillary 96 Samples

Gly-Q: Platform for Rapid Sample Preparation, Separation & Analysis of N-Glycans Cell culture media Protein A purification Gly-Q instrument 40ug glycoprotein Released N-glycans 5 minute in-solution N-Glycanase (PNGase F) digest Gly-Q InstantDye Replaceable cartridge with gel matrix & capillary 96 Samples Labeled N-glycans + dye Labeled N-glycans Cleanup 2 min separation on Gly-Q CE Data analysis

Gly-Q 5-Minute N-Glycanase In-solution Digestion kda 1 2 3 4 1 2 3 4 1 2 3 4 50-37 - 50-37 - 50-37 - PNGase F 25-20 - 25-25 - 20 - Rituxan higg Cetuximab 75-50 - 37-20 - 1 2 3 4 Enbrel 1: No enzyme 2: Gly-Q N-Glycanase, 5 mins 3: SDS + detergent, N-Glycanase, 2 hours 4: RapiGest/Rapid PNGase F, 5 mins

Separation of MabThera N-Glycans on Gly-Q

Separation of Enbrel N-Glycans on Gly-Q

Gly-Q relative migration time (RMT) library

Alignment of 72 samples on Gly-Q

Gly-Q

Comparability of Gly-Q to HILIC methods Gly-Q MabThera Lot B6069B03 n = 24 GlykoPrep/InstantAB HILIC-UHPLC

Separation of APTS N-Glycans by Gly-Q APTS Partitioned Biantennary N-Glycan Standard Library (GKSP-520) Afucosyl Biantennary Fucosyl Biantennary High Mannose

Summary Gly-Q Novel system for rapid N-glycan analysis Rapid, simple, automatable sample prep Fast (2 min) separation Potential for automated data analysis Suited to applications requiring N-glycan profiling of large numbers of samples (biotherapeutic cell line development)

InstantPC Novel Glycan Label for MS Increased MS and FLR sensitivity Amenable to rapid LC methods Developmental workflow, rapid in-solution digestion Kimzey et al., ASMS 2015, P603 Q1 2016 GlykoPrep-compatible workflow Beta testing ongoing

InstantPC Dye (IPC) Tertiary amine Procaine Tertiary amine generates high MS signal in positive mode Less polar than 2-AB & Procainamide: shorter HILIC retention time for IPC-labeled glycans

InstantPC Dye (IPC) Tertiary amine Procaine Glycan-NH 2

FLR Response Comparison

MS Response Comparison

60-Minute Separation of IPC-Glycans A. Mixture of high mannose and asialo biantennary N-glycans Agilent AdvanceBio Glycan Mapping column (HILIC) 2.1 x 150 mm, 2.7 μm B. High mannose and biantennary complex N-glycans with 2,6-linked sialic acid C. Enbrel N-Glycans

5-Minute Separation of IPC-Glycans A. Mixture of high mannose and asialo biantennary N- glycan standards Agilent AdvanceBio Glycan Mapping column (HILIC) 2.1 x 100 mm, 2.7 μm B. Enbrel N-Glycans C. Herceptin N-Glycans

Run Time and Resolution Comparison InstantPC-labeled Enbrel N-glycans, 5-minute method (AdvanceBio) RapiFluor-MS-labeled Enbrel N-glycans, 55- minute method (BEH)

Run Time and Resolution Comparison InstantPC-glycans 5-minute method (AdvanceBio column) InstantPC RapiFluor-MS-labeled Enbrel N-glycans 55-minute method (BEH column) Competitor X

CID MS/MS of IPC-labeled A2F IPC is suitable for Collision Induced Dissociation (CID) MS/MS CID profile contains mostly glycosidic cleavages with some cross-ring fragmentation.

Summary - InstantPC Highest fluorescence of any glycan label tested High MS response Flexible workflows in development

Summary GlykoPrep is a fast and accurate, high-throughput platform for the preparation of N-linked glycans The Agilent AssayMAP Bravo adds precise, automated liquid handling Manual and automation results are comparable for N-glycan distribution Reproducibility is improved with GlykoPrep-plus

Summary GlykoPrep/APTS label for CE-LIF offers option for orthogonal analysis, particularly suited for separation of α-gal-containing N-glycans Developmental Gly-Q system: rapid CE platform for user-friendly glycoanalysis Developmental InstantPC label provides greater MS sensitivity, allows for rapid (5-minute) and high-resolution (60-minute) HILIC methods for N-glycan separation Gly-Q system and InstantPC workflows coming in 2016! Talk to us about beta testing

Acknowledgements Ted Haxo Justin Hyche Mike Kimzey Steve Mast Shirley Ng Shiva Pourkaveh Tom Rice Vaishali Sharma Bopha Sun Zoltan Szabo Adele Taylor Samnang Tep Sergey Vlasenko Jo Wegstein info@prozyme.com Randy Bolger Steve Murphy Zach van den Heuvel Varouj Amirkhanian

N-Glycan Analysis: From High-Throughput Screening to In-Depth Characterization The AssayMAP technology is licensed from Agilent Technologies. ProZyme, GlykoPrep, Glyko, Gly-Q InstantDye, InstantAB, InstantAA, InstantPC, Sialidase A, Sialidase S, N- Glycanase and Rapid-Reductive-Amination are trademarks or registered trademarks of ProZyme, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.