Keywords: ADCP Antibody-dependent cellular phagocytosis Ag Antigen Ab Antibody α1, 3GT α1, 3galactosyl-transferase

First of all, I sincerely thank Dr. Annabelle Varrot and Dr. Jan Tkac for accepting to review my manuscript, as well as Prof. Chrystel Lopin-Bon and Dr. Julie Buckaert for accepting to be part of my thesis defence committee.

My gratitude to Dr. Ludovic Landemarre for giving me opportunity to be part of his GLYcoDiag team and to be part of the synBIOcarb project. Thank you for guiding me through my research but also for showing me how is it to work in a scientific company and for finding always the time to answer my questions.

A special thanks goes to Dr. Benoît Roubinet for his support, advices, patience, and for sharing is experience as an organic chemist. It was a pleasure working with you.

Thanks to my former voisin de bureau and friend Yasmina Traore, I am really happy to have gotten to know you (and petite Cassandre!!) during these three years and to have shared a lot of snacks.

Thank to Dr. Mateja Senicar, who is a great colleague and an amazing human being. I miss already our lunch break in the sun (or not) and all the adventures, conversations, laughs (or not), we have shared. I am sure there will be many more. Thank you for supporting me and sharing your knowledge. Thanks to Prof. Richard Daniellou and the amazing Glycobiochimie team Dr. Pierre Lafite, Arnaud Paris, Damien Bretagne, Nastassja Burrini. You have truly helped me during the course of my thesis and you've always been available to answer my questions and to help me solve my doubts.

I would love to thank all synBIOcarb PIs and PhD students: unfortunately we did not have many occasions to get together in person, but when we did it was really great. Thanks to Dr.

Simona Notova and Juvissan Aguedo with whom I collaborated and as well a thank you to

Dr. Anne Imberty, who welcomed me in her laboratory, and Dr. Jan Tcak that agreed for the collaboration with Juvi.

PhD is a great life experience but in order to be awesome, you have to have the right people around you. I was lucky enough to be surrounded by very good friends who turned into a family and with whom we had the greatest fun in the petite Orléans:

The itañol gang: mi tocaya e moglie Fede (e Gateau ovviamente) grazie per essere stata al mio fianco, come ci insegnano, nella gioia e nel dolore. Fe, Moni, Cla, Elenita: grazie per la compagnia, la spesa, le chiacchierate, le mangiate (special mention to carbonara), viaggi, bevute, passeggiate, risate, feste. Siete stati come una "home away from home" e un grande supporto nei momenti di sconforto.

El grupo de los latinos: Hectorcito y los colombianos, Fabiancito, Alfredo y los mexicanos.

Gracias por la música (no solo reguetón jaja), las fiestas, el baile, el chisme y el desorden que han traído en nuestra vida. Orléans es un lugar más feliz gracias a ustedes.

Los hijos de Orléans (or should I say "Los hijos de les Enfantes Terribles"): you all know who you are. I will never forget Rue du Grenier à Sel. A thank you to my dear friend Mahdi, who is always there for me.

The ADSO crew: it was a honour to be vice-president and I know I left the organization in very good hands. Thanks Pierre-Yves for the good memories, after all, we met for the first time at ADSO! Simo, perché dice te l'avevo detto ma poi alla fine mi aiuta sempre.

Federico, che mi ha accolta a Manchester quando avevo bisogno e ancora adesso riesce sempre ad essere presente al momento giusto. Giulia e Carme con cui abbiamo scattato le migliori foto da trentenni a Parigi: queste sono solo le ultime di una lunga serie di momenti passati insieme e spero le prime di altri che verranno.

Bice, che è venuta fino ad Orléans con me per assicurarsi che sopravvivessi.

Vale, che ovunque io sia riesce sempre a trovare qualche giorno per venire a trovarmi.

Paola, Roberto e Gianluca che sono la mia famiglia della porta accanto.

Zia Pina e Zio Franco che da molti km di distanza riescono sempre ad esserci col loro affetto.

Mia madre e mio padre a cui è piaciuta molto Orléans, mi dispiace adesso vi toccherà venire fino a Lille. Grazie di supportare sempre le mie scelte vagabonde.

Mia sorella Berry, che, insieme a Sirius, è la mia persona preferita. E infine Andrea: non ci sono abbastanza parole per ringraziarti, in tutte le lingue che conosciamo. Grazie per i km fatti e per avere sempre creduto in me e soprattutto in noi.

