RFMS Glycan Characterization Techniques for Biotherapeutics Dr Mark Hilliard, NIBRT Waters THE SCIENCE OF WHAT S POSSIBLE TM
The Complexity of Glycosylation Glycosylation is the most common posttranslational modification. Glycosylation of proteins is a complex process leading to glycoforms of the same protein. Glycans are branched, therefore leading to a higher degree of structural complexity (unlike DNA and proteins ). Sugar chains can be linked to the protein through either the nitrogen atom of asparagine residues (N-linked glycans) or via the hydroxyl group of serine and threonine residues (O-linked glycans).
Motivation for Understanding Glycosylation Biopharmaceuticals and recombinant proteins: Quality control the determination of correct product glycosylation is essential in order to ensure the efficacy and safety of therapeutic products. International Conference on Harmonisation (ICH) Guideline Q6B requires carbohydrate content, structure, and glycosylation sites present on therapeutic proteins to be characterised as extensively as possible
Classes of N-Linked Glycans High Mannose Complex Hybrid
Influence on Biopharmaceutical Production Asn Loss of sialylation decreases EPO half-life from 2 h to 10 min Fukuda et al (1989). Blood; 73(1): 84-89 Asn Desialylation of intravenous immunoglobulin abrogates its anti-inflammatory properties Kaneko et al (2006). Science; 313(5787): 670-673 Asn Presence of gal-α(1,3)-gal can induce anaphylaxis (shock) and can be present on biotherapeutics Chung et al (2006). N Engl J Med; 358(11): 1109-1117 Asn Asn Half of the population have antibodies against β(1,2)-xylose and α(1,3)-core fucose Bardor et al (1995). Glycobiology; 13(6): 427-434
Glycosylated Therapeutic Proteins Over 190 EMA/FDA Approved Therapeutic Proteins. Over 127 are Glycoproteins (>66%) (Walsh: Nature Biotechnology 28, 917 924 (2010)
Glycan Release Considerations Different enzymes exist that facilitate the easy release of N- glycans from glycoproteins. High Mannose N-glycans Endo D cleaves paucimannose sugars Endoglycosidase cleavage site: Endo F1, Endo H, Endo M, Endo B N X P S/T PNGase F / PNGase A cleavage site
UPLC (Ultra performance liquid chromatography ): See More, Faster 5 µm Tosoh TSKgel Amide-80 4.6 mm x 250 mm 3 h gradient 2AB 70 80 90 100 110 120 Minutes Increased resolution Deceased Run times BEH material less retentive 1.7 µm Waters BEH Glycan 2.1 mm x 150 mm 30 min gradient 2AB 0 2 4 6 8 10 12 14 Minutes
How Are Glucose Unit (GU) Values Generated? Part 1 The elution times of glycans are expressed in glucose units (GU) by reference to a dextran ladder. 4.0 Each individual glycan structure has a GU value that is related to the number, linkage of its monosaccharides. 5.0 6.0 7.0 y=ax+bx 2 +cx 3 +dx 4 +ex 5 % 50 mm Ammonium formate ph 4.4 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0
How Are Glucose Unit (GU) Values Generated? Part 2 5.0 Derived GU value 6.0 Dextran ladder 7.0 8.0 9.0 10.0 11.0 F(6)A2 5.41 5.77 6.25 7.08 7.20 6.95 7.52 7.76 8.05 8.41 9.04 9.67 10.0 10.2 6.84 8.81 6.71 7.63 5.89 Herceptin N-linked glycans 0 Retention Time (min) 16
GU Value Caveats Whilst GU values are helpful, their validity is dependent upon: The column chemistry and separation conditions used for their generation, The sugar ladder used for their annotation, The method used to fit the regression line, Associated R 2 value. Glc (α1 6) n Glc (α1 4) n 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 GU Mittermayr & Guttman, Electrophoresis, 33, 2012, 1000-7 10 20 30 40 50 60 70 80 90 100 110 120 130 140 Retention Time [min]
GU Increments with Sugar Addition Linkage type β - linkage α - linkage Linkage position 6 8 4 unknown linkage 3 Symbol for sugar 2 Glc GlcNAc Gal GalNAc Fuc Man NeuNAc Xylose
Exoglycosidase Digestions Exoglycosidases are enzymes that remove monosaccharides from glycans with a defined linkage specificity. ABS GUH AMF BKF SPG BTG BTG ABS α2-3/6/8 NAN 1 α2-3/8 Sialidase ABS NAN 1 AMF GUH SPG BTG BKF X1-2F BKF α1-6/2 X1-2F α1-2 AMF α1-3/4 BTG β1-3/4/6 SPG β1-4 GUH β1-2/4/6 Fucosidase Galactosidase Hexosaminidase
Overview of N-linked Glycan Release with UPLC Analysis Exoglycosidase array of Etanercept by UPLC analysis RELEASED LABELLED GLYCANS Biotherapeutic denaturation with RapiGest SF PNGase F Dry Elute Formic Acid Resuspend Elute Elute Dry Wash Glycan identity confirmed by mass spectrometry HILIC-FLR-MS/MS 2-AB Label FLR Search Glycobase 3.2 for preliminary identification MS1 Identification
Good analysis is not enough, how does all the data fit together that is the real question A classic approach is to release the glycan's and then analyse them separately to the peptide sequence Biotherapeutic Protein Core Released Glycan structures Glycan characterization Sialylated glycans speciation Immunogenic glycans Mr. Potato Head Biotherapeutic Biotherapeutic Protein Core Peptide sequence Intact mass analysis Glycan PTM s Structures We tend to analyse these in isolation. This can lead to a miss understanding of how the structure exists, even though our data is correct Peptide mapping Intact m/z Glycan analysis Taking into account how all of these structures work together will deliver a true understanding of the biotherapeutic. Site Specific /Site Heterogeneity PTM Analysis Glycoform/Isoform Intact Mass Analysis 3 Dimensional Structure
RapiFluor-MS (RFMS) label provides over X10 FLR and X100 MS sensitivity compared to the 2AB label MS1 analysis A MS1 analysis B
UPLC-HILIC-FLR analysis of Ribonuclease B glycans Ribonuclease B 2AB Ribonuclease B 2AA Ribonuclease B RFMS
2AB/2AA N-Glycan sample preparation There are a number of Tags available, but the standard labeling method can take days to complete! 2-aminobenzoic acid 2-aminobenzamide Aniline 2-aminopyridine 2-aminoacridone Labeling can take 1-2 days, sometimes longer!!
