Dr Mark Hilliard, NIBRT. Waters THE SCIENCE OF WHAT S POSSIBLE TM

Transcription

1 RFMS Glycan Characterization Techniques for Biotherapeutics Dr Mark Hilliard, NIBRT Waters THE SCIENCE OF WHAT S POSSIBLE TM

2 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).

3 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

4 Classes of N-Linked Glycans High Mannose Complex Hybrid

5 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): Asn Desialylation of intravenous immunoglobulin abrogates its anti-inflammatory properties Kaneko et al (2006). Science; 313(5787): 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): 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):

6 Glycosylated Therapeutic Proteins Over 190 EMA/FDA Approved Therapeutic Proteins. Over 127 are Glycoproteins (>66%) (Walsh: Nature Biotechnology 28, (2010)

7 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

8 UPLC (Ultra performance liquid chromatography ): See More, Faster 5 µm Tosoh TSKgel Amide mm x 250 mm 3 h gradient 2AB 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 Minutes

9 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 y=ax+bx 2 +cx 3 +dx 4 +ex 5 % 50 mm Ammonium formate ph

10 How Are Glucose Unit (GU) Values Generated? Part Derived GU value 6.0 Dextran ladder F(6)A Herceptin N-linked glycans 0 Retention Time (min) 16

11 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 GU Mittermayr & Guttman, Electrophoresis, 33, 2012, Retention Time [min]

12 GU Increments with Sugar Addition Linkage type β - linkage α - linkage Linkage position unknown linkage 3 Symbol for sugar 2 Glc GlcNAc Gal GalNAc Fuc Man NeuNAc Xylose

13 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

14 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

15 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

16 RapiFluor-MS (RFMS) label provides over X10 FLR and X100 MS sensitivity compared to the 2AB label MS1 analysis A MS1 analysis B

17 UPLC-HILIC-FLR analysis of Ribonuclease B glycans Ribonuclease B 2AB Ribonuclease B 2AA Ribonuclease B RFMS

18 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!!

19 RapiFluor-MS N-Glycan sample preparation From Glycoprotein to labeled N-glycans, to GU values with m/z data in 2 HOURS!

20 Herceptin (Trastuzumab) RFMS labeled N-linked Glycan Structural Assignments Dextran ladder Undigested 0.6 GU = 1 Sialic acids + Sialidase (ABS) GU = x Galactose GU = 2 x Galactose 1.2 GU = 2 Sialic acids + Galactosidase (ABS+BTG) GU = 2 x N-Acetylgalactosamine +β-hexosaminidase (ABS+BTG+GUH)

21 Herceptin (Trastuzumab) N-linked Glycan Structural Assignment using Glycobase NIBRT GlycoBase accessed through or through Biopharmaceutical Platform Solution with UNIFI : Waters

22 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

23 Herceptin RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa UPLC-HILIC-FLR-QDa analysis EU FLR* *GU values can also be determined with an appropriate processing method and dextran ladder x x x x10 7 QDa 1.6x10 7 Intensity 1.4x x x x x x x Minutes Overlay of raw FLR and QDa TIC data

24 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

25 Enbrel (etanercept) RFMS labeled glycans analysed by UPLC-HILIC-FLR-QDa UPLC-HILIC-FLR-QDa analysis FLR* EU *GU values can also be determined with an appropriate processing method and dextran ladder x10 7 Intensity 2.2x x x x x x x x x x x10 6 QDa Minutes Overlay of raw FLR and QDa TIC data

26 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

27 Herceptin RFMS N-linked Glycans and UPLC-QDa Exoglycosidase digestion array TIC ( m/z) Undigested

28 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

29 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

30 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

31 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

32 UPLC-HILIC-FLR-QDa Analysis of Procainamide labeled Bovine Fetuin O-Glycans FLR Peeling product N glycan source QDa

33 Acknowledgments Waters THE SCIENCE OF WHAT S POSSIBLE TM