M E T H O D S I N M O L E C U L A R B I O L O G Y ™

Series EditorJohn M. Walker

School of Life SciencesUniversity of Hertfordshire

Hat fi eld, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Glycosylation Engineering of Biopharmaceuticals

Methods and Protocols

Edited by

Alain Beck

Antibody Physico-Chemistry Department, Centre d’Immunologie Pierre-Fabre, 5 Avenue Napoléon III, BP 60497, Saint Julien-en-Genevois, France

EditorAlain Beck, Ph.D.Antibody Physico-Chemistry Department Centre d’Immunologie Pierre-Fabre5 Avenue Napoléon III, BP 60497Saint Julien-en-Genevois, France

ISSN 1064-3745 ISSN 1940-6029 (electronic)ISBN 978-1-62703-326-8 ISBN 978-1-62703-327-5 (eBook)DOI 10.1007/978-1-62703-327-5Springer New York Heidelberg Dordrecht London

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v

Foreword: Glyco-Engineering of Monoclonal Antibodies

Monoclonal antibodies (mAbs) have become increasingly important as innovative thera-peutic agents during the past two decades. Interest by biopharmaceutical fi rms in develop-ing these products has been driven by critical advances in the engineering, design and manufacturing of mAbs, as well as improved understanding of target biology and antibody mechanisms of action. As of 2012, the rate at which new mAb product candidates enter clinical study has risen to ~60 per year, a total of ~350 mAbs are undergoing evaluation in clinical studies, over 30 mAbs have been approved in either the United States or European Union, and annual global sales of mAb therapeutics total nearly $60 billion.

Although canonical antibodies (i.e., unmodi fi ed full-length molecules) have a notable record of success, the biopharmaceutical industry, often in collaboration with academic and government organizations, is now dedicating substantial resources to the exploration of new types of mAbs that may ful fi ll unmet medical need, or prove safer and more ef fi cacious com-pared with the currently marketed products. In particular, research has focused on improv-ing critical antibody properties (e.g., potency) by controlling the composition of carbohydrates on the molecule. A commonly used approach to glyco-engineering antibodies involves reduction of fucose, which has been shown to enhance antibody-dependent cell-mediated cytotoxicity (ADCC). Two companies, BioWa/Kyowa Hakko Kirin and GlycArt/Roche, have developed proprietary cell lines that yield defucosylated antibodies. To achieve this result, BioWa/Kyowa Kirin Hakko established α -1,6-fucosyltransferase (FUT8) enzyme knockout Chinese hamster ovary cell line (POTELLIGENT ® technology), while GlycArt/Roche chose to over-express heterologous β 1,4-N-acetylglucosaminyltransferase III in anti-body-producing cells (GlycoMab™ technology). This glyco-engineering approach was vali-dated with the approval in Japan of mogamulizumab (POTELIGEO ® ), which was derived from the POTELLIGENT ® technology. Mogamulizumab’s approval as a treatment for patients with relapsed or refractory CCR4-positive adult T-cell leukemia-lymphoma, which was granted March 30, 2012 by the Japanese Ministry of Health, Labour and Welfare, is a triumph for the glyco-engineering fi eld.

To my knowledge, a total of 15 glyco-engineered mAbs are in the commercial clinical pipeline as of mid-2012. Six POTELLIGENT ® -derived mAbs are in clinical studies, with three (benralizumab, MEDI-551, BIW-8962) in Phase 2 studies and three (KHK2898, KHK2804, KHK2866) in Phase 1 studies. Benralizumab targets interleukin-5 receptor alpha chain, and it is undergoing evaluation as a treatment for asthma and for chronic obstructive pulmonary disease. MEDI-551, which targets CD19 on B cells, is currently in Phase 2 studies in adults with diffuse large B cell lymphoma or adults with chronic lympho-cytic leukemia, Phase 1/2 studies of patients with scleroderma or relapsing-remitting mul-tiple sclerosis, and Phase 1 studies in adults with advanced B cell malignancies. Anti-GM2 ganglioside BIW-8962 was undergoing evaluation as monotherapy in a Phase1/2 study of patients with previously treated multiple myeloma, but the study was terminated due to lack of ef fi cacy. KHK2898, KHK2804, and KHK2866 target CD98, a tumor glycan, and heparin-binding epidermal growth factor-like growth factor, respectively; these mAbs are undergo-ing evaluation in Phase 1 studies as therapies for patients with advanced solid tumors.

