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1 UPLC and HPLC Separation Strategies for Successful Characterization of Glycans Derived from Therapeutic Proteins Thank you for joining us! Our session will begin shortly 2013 Waters Corporation 1

2 Friendly Reminders Please use text chat functionality to submit your questions today. Poll Questions Audience participation Providing Live Technical Support during today s event Upon conclusion, follow up information will be available: Recorded version of today s presentation PDF Copy of today s slides Product discount offers Product specific information and reference materials 2013 Waters Corporation 2

3 Today s Speaker Bill Warren has been with Waters Corporation for more than 20 years, having worked in both technical and marketing capacities. He is currently responsible for strategic and tactical implementation of programs that support new bioseparationsproducts and technologies which help accelerate customer productivity in the biopharmaceutical market segment Waters Corporation 3

4 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 4

5 N-Linked Glycosylation & Biopharma Significance 2013 Waters Corporation 5

6 Glycosylation plays a critical role in biology Nearly 50% of all proteins are glycosylated. What do the glycans do? N-linked glycans play a role in Protein folding Cell-cell communication Biological activity Protein half-life Cell attachment etc IgG-FcγRIIIa Interaction PDB file 3SGJ 2013 Waters Corporation 6

7 N-linked glycan biosynthesis is a highly complex process Struwe WB, Cosgrave EFJ, and Rudd PM. (2011). Glycoproteomics in Health and Disease. Functional and Structural Proteomics of Glycoproteins Waters Corporation 7

8 Glycans are Highly Heterogeneous High Mannose Complex Hybrid 2013 Waters Corporation 8

9 Number of Approved Biotherapeutics in Europe and US 190 EMA and/or FDA Approved Walsh (2010). Nature Biotech; 28(9): Waters Corporation 9

10 127 of 190 approved are Glycoproteins Walsh (2010). Nature Biotech; 28(9): are Glycoproteins (>66%) 2013 Waters Corporation 10

11 2013 Waters Corporation 11

12 N-Linked Glycosylation Is Critical to Mab Biological Activity Asn-297(a) Asn-297(b) C H 2b C H 2a C H 3a C H 3b Asn-297 IgG Fc region IgG Glycosylation Asn-297 Structure based on PDB file 1H3Y from Krapp S et al. J Mol Biol, 2003, 325(5): Waters Corporation 12

13 Terminal Glycan Function: Sialic Acid Desialylation of IVIg abrogates antiinflammatory properties in K/N mice Kaneko et al (2006). Science; 313(5787): Asn 2013 Waters Corporation 13

14 Terminal Glycan Function: Galactose Involved with placental transport of IgG IgG galactosylation increased in pregnant women Kibe et al (1996). J Clin Biochem Nutr; 21(1): Asn 2013 Waters Corporation 14

15 Terminal Glycan Function: Core Fucose Loss of core α(1,6) fucose on IgG results in enhanced ADCC activity Okazaki et al (2004). J Mol Biol; 336(5): Asn 2013 Waters Corporation 15

16 Biopharma Attempts to Replicate Human Glycosylation Struwe WB, Cosgrave EFJ, and Rudd PM. (2011). Glycoproteomics in Health and Disease. Functional and Structural Proteomics of Glycoproteins Waters Corporation 16

17 Negative Attributes to Mammalian Cell Culture Glycosylation Asn Asn 50% of non-allergic blood donors contain antibodies against β(1,2)-xylose and α(1,3)-core fucose Bardor et al (1995). Glycobiology; 13(6): Asn N-glycolylneuraminic acid is an oncofetal antigen in humans Muchmore et al (1989). J Biol Chem; 264(34): Asn Presence of gal-α(1,3)-gal can induce anaphylaxis Chung et al (2006). N Engl J Med; 358(11): Waters Corporation 17

18 Regulatory Agencies Are Dutifully Aware of Glycosylation 2013 Waters Corporation 18

19 Effects that Augment Glycosylation Can Have Consequences 2013 Waters Corporation 19

20 2013 Waters Glycan Analysis Survey (N = 176) Question: For what purpose(s) is your laboratory performing glycan / monosaccharide analyses? (Multiple Responses Allowed) 2013 Waters Corporation 20

21 Agenda: Overview of Glycan Characterization Strategies Intact Glycoprotein LC/MS Analysis Released, N-Glycan Profiling by UPLC and HPLC and Effective Use of GU-Based Data Reduction Structural / Linkage Data Using Exoglycosidase Digestions Summary 2013 Waters Corporation 21

22 Fractionation Methods to Characterize Glycoproteins 6 3 Glycan Release 6 3 Protease Digestion 6 3 Glycoprotein Oligosaccharides (Glycans) 6 3 Glycopeptides Monosaccharides 2013 Waters Corporation 22

23 2013 Waters Glycan Analysis Survey (N = 176) Which of the techniques listed below does your laboratory perform? (Multiple Responses Allowed) 2013 Waters Corporation 23

