Transzlációs glikomika

Transcription

1 Ezerarcú szénhidrátok Az MTA Kémiai Tudományok Osztályának tudományos ülése Transzlációs glikomika Olajos Marcell és Guttman András MTA-PE Transzlációs Glikomika Kutatócsoport, Pannon Egyetem, Veszprém Horváth Laboratory of Bioseparation Sciences, Debreceni Egyetem Magyar Tudományos Akadémia, Budapest, november 5.

2 GLYCOMICS Glycome: the entire complement of sugars, whether free or in complex molecules. Glycomics (a subset of glycobiology) is the systematic study of all glycan structures of a given cell type or organism. Unlike genomes, glycome is highly dynamic. Protein glycosylation involves the interplay of > 600 enzymes. Mutations in glycosylation processing enzymes can be lethal or severely compromise health. Glycosylation is cell, protein and site specific (modulated by bioprocessing conditions and responsive to cell culture conditions). Specific monosaccharides / motifs provide recognition epitope/s (type, position, linkage) DNA RNA Polypeptide Glycoprotein

3 Symbolic representation of glycans Chemical structure of a complex core fucosylated biantennary galactosylated and sialylated glycan FA2BG2S2

4 Significance of Glycosylation Glycocalyx at the surface of an erythrocyte (Taylor and Drickamer, Glycobiology, Oxford) Glycans protect proteins, orient binding faces, prevent non-specific interactions, increase protein stability, e.g., N-glycans shield large areas of protein surfaces from proteases. Main glycosylation types on glycoproteins: - N-linked (Asn) Asn Asn Rillahan, C.; Paulson, J. Annu. Rev. Biochem. 2011, 80, O-linked (Ser/Thr) Ser/ Thr Microheterogeneity + site occupancy biological activity changes Rillahan, C.; Paulson, J. Annu. Rev. Biochem. 2011, 80,

5 Glycan analysis options CHALLENGE: complex, diversified structures; no chromophore / fluorophore groups; mostly not charged Analytical methods in glycan analysis: Structural characterization options: MS and NMR HPLC: - HPAE/PAD - Normal phase and HILIC (HPLC and UPLC) - Graphitized carbon (HPLC and chiplc) Capillary Electrophoresis Hyphenated Methods: LC-MS, CE-MS, CESI-MS

6 Glycan labeling by APTS Purpose: Introduction of label and charge Reductive amination Sugar reducing ends only ex 488 / em 520 nm LIF/LedIF, excellent sensitivity Simple, one step reaction Great efficiency (over 90%) under optimized conditions (reagent concentration, time, temperature, ph, solvent) Non-selective: uniform labeling for most structures Easy quantification: one fluorophore per sugar molecule

7 RFU RFU RFU RFU RFU Magnetic bead based sample preparation for Minute N-glycosylation analysis by CE-LIF A B PNGase F digestion Glycan capture C D E APTS labeling 50 IgG Clean-up A Elution with water 50 Fetuin F CE-LIF analysis of the supernatant B 40 APTS 30 Picoleneborane Unpurified Water Unpurified COOH COOH COOH Magbead purified COOH COOH COOH Magbead purified 1h/50 C magnet 0 magnet magnet h/37 C Minute Minute IgG A Fetuin B RNAse B C Unpurified 30 Unpurified 30 Unpurified Magbead purified Magbead purified Magbead purified Minute Minute Minute

8 RFU Charge / Hydrodynamic-volume based separation by CE APTS 2.0 A) Glu(α1 4) n B) Glu(β1 4) n C) GluNAc(β1 4) n Time (min)

9 Sequential Exoglycosidase Digestion

10 Bottom-up Identification + ~ 1 GU ~ 1.8 GU + ~ GU ~ 2.4 GU

11 AEC Fractionation

12 TRANSLATIONAL ASPECTS BIOTHERAPEUTICS BIOMEDICAL Immunoglobulin G 1

13 The manufacturing process of recombinant therapeutic proteins Cell expansion Process Development Transfection/Selection Production/Bioreactors Cloning Characterization and validation Purification

14 Alterations in glycosylation processing Many factors contribute to alterations in glycan processing on recombinant glycoproteins Expression levels of the processing enzymes in the host cell line Monosaccharide nucleotide donor levels Cell signaling pathways: cytokines/hormones, drugs, media components Loss of cellular organelle organization due to e.g., ph changes Mutations in genes, gene silencing, overexpression Bioprocessing environment such as temperature, oxygen level, etc. Sugars are involved in key aspects of bioprocessing Importance of Process Analytical Technology Quality by design: Importance of sugar function relationship Selection of cell lines with appropriate glycosylation Proper analytical toolset to ensure that the product has correct glycosylation for optimal activity

15 IgG1 Wormald, Rudd & Dwek. Oxford Glycobiology Institute. Fab Fc One N-linked glycosylation site ~Asn 297. Variability depending on cell line and expression conditions. Structural diversity: Fuc, Gal, Neu5AC, GlcNAc Glycosylation determines: - biological activity, - physicochemical properties, - ADCC and CDC functions. - Immunogenicity: α1-3 Gal, NGNA Importance of IgG glycosylation analysis in: - clone selection, - product characterization, - lot release

16 Interpretation of IgG glycosylation Half Life (galactose) Anti-Inflammatory (sialic acid) CDC Activation (GlcNAc) ADCC Suppression (core fucose)

17 Malignant cells release glycoproteins carrying disease-related glycans into the interstitial space, where they can reach the circulation The glycoprotein products of tumor cells carry aberrant carbohydrate structures compared to their normal counterparts. Typical changes include increased levels of fucose (red triangle) and sialic acid (purple diamond), the addition of polylactosamine units [repeating sequences of galactose (yellow circle) and N- acetylglucosamine (blue square)], and higher-order branching of N-linked glycans. Drake, P. M. et al. Clin Chem 2010;56:

18 The increased size of N-glycans that occurs upon malignant transformation can be explained by an elevation in GlcNAc transferase-v (GNT-V) activity Essentials of Glycobiology Second Edition Chapter 44, Figure 1

19 Human Serum Glycome Profiling by CE

20 N-linked glycan profiling of pooled healthy and prostate cancer patient sera after BLAC partitioning Fuc Olajos et al, Anal. Chem. 2008, 80,

21 Acknowledgment MTA-PE Translational Glycomics Group, Veszprém, HU Bodnár Judit Hajba László Járvás Gábor Patakiné Ostoros Györgyi Schmidt Anikó Szigeti Márton Horváth Laboratory of Bioseparation Sciences, University of Debrecen, HU Dönczö Boglárka Kalmár László Kerékgyártó Márta Kovács Zsuzsanna Váradi Csaba Beckman Coulter, ProZyme, PhyNexus