vi

Three GlycoMab™-derived mAbs (obinutuzumab, GA201, RO5479599) are in clinical study. Anti-CD20 obinutuzumab, which is also known as GA101, is undergoing evaluation in four Phase 3 studies, four Phase 2 studies, and two Phase 1 studies, all of which include patients with hematological malignancies such as non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. The anti-epidermal growth factor receptor (EGFR) GA201 is currently being evaluated in Phase 2 studies of patients with non-small cell lung cancer or colorectal cancer, and a Phase 1 study of patients with head and neck squamous cell carcinoma. RO5479599, which targets human epidermal growth factor receptor (HER)-3, is in a Phase 1 dose-escalation study in patients with HER3-positive solid tumors.

The remaining six of the 15 glyco-engineered mAbs in the clinical pipeline are either derived from YB2/0 rat cells or Glycotope’s GlycoExpress™ technology. The YB2/0 cell line expresses FUT8 at a low level, and thus it produces recombinant mAbs with low fucose content. Three mAbs (ecromeximab, roledumab, ublituximab) in clinical studies are derived from YB2/0 cells. The safety and effectiveness of anti-GD3 ecromeximab, developed by Kyowa Hakko and licensed by Life Science Pharmaceuticals, is being evaluated in a Phase 2 study of patients with metastatic melanoma. LFB is developing two low-fucose mAbs, roledumab, and ublituximab. Anti-rhesus (Rh) D roledumab was evaluated in a Phase 2 study designed to demonstrate the ability of LFB-R593 to effectively eliminate exogenously administered RhD-positive red blood cells from the circulation of an RhD-negative indi-vidual, thereby preventing RhD alloimmunization. Anti-CD20 ublituximab, which is licensed to TG Therapeutics, is being evaluated in a Phase 1/2 study of patients with relapsed or refractory B-cell lymphoma who were previously treated with rituximab. The GlycoExpress™ technology utilizes glyco-engineered human cell lines that allow expression of antibodies with varying percentages of sialylation, fucosylation, and galactosylation. Three GlycoExpress™-derived mAbs (GT-MAB2.5GEX, GT-MAB5.2GEX, GT-MAB7. 3GEX) are in Phase 1 studies. The safety and tolerability of GT-MAB2.5GEX, which targets MUC1, is being evaluated in a dose escalation study in patients with advanced MUC1-positive solid malignancies. Anti-EGFR GT-MAB5.2GEX and anti-HER2 GT-MAB7.3GEX are undergoing evaluation in Phase 1 studies of patients with EGFR-positive and HER2-positive solid tumors, respectively.

Enhancing ADCC through reduction of fucose in antibodies produced in mammalian cells has proven valuable, but there are numerous other approaches to antibody glyco- engineering that are also being explored. Antibodies with humanized glycoforms can now be produced in non-mammalian cell lines such as yeast ( Pichia pastoris and Saccharomyces pombe and cerevisiae ), fi lamentous fungi ( Aspergillus niger and nidulans ), duck embry-onic stem cells, and plants. Galactosylation levels, which are correlated with complement-dependent cytotoxicity activity, can be manipulated via the concentrations of components of the cell culture medium. In yet another approach, alterations in antibody sialyation, which have been shown to affect anti-in fl ammatory properties and ADCC, can be achieved through glyco-engineering. These approaches are all being applied to the development of glyco-engineered biobetter or next-generation mAbs that may enter the clinical pipeline in the near future.

Because of the importance of advances in glyco-engineering to antibody development and the obvious promise of designed mAbs as therapeutics, the publication of Glycosylation Engineering of Biopharmaceuticals , edited by Alain Beck, is notable and timely. The current methodologies for therapeutic glycoprotein engineering used to achieve a variety of effects, including strategies to enhance cytotoxicity or in fl ammatory properties, are explained in a clear and concise manner. Production of glyco-engineered biopharmaceuticals from various

Foreword

vii

cell types, including mammalian, yeast or fungi, baculovirus/insect, prokaryote and plant, and via biochemistry- or chemical-based methods, is discussed in detail. In addition, critical protocols for glyco-analysis, functional assays, and pharmacokinetic, pharmacodynamic and toxicology assessment of glyco-engineered biopharmaceuticals are included. Glycosylation Engineering of Biopharmaceuticals thus provides comprehensive coverage of this important topic and will most certainly prove to be a valuable resource for both experts and those new to the fi eld.