Table of figures

Chapter 1 -Introduction and thesis objective

Fig. 1. 1 synBIOcarb network. On the left, highlighted in yellow all countries where the beneficiaries and partners are located. On the right, the logos of all beneficiaries and partners involved in the network. __________________________________________________________________________________ 2 Fig. 1. 2 synBIOcarb work packages _________________________________________________________ 3 Fig. 1. 3 Thesis workflow. ___________________________________________________________________ 5 Fig. 1. 4 The non-template glycosylation drastically increases biological functional diversity, adapted from [5]. __________________________________________________________________________________ 9 Fig. 1. 5 Classification of lectin based on their CRDs and their presence across the kingdoms of life adapted from [4] __________________________________________________________________________ 10 Fig. 1. 6 Lectin array design, adapted from [START_REF] Gupta | Lectin microarrays for glycomic analysis[END_REF]. ______________________________________________ 12 Fig. 1. 7 Glycan array principle, adapted from [START_REF] Muthana | Glycan microarrays: Powerful tools for biomarker discovery[END_REF] ____________________________________________ 14 Fig. 1. 8 SPR principle adapted from https://bit.ly/2Es3k2s. ____________________________________ 15 Fig. 1. 9 ITC principle adapted from https://bit.ly/2rP1OAL _____________________________________ 15 Fig. 1. 10 Principle of HPLC, adapted from https://bit.ly/3UWsiMH ______________________________ 16 Fig. 1. 11 Schematic representation of CE system, adapted from https://bit.ly/3SHSR64 ___________ 17 Fig. 1. 12 Derivatization of sialic acid linkages.Adapted from the poster "Rapid and high-throughput methods for discrimination of sialic acids linkages in glycoproteins" J. Aguedo and F. Vena. _______ 18 Fig. 1. 13 MALDI-TOF/MS for the discrimination of α2,3 and α2,6 sialic acid linkages workflow. Adapted from the poster "Rapid and high-throughput methods for discrimination of sialic acids linkages in glycoproteins" J. Aguedo and F. Vena. ____________________________________________ 18 Fig. 1. 14 A 2020 best-selling drugs, adapted from [START_REF] Community | Glyco@ALPS -Glyco 2030: A Roadmap for Glycoscience in Europe[END_REF]. B Biotherapeutics global revenues from 2005 to 2006 adapted from [START_REF] Dhara | Recombinant Antibody Production in CHO and NS0 Cells: Differences and Similarities[END_REF]. _________________________________________________________________ 19 Fig. 1. 15 Different glycosylation according to the kind of expression system. ____________________ 23 Fig. 1. [START_REF] Angeloni | Glycoprofiling with micro-arrays of glycoconjugates and lectins[END_REF] A GLYcoPROFILE in direct binding. B GLYcoPROFILE in inhibition. ___________________ 27 Chapter 2 -Neoglycoproteins Fig. 2. 1 A structure of the neoRSLVI in complex with aMetilFucose (spheres), all binding sites result occupied, adapted from [START_REF] Arnaud | Membrane deformation by neolectins with engineered glycolipid binding sites[END_REF]. B Cartoon representation of a Janus lectin, able to cross-link biological surfaces thanks to its double specificity for fucosylated glycans on one side and sialylated ones on the other side, adapted from [START_REF] Ribeiro | Tailor-made Janus lectin with dual avidity assembles glycoconjugate multilayers and crosslinks protocells[END_REF]. ______________________________________________________________ 31 Fig. 2. 2 Mechanisms of interaction of multivalent partners, adapted from [8] _____________________ 32 Fig. 2. 3 Types of multivalent synthetic glycoconjuagtes, adapted from [8] _______________________ 33 Fig. 2. 4 A CuAAC via 1,3-dipolar cycloaddition of azides and alkynes reaction resulting in 1,2,3triazoles, adapted from [START_REF] Haldón | Copper-catalysed azide-alkyne cycloadditions (CuAAC): An update[END_REF]. B CuAAC reaction mechanism (adapted from, http://bitly.ws/uSiZ) ____ 36 Fig. 2. 5 A Reaction between the primary ammines of the BSA and PNHS ester, followed by the clickchemistry reaction (CuAAC) between BSA-alkyne and the glycan functionalized with an azide group. B Reaction between the primary ammines of the BSA and the NHS, followed by the click-chemistry reaction (CuAAC) between BSA-azide and the glycan functionalized with an alkyne group. ________ 38 Fig. 2. 