RapiFluor-MS N-Glycan sample preparation From Glycoprotein to labeled N-glycans, to GU values with m/z data in 2 HOURS!
Herceptin (Trastuzumab) RFMS labeled N-linked Glycan Structural Assignments 5.0 6.0 7.0 8.0 9.0 10.0 Dextran ladder 5.29 5.45 6.52 6.65 7.40 7.97 8.52 5.73 6.48 5.77 Undigested 0.6 GU = 1 Sialic acids + Sialidase (ABS) 5.25 5.41 0.7 GU = x Galactose 5.28 5.44 5.76 6.61 7.37 1.62 GU = 2 x Galactose 1.2 GU = 2 Sialic acids + Galactosidase (ABS+BTG) 4.39 4.76 1.1 GU = 2 x N-Acetylgalactosamine +β-hexosaminidase (ABS+BTG+GUH)
Herceptin (Trastuzumab) N-linked Glycan Structural Assignment using Glycobase NIBRT GlycoBase accessed through www.glycoase.nibrt.ie or through Biopharmaceutical Platform Solution with UNIFI : Waters https://shar.es/1juhbx
RFMS labeled N-glycans analysed by UPLC-HILIC-FLR-QDa at NIBRT UPLC Stack FLR detector QDa The NIBRT QDa was donated by Waters as part of the Enterprise Ireland (EI) NIBRT-Waters project
Herceptin RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa UPLC-HILIC-FLR-QDa analysis 1200.00 EU 1100.00 1000.00 900.00 800.00 700.00 600.00 500.00 FLR* *GU values can also be determined with an appropriate processing method and dextran ladder 400.00 300.00 200.00 100.00 0.00 2.4x10 7 2.2x10 7 2.0x10 7 1.8x10 7 QDa 1.6x10 7 Intensity 1.4x10 7 1.2x10 7 1.0x10 7 8.0x10 6 6.0x10 6 4.0x10 6 2.0x10 6 0.0 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 Minutes Overlay of raw FLR and QDa TIC data
Herceptin RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa UPLC-HILIC-FLR-QDa analysis Extracted glycan m/z values from QDa data FLR* *GU values can also be determined with an appropriate processing method and dextran ladder QDa FLR with GU values
Enbrel (etanercept) RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa UPLC-HILIC-FLR-QDa analysis 1300.00 1200.00 1100.00 1000.00 FLR* EU 900.00 800.00 700.00 600.00 500.00 400.00 *GU values can also be determined with an appropriate processing method and dextran ladder 300.00 200.00 100.00 0.00 2.4x10 7 Intensity 2.2x10 7 2.0x10 7 1.8x10 7 1.6x10 7 1.4x10 7 1.2x10 7 1.0x10 7 8.0x10 6 6.0x10 6 4.0x10 6 2.0x10 6 QDa 0.0 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 Minutes Overlay of raw FLR and QDa TIC data
FLR* Enbrel (etanercept) RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa UPLC-HILIC-FLR-QDa analysis Extracted glycan m/z values from QDa data QDa FLR with GU values
Herceptin RFMS N-linked Glycans and UPLC-QDa Exoglycosidase digestion array TIC (500-1200 m/z) Undigested
Development of a novel workflow for monitoring O-Glycans utilising the new Waters QDa system Etanercept, Enbrel O-linked sites=13, hinge region. Very challenging. TNF-α receptor N-linked sites= 3 2 in the TNF-α receptor 1 in the Fc region Fc region
What about O-Glycans? Characterization of O-glycosylation is considerably more complicated than the analysis of their N-linked counterparts: Generally lower carbohydrate to protein ratio, sensitivity considerations Multicore structures, see below Chemical release often necessary * T S P S *Typical CHO O-glycans: Core 1, Mono- and Disialyl Core 1. Found on Enbrel, FC-Fusion and EPO biotherapeutics
How do we release O-Glycans? Chemical release is also possible, normally base catalyzed beta elimination type reactions. But there can be issues with chemical artifacts (peeling products). LC-MS-UNIFI O-Glycan database (Waters) UPLC-HILIC-FLR-MS (MS friendly tags preferred) Base: Hydrazine, hydroxide, ammonia LC-PGC-MS (Agilent /Thermo Fisher) Reducing agents OH Reducing sugars possible (labeling), peeling reactions problematic More stable alditols formed, subsequent labeling not possible
UPLC Analysis of Procainamide labeled Bovine Fetuin O-Glycans (HILIC Plate clean up) Ammonia-based β-elimination release (16hrs 65c) Desalting via evaporation (1 day) Procainamide labeling (or other MS friendly tag) O-linked Glycans HILCI plate clean up vs traditional paper chorography Peeling product! UPLC method optimization for QDA N-linked Glycans Preliminary assignments UPLC-HILIC- FLR
UPLC-HILIC-FLR-QDa Analysis of Procainamide labeled Bovine Fetuin O-Glycans FLR 529.03 Peeling product N glycan source QDa
Acknowledgments Waters THE SCIENCE OF WHAT S POSSIBLE TM