Janice M. Reichert, Ph.D.

Foreword

ix

Preface

Therapeutic properties of monoclonal antibodies (mAbs) and other glycoproteins strongly depend on the composition of their glycans. Most of the currently approved biopharmaceu-ticals are produced in mammalian cell lines, which yield mixtures of different glycoforms that are close to those of humans but not fully identical. Glyco-engineering is being developed as a method to control the composition of carbohydrates and to enhance the pharmacological properties of mAbs and other proteins. The approval, on April 30, 2012, in Japan, of moga-mulizumab, the fi rst glyco-engineered antibody to reach the market, was a landmark in the fi eld of engineered biopharmaceuticals. Mogamulizumab is a humanized mAb with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity linked to opti-mized a-fucosylated glycoforms and an illustration of the therapeutic importance of a tailored glycosylation. The antibody is indicated for patients with relapsed or refractory CCR4-positive adult T-cell leukemia-lymphoma.

The aim of this present volume of Methods in Molecular Biology is to provide readers with production and characterization protocols of glycoproteins and glyco-engineered bio-pharmaceuticals with a focus on mAbs. The volume is divided in four complementary parts dealing with Glyco-engineering of therapeutic proteins (Part I), Glycoanalytics (Part II), Glycoprotein complexes characterization (Part III), and PK/PD assays for therapeutic anti-bodies (Part IV).

The fi rst two chapters deal with recombinant glycoproteins produced in Chinese Hamster Ovary (CHO) cells, the most frequently used cell line to produce biopharmaceu-ticals. J. Holgersson (University of Gothenburg, SE) and colleagues report methods to engi-neer therapeutic and diagnostic O-glycans on recombinant mucin-type immunoglobulin fusion proteins. C. Ronin et al. (Siamed’Xpress, FR) follow up with protocols to engineer human-like glycosylation of therapeutic glycoproteins based on 6-linked sialylation. The next three chapters discuss the use of non-mammalian cell line to produce glycoproteins including yeasts ( Pichia pastoris and Saccharomyces cerevisiae ) and insect cells infected with baculoviruses. First, D. Zha (Merck-GlycoFi, US) describes the production of glyco-engineered Pichia-based expression of Monoclonal Antibodies. Then C. Javaud (Glycode, FR) presents the humanization of N-glycosylation of antibodies produced in S. cerevisiae . Finally, M. Cérutti (CNRS, FR) and colleagues report methods to engineer the baculovirus genome to produce galactosylated antibodies.

To assess the structure of glycoprotein and glyco-engineered biopharmaceuticals, state-of-the art orthogonal analytical methods are needed. E. Wagner-Rousset (CIPF, FR), C. Schaeffer-Reiss (CNRS-LSMBO), and colleagues describe nanoLC-chips-MS/MS methods for the characterization on N-glycopeptides generated from trypsin digestion of monoclonal antibodies. Alternatively, M.C-Janin Bussat, L. Tonini, and colleagues (CIPF, FR) propose the use of IdeS proteolytic digestion and electrospray ionization—time-of- fl ight mass spectrometry for antibody fast differential glycopro fi lling of cetuximab Fab and Fc glycans. Then, A. Delobel, G. Van Vyncht, et al. (Quality Assistance, BE) report a panel of analytical methods that are used to characterize therapeutic antibody glycosylation for batch release or comparability support, tacking the case of trastuzumab. To have a complete