6 SDS-PAGE of BSA functionalization with alkyne and azide groups. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA, Line 3: BSA-alkyne, Line 4: BSA-azide. ______________________________________________________ 39 Fig. 2. 7 A MALD-TOF/MS spectrum of BSA B MALD-TOF/MS spectrum of BSA-alkyne C MALD-TOF spectrum of BSA-azide. ____________________________________________________________________ 40 Fig. 2. 8 CuAAC reaction between BSA-azide and Galα1-3Gal-N-Acetyl-Propargyl. _______________ 40 Fig. 2. 9 SDS-PAGE of NeoGalili synthesis. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA, Line 3: BSA-alkyne, Line 4: BSA-azide, Line5: NeoGalili neoglycoprotein. ______________________________________________ 41 Fig. 2. 10 MALD-TOF spectrum of NeoGalili neoglycoprotein. __________________________________ 41 Fig. 2. 11 GLYcoPROFILE in direct binding of the NeoGalili neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with six different lectins. The data shown are the average of experiments performed in triplicate. _________________________ 42 Fig. 2. 12 CuAAC reaction between BSA-alkyne and 6'-sialylactose-N3 or 3'-sialylactose-N3. ______ 43 Fig. 2. 13 SDS-PAGE of Neo6'SL and Neo3'SL synthesis. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA-alkyne, Line 3: Neo6'SL, Line 4: Neo3'SL. _________________________________________________________________ [START_REF] Biolabs | Glycoproteomics Technical Guide[END_REF]. B, the cetuximab mechanism: it binds to the EGFR and prevents its dimerization, blocking all its pro-tumour functions and promoting apoptosis, adapted from [START_REF] Patil | Cetuximab and biomarkers in non-small-cell lung carcinoma[END_REF]. ________________________________________________________________________ 59 Fig. 3. 3 Schematic representation of the distribution of hypersensitivity reactions to Cetuximab and red meat in the southeast region of North America and its connection to the increasing deer population carrying ticks and tick bites, adapted from [START_REF] Steinke | The alpha-gal story: Lessons learned from connecting the dots[END_REF]]. ____________________________________ 61 Fig. 3. 4 Galsafe engineered pigs used for xenotraspantation, adapted from [START_REF] Wang | First pig-to-human heart transplantation[END_REF]. _________________ 62 Fig. 3. 5 Schematic representation of the viral vaccine mechanism of action, adapted from [START_REF] Sokolova | The potential of nanoparticles for the immunization against viral infections[END_REF]. ____ 63 Fig. 3. 6 Rrepresentation of the α-Gal glycolipids and how they are recognized by anti-gal Abs when injected in tumour tissues, adapted from [START_REF] Galili | Anti-Gal: An abundant human natural antibody of multiple pathogeneses and clinical benefits[END_REF]. ________________________________________________ 64 Fig. 3. 7 MOA β-trefoil portion crystal structure in complex with the Galili antigen. This figure was created with Pymol, adapted from the crystal structure of Grahn et al,[START_REF] Grahn | Crystal Structure of the Marasmius Oreades Mushroom Lectin in Complex with a Xenotransplantation Epitope[END_REF] PDB: 2IHO). __________ 65 Fig. 3. 8 Crystal structure of GSLI-B4 in complex with the xenograft antigen. PDB code 1HQL, adapted from [START_REF] Tempel | The xenograft antigen in complex with GS-1-B 4 lectin: crystallization and preliminary X-ray analysis[END_REF]. ________________________________________________________________________________ 66 Fig. 3. 9 A SDS-PAGE analysis of MOAβT The protein sample was analyzed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: MOAβT [START_REF] Chen | Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays[END_REF]2 kDa). B GLYcoPROFILE in direct binding of MOAβT interacting with labelled Porcine Thyroglobulin, Bovine Thyroglobulin, Invertase, NeoGalili and NeoβGal, NeoF. _______________________________________ 67 Fig. 3. 10 GLYcoPROFILE in direct binding of GSLI-B4 interacting with labelled Porcine Thyroglobulin, Bovine Thyroglobulin, Invertase, NeoGalili, NeoL, NeoF. ______________________________________ Fig. 3. 11 A Comparison of the binding of MOAβT at decreasing quantity/well to the NeoGalili neoglycoprotein at a fixed concentration (1.15

