New Methods for Characterization of N-type Glycosylation of Proteins by Integration of LC-MS/MS and NMR Thesis Submitted in fulfillment of the requirements of the degree Doctor rer. nat. at the Department of Chemistry, Faculty of Sciences, University of Hamburg by Alena Wiegandt from Hamburg Hamburg, 2016 This thesis was prepared at the Institute for Organic Chemistry from October 2012 to July 2016 (managing director: Prof. Dr. C.B.W. Stark). I would like to thank Prof. Dr. Bernd Meyer for his continuous and motivating support during the work on my PhD thesis. I would like to thank Prof. Dr. Dr. h. c. mult. Wittko Francke for being the second reviewer of this thesis. 1st Reviewer: Prof. Dr. Bernd Meyer 2nd Reviewer: Prof. Dr. Dr. h.c. mult. Wittko Francke Date of defense: 07.04.2017 OUTLINE Outline ABBREVIATIONS ..................................................................................................................... VIII 1 SUMMARY ............................................................................................................................ 1 2 ZUSAMMENFASSUNG .......................................................................................................... 4 3 INTRODUCTION ................................................................................................................... 7 3.1 Glycosylation of Proteins - Biosynthesis, Structures, Functions .................................... 8 3.2 Glycans in Human Diseases .......................................................................................... 10 Potential of Glycans as Specific Biomarkers for Human Diseases ...................... 11 Limits of Glyco-Based Biopharmaceuticals - Quality Control ............................ 13 3.3 Analytics of Protein Glycosylation ............................................................................... 14 4 OBJECTIVE ........................................................................................................................ 19 5 RESULTS AND DISCUSSION ............................................................................................... 20 5.1 Glycosylation of Biopharmaceuticals ........................................................................... 20 Three-Dimensional Cross Correlation of Cetuximab ........................................... 22 Glycosylation Analysis on the Level of Intact Proteins ....................................... 38 Interlaboratory Study on Glycan Analysis ........................................................... 59 5.2 Histidine-Rich Glycoprotein ......................................................................................... 71 Development of a Purification Strategy for HRG from Human Blood Plasma ... 72 Mass Spectrometric Analysis of Histidine-Rich Glycoprotein ............................ 79 5.3 Prolactin-Inducible Protein ........................................................................................... 86 Structural Analysis of the N-Glycosylation of Human Salivary PIP ................... 87 Enrichment of Prolactin-Inducible Protein from Human Saliva .......................... 99 Nonsynonymous Single Nucleotide Polymorphism in Salivary PIP ................. 103 Comparison of the N-Glycosylation of Salivary PIP from Healthy Individuals 107 N-Glycosylation of Salivary PIP during Pregnancy ........................................... 128 Comparative Study of N-Glycosylation of Salivary PIP between Breast ............... Cancer Patients and Healthy Donors .................................................................. 129 V OUTLINE 6 EXPERIMENTAL PROCEDURES ....................................................................................... 132 6.1 Instruments, Material and Software ............................................................................ 132 6.2 Analysis of Released N-Glycans of Cetuximab .......................................................... 133 N-Glycan Release from Cetuximab and Purification ......................................... 133 Chromatographic Separation, Mass Spectrometric Analysis and Fractionation ...... of Released N-Glycans from Cetuximab by PGC-LC-ESI-q/TOF ..................... 134 1H-NMR Analysis of Released N-Glycans from Cetuximab .............................. 135 Three-Dimensional Cross Correlation (3DCC) of Released N-Glycans ................. from Cetuximab .................................................................................................. 135 6.3 Analysis of Intact mAbs and mAb Sub-Domains ....................................................... 135 MS Analysis of Intact mAbs for N-Glycosylation Analysis ............................... 135 Middle Up Approach for Analyzing the N-Glycosylation of Cetuximab ........... 136 Middle Up Approach for Analyzing the N-Glycosylation of Bevacizumab ....... 137 6.4 Interlaboratory Study on Glycan Analysis .................................................................. 139 MS Analyses of Intact NIST Monoclonal Antibodies ........................................ 139 Middle Up Analyses of NIST Monoclonal Antibodies ...................................... 139 Bottom Up Analyses of NIST Monoclonal Antibodies ...................................... 140 6.5 Histidine-Rich Glycoprotein ....................................................................................... 142 Immobilized Metal Affinity Chromatography by Ni2+-NTA Resin for ................... Isolation of Human Histidine-Rich Glycoprotein ............................................... 143 Size-Exclusion Chromatography of IMAC Enriched Blood Components .............. for Isolation of Human Histidine-Rich Glycoprotein ......................................... 143 SDS-PAGE for Control of HRG Enrichment ..................................................... 143 Dynamic Light Scattering for Determination of Size Distribution of ..................... Enriched HRG .................................................................................................... 144 Acquisition of MALDI Spectra of Intact Histidine-Rich Glycoprotein ............. 144 Plasmin Cleavage of Histidine-Rich Glycoprotein ............................................. 144 6.6 Prolactin-Inducible Protein .......................................................................................... 145 VI OUTLINE Saliva Preparation .............................................................................................. 145 Antibody Affinity Chromatography for Isolation of Salivary PIP ..................... 145 Optimizing Parameters for Isolation of Salivary PIP by AIEX ......................... 146 Comparison of Human PIP from Multiple Saliva Samples by 2D-LC-MS ............ (AIEX-RP) ......................................................................................................... 146 Comparison of Human PIP from Multiple Saliva Samples by 2D-LC-MS ............ (SEC-RP) ............................................................................................................ 147 Combination of AIEX and SEC for PIP Enrichment ......................................... 147 Neuraminidase Digest ........................................................................................ 148 Release of N-Glycans from Human Salivary PIP .............................................. 148 Mass Spectrometric Analyses of PIP Samples ................................................... 148 7 HAZARDS ......................................................................................................................... 150 8 APPENDIX ........................................................................................................................ 152 9 REFERENCES ................................................................................................................... 174 ACKNOWLEDGEMENTS........................................................................................................... 184 DANKSAGUNG ......................................................................................................................... 185 CURRICULUM VITAE .............................................................................................................. 186 AFFIDAVIT ............................................................................................................................... 188 VII ABBREVIATIONS ABBREVIATIONS 1D one-dimensional 3DCC three-dimensional cross correlation aa amino acid ACN acetonitrile ADCC antibody-dependent cellular cytotoxicity AFP α-Fetoprotein AIEX anion exchange chromatography BPC base peak chromatogram CDG congenital disorder of glycosylation CCSD complex carbohydrate structure database CFG Consortium for Functional Glycomics CHO chinese hamster ovary CID collision-induced dissociation COSY correlation spectroscopy CRC colorectal cancer CRD carbohydrate-recognition domain CV column volume DAC diammonium citrate DHAP 2,5-dihydroxyacetophenone DHB 2,5-dihydroxybenzoic acid DLS dynamic light scattering DTT 1,4-D/L-dithiothreitol EDC extracted delta chromatogram EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor EGFR epidermal growth factor receptor EIC extracted ion chromatogram ER endoplasmic reticulum ESI electrospray ionization FA formic acid GBP glycan-binding protein VIII ABBREVIATIONS GCDFP-15 gross cystic disease fluid protein 15 gp17 glycoprotein 17 hc heavy chain HCC hepatocellular cancer HPLC high performance liquid chromatography HR high resolution HRG histidine-rich glycoprotein IAA iodoacetamide IEX