x

picture, mass spectrometric analysis of O -linked oligosaccharides from various recombinant expression systems are described by J. Holgersson, N.G. Karlsson, and colleagues (University of Gothenburg, SE). In complement to mass spectrometry, glycopro fi lling can also be per-formed by liquid chromatography and by electrophoresis based methods. This is illustrated by T.S. Raju (Janssen R&D, US) who reports the assessment of Fc Glycan heterogeneity of therapeutic recombinant mAbs by Normal Phase—HPLC, and by R.R. Rustandi and col-leagues (Merck) who report two different Capillary Electrophoresis systems. Alternatively glycopro fi lling may be performed based on lectins as illustrated by L. Landemarre (GLYcodiag, FR) and E. Duverger (Université d’Orléans, FR) for recombinant therapeutic Interleukin-7. Glycosylation also has a deep impact on glycoprotein solubility and limits the propensity to aggregate. This can be assessed either by Hydrophobic Interaction Chromatography, as illustrated by R.R. Rustandi (Merck, US), or by Sedimentation Velocity Analytical Ultracentrifugation as reported by W.B. Stine (Abbott, US).

To go a step forward, glycoprotein complexes can be characterized by emerging mass spectrometry methods. S. Sanglier-Cianferani (CNRS-LSMBO), E. Wagner-Rousset (CIPF, FR), and colleagues report the use of non-covalent mass spectrometry for the characteriza-tion of antibody/antigen complexes. In addition, conformational analysis of recombinant mAbs can be performed by Hydrogen/Deuterium Exchange Mass Spectrometry as reported by D. Houde (BiogenIdec, US) and J.R. Engen (Northeastern University, Boston, MA). To gain insights on the interaction of antibodies with their target antigens, epitope and paratope can be mapped by different Mass Spectroscopy methods as described by Victor Obungu and colleagues (Lilly, US).

Last, but not least, PK/PD assays for therapeutic antibodies are mandatory to explore the impact of glycosylation or glyco-engineering on pharmacokinetics and potency. For this purpose, M. Broussas, L. Goetsch, and L. Broyer (CIPF, FR) describe a method for the evaluation of antibody-dependent cell cytotoxicity (ADCC) using Lactate Dehydrogenase Measurement and a method for complement-dependent cytotoxicity (CDC) determination using ATP measurement and C1q/C4b binding. As a surrogate in vitro assay, the capture of the human IgG1 antibodies by protein A for the kinetic study of h-IgG/FcgammaR interaction using SPR-based biosensor technology is reported by T. Champion (CIPF, FR). To evaluate the antibody clearance, a mass spectrometry protocol for the absolute quanti fi cation of a mAbs in serum with immuno-puri fi cation is described by F. Becher and colleagues (CEA, FR).

I would like to acknowledge Janice M. Reichert for her foreword as well as John M. Walker for his invitation to edit this volume of Methods in Molecular Biology and for his enthusiasm and his support.

The book is dedicated to my wife Nathalie, to my daughters Juliette and Louise, and to my parents Paulette and Norbert. Thanks also to Claire Catry for her help in some logistics aspect concerning this book.

Saint Julien-en-Genevois, France Alain Beck

Preface

xi

Contents

Foreword: Glyco-Engineering of Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . vPreface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ixContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

PART I GLYCO-ENGINEERING OF THERAPEUTIC PROTEINS

1 Engineering of Therapeutic and Diagnostic O-Glycans on Recombinant Mucin-Type Immunoglobulin Fusion Proteins Expressed in CHO Cells . . . . . 3Linda Lindberg, Jining Liu, and Jan Holgersson

2 Engineering a Human-Like Glycosylation to Produce Therapeutic Glycoproteins Based on 6-Linked Sialylation in CHO Cells . . . . . . . . . . . . . . . 19Nassimal El Maï, Sandrine Donadio-Andréi, Chloé Iss, Valérie Calabro, and Catherine Ronin

3 Glycoengineered Pichia-Based Expression of Monoclonal Antibodies . . . . . . . 31Dongxing Zha

4 N-Glycosylation Humanization for Production of Therapeutic Recombinant Glycoproteins in Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . 45Christelle Arico, Christine Bonnet, and Christophe Javaud

5 Engineering the Baculovirus Genome to Produce Galactosylated Antibodies in Lepidopteran Cells . . . . . . . . . . . . . . . . . . . . . . . 59Sylvie Juliant, Marylêne Lévêque, Pierre Cérutti, Annick Ozil, Sylvie Choblet, Marie-Luce Violet, Marie-Christine Slomianny, Anne Harduin-Lepers, and Martine Cérutti