24 Waters Glycan Separation Technology Sample Preparation Informatics UPLC-based HPLC-based Separations Chemistry Mass Spectrometry 2013 Waters Corporation 24

25 Intact Glycoprotein LC/MS Analysis 2013 Waters Corporation 25

26 Intact Glycoprotein LC/MS Analysis TIC ( min) Pre-run Blank G0F/G1F 0.5 µg IgG1 GOF/G0F G1F/G1F G0F/G2F Post-run Blank G0/GOF G1F/G2F G2F/G2F 2013 Waters Corporation 26

27 LC/MS Analyses of four batches of mab marketed Trastuzumab) showing variations in the proportions of major glycoforms Intact protein glycosylation profile 2013 Waters Corporation 27

28 BiopharmaLynx mirror plot for comparison to Reference compound Intact protein glycosylation profile of a monoclonal Antibody in mirror mode Automated Processing using deconvolution and mass assignment Relative quantification Reference and sample easily compared Batch variations directly measurable Tabular assignment of Glycoforms in BiopharmaLynx 2013 Waters Corporation 28

29 MaxEnt1 Deconvoluted Spectra of Intact IgG1 from three Batches Processed by BiopharmaLynx (Man5)2 G0/G0F (G0F)2 G0F/G1F (G1F)2 G0F/G2F G1F/G2F (G2F)2 Batch 1 Man5/Man6 Method is fast, adapted to high-throughput Batch 2 Batch Waters Corporation 29

30 Released, N-Glycan Profiling by UPLC and HPLC 2013 Waters Corporation 30

31 Challenges: Glycan Analyses Sample Preparation A Difficult and Complex Analytical Problem o Complex frequently involving enzymatic glycan release from isolated glycoproteins followed by labeling Separation o May involve intact protein, peptide, and / or isolated glycan analysis o Complete characterization MOST difficult and time consuming Detection o No chromophore on isolated glycans o LC/MS for compound identification 2013 Waters Corporation 31

32 Waters GlycoWorks TM And Sample Preparation Workflow Step 1 Step 1 Step 2 Step 5 Step 3 Step Waters Corporation 32

33 GlycoWorks HILIC SPE with the optimized elution conditions A Control Before SPE C 20 B GlycoWorks HILIC SPE Processed Elution with 100 mm NH 4 OAc, 5% ACN % Abundan nce Peak 3 G0F Peak 16 A Waters Corporation 33

34 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 34

35 ACQUITY UPLC BEH Glycan Column Chemistry BEH Particle Ligand type: Trifunctional Amide BEH Particle size: 1.7 µm, 2.5um, and 3.5um Endcap style: None Recommended ph range: 2 to Waters Corporation 35

36 Effective Separation of Neutral and Charged 2-AB Labeled Glycans on Waters BEH Glycan, 1.7um Column Glycan Performance Test Standard 1x +A3 (Trisialylated) Glycans 3x Peak 2-AB Labeled Glycan 1 G0-GN 2 G0 3 G0F 4 Man5 5 G0FN 6 G1F 7 G1F 8 G1FN 9 Man6 10 G2 11 G2F 12 G2FN 13 G1FS1 14 G2FS1 15 A3 16 A , ,16 Neutral Glycans Acidic Glycans Nature 446, (26 April 2007) 2013 Waters Corporation 36

37 UPLC and HPLC-based, BEH Glycan Column Certificate of Analysis Chemical Tests Chromatographic Test with Glycan Performance Test Standard Individual Column Tests 2013 Waters Corporation 37

38 Particle Size Evolution: Increased Rs with Smaller Particles Early 1970 s 40µ pellicular non-porous coated psi 1000 plates/meter 1m columns 10 min Late 1970 s 10µ Irregular micro-porous psi 25,000 plates/meter 3.9 x 300mm 10 min 1980 s thru µ spherical micro-porous psi 50,000-80,000 plates/meter 3.9 x 150mm 10 min 2013 Waters Corporation 38

39 Advantages of UPLC vs. HPLC- Based Particle and Instrument Technologies for Bioseparations HPLC Broad Band Broad Peak Less Sensitivity Less Resolving Power Waters UPLC Technology Narrow Peak Increased Sensitivity Increased Resolving Power 2013 Waters Corporation 39

40 UPLC and HPLC-based, 2-AB Labeled Glycan Analyses EU ACQUITY BEH Glycan 1.7 µm 6 UPLC EU x 150 mm 0.50 ml/min XBridge BEH Glycan 2.5 µm XP 4 Alliance HPLC 2.1 x 150 mm 0.34 ml/min P c * half-height = psi (Column, Max) P c * half-height = psi (Column, Max) UPLC-based HPLC-based EU 5.00 XBridge BEH Glycan 3.5 µm Alliance HPLC 2.1 x 150 mm 0.24 ml/min P c * half-height = psi (Column, Max) HPLC-based Minutes 2013 Waters Corporation 40