PART II GLYCOANALYTICS

6 NanoLC Chips MS/MS for the Characterization of N-Glycopeptides Generated from Trypsin Digestion of a Monoclonal Antibody. . . . . . . . . . . . . 81Elsa Wagner-Rousset, Christine Schaeffer-Reiss, Audrey Bednarczyk, Nathalie Corvaïa, Alain Van Dorsselaer, and Alain Beck

7 Cetuximab Fab and Fc N -Glycan Fast Characterization Using IdeS Digestion and Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . 93Marie-Claire Janin-Bussat, Laure Tonini, Céline Huillet, Olivier Colas, Christine Klinguer-Hamour, Nathalie Corvaïa, and Alain Beck

8 Therapeutic Antibody Glycosylation Analysis: A Contract Research Organization Perspective in the Frame of Batch Release or Comparability Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115Arnaud Delobel, Fabrice Cantais, Anicet Catrain, Erell Dereux, and Géry Van Vyncht

xii

9 Mass Spectrometric Analysis of O-Linked Oligosaccharides from Various Recombinant Expression Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145Diarmuid T. Kenny, Stefan Gaunitz, Catherine A. Hayes, Anki Gustafsson, Magnus Sjöblom, Jan Holgersson, and Niclas G. Karlsson

10 Assessing Fc Glycan Heterogeneity of Therapeutic Recombinant Monoclonal Antibodies Using NP-HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169T. Shantha Raju

11 Application of Capillary Electrophoresis in Glycoprotein Analysis . . . . . . . . . . 181Richard R. Rustandi, Carrie Anderson, and Melissa Hamm

12 Characterization of Glycoprotein Biopharmaceutical Products by Caliper LC90 CE-SDS Gel Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . 199Grace Chen, Sha Ha, and Richard R. Rustandi

13 Hydrophobic Interaction Chromatography to Analyze Glycoproteins . . . . . . . 211Richard R. Rustandi

14 Lectin Glycoprofiling of Recombinant Therapeutic Interleukin-7 . . . . . . . . . . 221Ludovic Landemarre and Eric Duverger

15 Analysis of Monoclonal Antibodies by Sedimentation Velocity Analytical Ultracentrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227W. Blaine Stine Jr.

PART III GLYCOPROTEIN COMPLEXES CHARACTERIZATION

16 Noncovalent Mass Spectrometry for the Characterizationof Antibody/Antigen Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243Cédric Atmanene, Elsa Wagner-Rousset, Nathalie Corvaïa, Alain Van Dorsselaer, Alain Beck, and Sarah Sanglier-Cianferani

17 Conformational Analysis of Recombinant Monoclonal Antibodies with Hydrogen/Deuterium Exchange Mass Spectrometry. . . . . . . . . . . . . . . . 269Damian Houde and John R. Engen

18 Epitope Mapping of Antibodies by Mass Spectroscopy: A Case Study . . . . . . . 291Victor H. Obungu, Valentina Gelfanova, and Lihua Huang

PART IV PK/PD ASSAYS FOR THERAPEUTIC ANTIBODIES

19 Evaluation of Antibody-Dependent Cell Cytotoxicity Using Lactate Dehydrogenase (LDH) Measurement . . . . . . . . . . . . . . . . . . . . . . . . . 305Matthieu Broussas, Lucile Broyer, and Liliane Goetsch

20 Evaluation of Complement-Dependent Cytotoxicity Using ATP Measurement and C1q/C4b Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319Lucile Broyer, Liliane Goetsch, and Matthieu Broussas

21 Capture of the Human IgG1 Antibodies by Protein A for the Kinetic Study of h-IgG/FcgR Interaction Using SPR-Based Biosensor Technology . . . 331Thierry Champion and Alain Beck

22 Mass Spectrometry Protocol for the Absolute Quantification of a Monoclonal Antibody in Serum with Immunopurification . . . . . . . . . . . . 345François Becher, Mathieu Dubois, François Fenaille, and Eric Ezan

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

Contents

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Contributors

CARRIE ANDERSON • Vaccine Analytical Development , Merck Research Laboratories , West Point , PA , USA

CHRISTELLE ARICO • Glycode S.A.S , Uzerche , France CÉDRIC ATMANENE • Laboratoire de Spectrométrie de Masse Bio-Organique ,

IPHC, CNRS, UMR7178, Université de Strasbourg , Strasbourg , France ALAIN BECK • Antibody Physico-Chemistry Department , Centre d’Immunologie

Pierre-Fabre , Saint Julien-en-Genevois , France FRANÇOIS BECHER • Service de Pharmacologie et d’Immunoanalyse , CEA ,

Gif-sur-Yvette , France AUDREY BEDNARCZYK • Laboratoire de Spectrométrie de Masse Bio-Organique ,

IPHC, CNRS, UMR7178, Université de Strasbourg , Strasbourg , France CHRISTINE BONNET • Glycode S.A.S , Uzerche , France MATTHIEU BROUSSAS • Experimental Oncology Department , Centre d’Immunologie

Pierre-Fabre , Saint Julien-en-Genevois , France LUCILE BROYER • Experimental Oncology Department , Centre d’Immunologie

Pierre-Fabre , Saint Julien-en-Genevois , France VALÉRIE CALABRO • Siamed’Xpress , University Aix-Marseille , Marseille , France FABRICE CANTAIS • Quality Assistance , Donstiennes , Belgium ANICET CATRAIN • Quality Assistance , Donstiennes , Belgium MARTINE CÉRUTTI • CNRS UPS3044 , Saint Christol-Lèz-Alès , France PIERRE CÉRUTTI • CNRS UPS3044 , Saint Christol-Lèz-Alès , France THIERRY CHAMPION • Physico-Chemistry Department , Centre d’Immunologie

Pierre-Fabre , Saint Julien-en-Genevois , France GRACE CHEN • Vaccine Analytical Development , Merck Research Laboratories ,

West Point , PA , USA SYLVIE CHOBLET • CNRS UPS3044 , Saint Christol-Lèz-Alès , France OLIVIER COLAS • Physico-Chemistry Department , Centre d’Immunologie Pierre-Fabre ,

Saint Julien-en-Genevois , France NATHALIE CORVAÏA • Centre d’Immunologie Pierre-Fabre , Saint-Julien-en-Genevois ,

France ARNAUD DELOBEL • Quality Assistance , Donstiennes , Belgium ERELL DEREUX • Quality Assistance , Donstiennes , Belgium SANDRINE DONADIO-ANDRÉI • Siamed’Xpress, University Aix-Marseille , Marseille ,

France MATHIEU DUBOIS • Service de Pharmacologie et d’Immunoanalyse , CEA ,

Gif-sur-Yvette , France ERIC DUVERGER • Laboratoire de Neurobiologie , Université d’Orléans , Orléans , France NASSIMAL EL MAÏ • Siamed’Xpress, University Aix-Marseille , Marseille , France JOHN R. ENGEN • Department of Chemistry and Chemical Biology , The Barnett

Institute of Chemical and Biological Analysis, Northeastern University , Boston , MA , USA

xiv Contributors

ERIC EZAN • Service de Pharmacologie et d’Immunoanalyse , CEA , Gif-sur-Yvette , France

FRANÇOIS FENAILLE • Service de Pharmacologie et d’Immunoanalyse , CEA , Gif-sur-Yvette , France

STEFAN GAUNITZ • Division of Clinical Immunology and Transfusion Medicine, Karolinska Institute, Stockholm, Sweden

VALENTINA GELFANOVA • Lilly Research Laboratories , Indianapolis , IN , USA LILIANE GOETSCH • Experimental Oncology Department , Centre d’Immunologie

Pierre-Fabre , Saint Julien-en-Genevois , France ANKI GUSTAFSSON • Recopharma AB , Stockholm , Sweden SHA HA • Vaccine Analytical Development , Merck Research Laboratories , West Point ,

PA , USA MELISSA HAMM • Vaccine Analytical Development , Merck Research Laboratories ,

West Point , PA , USA ANNE HARDUIN-LEPERS • Unité de Glycobiologie Structurale et Fonctionnelle, CNRS

UMR8576, Université Lille Nord de France, Lille1 Villeneuve-d’Ascq, France CATHERINE A. HAYES • Medical Biochemistry , University of Gothenburg , Gothenburg ,