41 Relative Abundance Determinations , , um, 0.50 ml/min) um XP, 0.34 ml/min % Abun ndance um, 0.24 ml/min Peak 1 G0-GN Peak 2 G0 Peak 3 G0F Peak 4 Man5 Peak 5 G0FN Peak 6 G1F Peak 7 G1F Peak 8 G1FN Peak 9 Man6 Peak 10 G2 Peak 11 G2F Peak 12 G2FN Peak 13 G1FS1 Peak 14 G2FS1 n=3 Peak 15 A3 Peak 16 A3 *Peaks 8 and µm resolution insufficient, accuracy of the integration is poor 2013 Waters Corporation 41

42 Glucose Unit Concept (Way to Normalize RT Variations) 2013 Waters Corporation 42

43 Dextran Calibration Ladder Standard and the Assignment of GU Values (UNIFI) 2013 Waters Corporation 43

44 Using GU Values: BEH Glycan Column Scaling 2013 Waters Corporation 44

45 Using UPLC and MS For Glycan Analysis 2013 Waters Corporation 45

46 Glycan Analysis by LC or MS Alone Is Insufficient for 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 46

47 Glycan Analysis by LC or MS Alone Is Insufficient for Characterization Co-elution of structures in LC makes identification challenging Fucosylated Sialylated High Mannose Structures Terminal Galactose Retention Time (min) Waters Glycan Performance Standard Waters Acquity H-Class Bio 1.7 µm Waters BEH Glycan 2.1 mm x 150 mm 30 min gradient 2013 Waters Corporation 47

48 Combining the techniques can answer many important questions UPLC-FLR-ESI-MS/MS Unifi Waters Corporation 48

49 Structural / Linkage Data Using Exoglycosidase Digestions 2013 Waters Corporation 49

50 Structural / Linkage Data Using Exoglycosidase Digestions Released glycan pool is highly complex Requires use of sequential enzymatic workflows to release glycans at known cleavage positions Presence of branching & linkage isomers Chromatographic resolution is a limiting factor HPLC separations w/ HILIC chemistries often yield coelutions Results interpretation are compromised Glycan identification Must first identify enzymatically-released glycan Then piece together the complete carbohydrate structure based on enzymatic workflow evidence 2013 Waters Corporation 50

51 Glycan Profiling Applying Exoglycosidase Enzymes 2-AB-labeled bovine feting glycans 1 No exoglycosidase digestion Intact feting 2-AB-labeled glycans Man3 A2 Mannose 2 α2-3,6,8 Sialidase A3 N-Acetylglucosamine Galactose 3 Sialidase + β1-4 Galactosidase A2G(4)2 4 4 Sialidase + β1-3,4 Galactosidase A3G(3)1 3 2 β-linkage 5 Sialidase + Galactosidase + Hexosaminidase A3G(3,4,4)3 A3G(4,4,4)3 α-linkage Workflow of the enzymatic array digestion and the structures of the released bovine fetuin N-glycans 2013 Waters Corporation 51

52 Glycan Profiling Meeting the Resolution Challenge 1 UPLC Intact 2 AB labeled fetuin N glycans HPLC 2 linkage isomers Removed: Sialic acid 3 Removed: Sialic acid, Galactose β1-4 4 Removed: Sialic acid, Galactose β1-3, β1-4 5 Removed: Sialic acid, Galactose β1-3, β1-4 acetylglucosamine 2013 Waters Corporation 52

53 Loss of Terminal Monosaccharides Can Be Tracked Using GU Values Approach: Exoglycosidase Arrays with UPLC-FLR Analysis Monosaccharide Linkage To GU Increment Core fucose α(1,6) Any structure 0.5 Outer arm fucose α(1,3) α(1,6) GlcNAc 0.8 Outer arm fucose α(1,2) Gal 0.5 Mannose α(1,2) Man α(1,3) α(1,6) GlcNAc β(1,2) β(1,4) β(1,3) β(1,6) Any structure Bisecting GlcNAc β(1,4) α-mannose Galactose α/β(1,3) α/β1,4) Any structure NeuAc α(2,3) Gal of any structure ~0.7 NeuAc α(2,6) Gal of any structure ~ Waters Corporation 53

54 SUMMARY SLIDE Glycosylation of many biotherapeutic proteins (e.g., mabs) is critically important since it effects the efficacy and potential undesired immunogenicity of the prescribed drug. The effective characterization of protein associated glycans is complex and frequently requires use of several different separation technologies. US and various international regulatory agencies require submission of glycan analysis data in order to approve / license a biotherapeutic or biosimilar drug. Waters offers a range of HPLC and UPLC-based sample preparation, column chemistries, and glycan / sialic acid / monosaccharide application solutions to help in the discovery, development, and manufacturing quality testing required to deliver safe and effective biotherapeutic proteins Waters Corporation 54

55 Thank You! Post-Event Landing Page Promotional Offer on Glycan Columns and other BioSeparations Products Full Webinar Recording of Today s Session PDF Slide Deck Compilation of TODAY S KEY Application Notes, Literature, Brochures etc Questions and to Submit your Ideas for our Next Topic Please - mychemrep@waters.com 2013 Waters Corporation 55