Sweden JAN HOLGERSSON • AbSorber AB , Stockholm , Sweden ;

Division of Clinical Chemistry and Transfusion Medicine , Sahlgrenska Academy, University of Gothenburg , Gothenburg , Sweden

DAMIAN HOUDE • Biogen Idec, Inc. , Cambridge , MA , USA LIHUA HUANG • Lilly Research Laboratories , Indianapolis , IN , USA CÉLINE HUILLET • Physico-Chemistry Department , Centre d’Immunologie

Pierre-Fabre , Saint Julien-en-Genevois , France CHLOÉ ISS • Siamed’Xpress, University Aix-Marseille , Marseille , France MARIE-CLAIRE JANIN-BUSSAT • Physico-Chemistry Department , Centre d’Immunologie

Pierre-Fabre , Saint Julien-en-Genevois , France CHRISTOPHE JAVAUD • Glycode S.A.S , Uzerche , France SYLVIE JULIANT • CNRS UPS3044 , Saint Christol-Lèz-Alès , France NICLAS G. KARLSSON • Medical Biochemistry , University of Gothenburg , Gothenburg ,

Sweden DIARMUID T. KENNY • School of Chemistry and Medical Biochemistry, National

University Ireland Galway and University of Gothenburg , Galway , Ireland and Gothenburg, Sweden

CHRISTINE KLINGUER-HAMOUR • Physico-Chemistry Department , Centre d’Immunologie Pierre-Fabre , Saint Julien-en-Genevois , France

LUDOVIC LANDEMARRE • GLYcoDiag , Orléans , France MARYLÊNE LÉVÊQUE • CNRS UPS3044 , Saint Christol-Lèz-Alès , France LINDA LINDBERG • AbSorber AB , Stockholm , Sweden JINING LIU • AbSorber AB , Stockholm , Sweden VICTOR H. OBUNGU • Lilly Research Laboratories , Indianapolis , IN , USA ANNICK OZIL • CNRS UPS3044 , Saint Christol-Lèz-Alès , France T. SHANTHA RAJU • Biologics Research , Janssen Research and Development, LLC ,

Radnor , PA , USA

xvContributors

JANICE M. REICHERT • Reichert Biotechnology Consulting LLC , Framingham , MA , USA

CATHERINE RONIN • Siamed’Xpress, University Aix-Marseille , Marseille , France RICHARD R. RUSTANDI • Vaccine Analytical Development , Merck Research Laboratories ,

West Point , PA , USA SARAH SANGLIER-CIANFÉRANI • Laboratoire de Spectrométrie de Masse Bio-Organique ,

IPHC, CNRS, UMR7178, Université de Strasbourg , Strasbourg , France CHRISTINE SCHAEFFER-REISS • Laboratoire de Spectrométrie de Masse Bio-Organique ,

IPHC, CNRS, UMR7178, Université de Strasbourg , Strasbourg , France MAGNUS SJÖBLOM • Department of Civil, Environmental and Natural Resources

Engineering , Luleå University of Technology , Luleå , Sweden MARIE-CHRISTINE SLOMIANNY • Unité de Glycobiologie Structurale et Fonctionnelle,

CNRS UMR8576, Université Lille Nord de France, Lille1 Villeneuve-d’Ascq, France W. BLAINE STINE JR. • Abbott Bioresearch Center , Worcester , MA , USA LAURE TONINI • Physico-Chemistry Department , Centre d’Immunologie Pierre-Fabre ,

Saint Julien-en-Genevois , France ALAIN VAN DORSSELAER • Laboratoire de Spectrométrie de Masse Bio-Organique ,

IPHC, CNRS, UMR7178, Université de Strasbourg , Strasbourg , France GÉRY VAN VYNCHT • Quality Assistance , Donstiennes , Belgium MARIE-LUCE VIOLET • CNRS UPS3044, Saint Christol-Lèz-Alès, France ELSA WAGNER-ROUSSET • Physico-Chemistry Department , Centre d’Immunologie

Pierre-Fabre , Saint Julien-en-Genevois , France DONGXING ZHA • GlycoFi Inc., Merck and Co , Lebanon , NH , USA