µM) on covalent plate (black) and adsorption plate (pink).

The two experiments can be compared because the signal in both cases is measured in absorbance. B Binding of MOAβT at decreasing quantity/well to the NeoGalili neoglycoprotein at a fixed concentration (1.15 µM) on adsorption plate, this time the interaction is reported as fluorescence intensity. _________________________________________________________________________________ Fig. 3. 12 A MOAβT decreasing quantity/well (4 µg/well to 0.12 µg/well) plotted against the absorbance at 450 nm resulting from the lectin interaction with the NeoGalili neoglycoprotein at a fixed concentration (0.3 µM) on covalent plate. B MOAβT decreasing quantity/well (1 µg/well to 0.1 µg/well) plotted against the absorbance at 450 nm resulting from the lectin interaction with the NeoGalili neoglycoprotein at a fixed concentration (0.3 µM) on covalent plate. ___________________ Fig. 3. [START_REF] Gupta | Lectin microarrays for glycomic analysis[END_REF] Comparison of MOAβT binding to NeoGalili neoglycoprotein when MOAβT is at a fixed quantity/well of either 1 µg/well or 0.5 µg/well with NeoGalili added in serial dilutions. _____________ Fig. 3. 14 Direct binding curve of MOAβT immobilized on covalent plate at the fixed quantity of 1 µg/well with NeoGalili neoglycoprotein applied to the plate at 1.15 µM and serially diluted. _________ Fig. 3. 15 Inhibition experiment performed with MOAβT at 1 µg/well interacting with labelled NeoGalili at the fixed concentration of 0.5 µg/mL and with the non-labelled inhibitors applied in serial dilution, from a starting concentration of Porcine Thyroglobulin=12µM, NeoGalili=12 µM, Melibiose=50 mM. Fig. 3. [START_REF] Angeloni | Glycoprofiling with micro-arrays of glycoconjugates and lectins[END_REF] Direct binding curve of MOAβT coated on adsorption plate at the fixed quantity of 1 µg/well with NeoGalili neoglycoprotein applied to the plate at 2.3 µM and serially diluted. _________________ Fig. 3. 17 Direct binding curve of MOA coated on adsorption plate at the fixed quantity of 1 µg/well with NeoGalili neoglycoprotein applied to the plate at 2.3 µM and serially diluted. _________________ Fig. 3. 18 Direct binding curve of GSLI-B4 coated on adsorption plate at 1µg/well interacting with NeoGalili neoglycoprotein applied to the plate at 2.3 µM and serially diluted. _____________________ Fig. 3. [START_REF] Bertok | Analysis of serum glycome by lectin microarrays for prostate cancer patients -a search for aberrant glycoforms[END_REF] Inhibition experiment performed with GSLI-B4 interacting with labelled NeoGalili at the fixed concentration of 35 µg/mL and with the non-labelled inhibitors applied in serial dilution, from a starting concentration of Porcine Thyroglobulin=12µM, NeoGalili=12 µM, Melibiose=100 mM, α-galactose= 100 mM. The IC50 for α-galactose and Melibiose could not be calculated because of the saturation of the signal. ________________________________________________________________________________ [START_REF]Serum sialylation changes in cancer[END_REF]. ________________________________________________________ Fig. 4. 2 Asialoglycoprotein receptors. ASGP receptors in the liver do not recognize desialylated proteins, targeting them for uptake and degradation [START_REF] Maverakis | Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: A critical review[END_REF] ______________________________________________ Fig. 4. 3 Roles of sialic acids. They can act as ligands for intrinsic receptors as well as extrinsic receptors [START_REF] Varki | Sialic acids in human health and disease[END_REF]. ___________________________________________________________________________ Fig. 4. 4 N-Acetyl neuraminic acid. The red circle highlights the acetyl functional group on C5 ______ Fig. 4. 5 Neu5Ac acid metabolism in mammalian cells [START_REF] Zhou | Biological Functions and Analytical Strategies of Sialic Acids in Tumor[END_REF]. ___________________________________ Fig. 4. 6 Human Transferrin glycoforms [START_REF] Porpiglia | A new sample treatment for asialo-Tf determination with capillary electrophoresis: an added value to the analysis of CDT[END_REF] _________________________________________________ Fig. 4. 7 Sialome organization [4]. __________________________________________________________ Fig. 4. 8 IgG structure and its glycosylation. The bottom domain is the Fc and the top one is the Fab. VL= variable region of the light chain, CL= constant region of the light chain, VH= variable region of the heavy chain, CH1,2,3= Constant region of the heavy chain, domain 1, 2, 3, Ag= antigen [START_REF] O'kennedy | Technology advancements in antibody purification[END_REF]. _____ 119 Fig. 4. 9 Different glycan structures that can be found on IgG. G= galactose, F= fucose, S= sialic acid [START_REF] Raymond | Production of α2,6sialylated IgG1 in CHO cells[END_REF] . __________________________________________________________________________________ 120 Fig. 4. 10 A N-Glycolyl neuraminic acid biosynthesis. B taxa are found in blue, not found in purple and purple and blue where only certain species have lost the capacity to make it [START_REF] Dhar | From 'Serum Sickness' to 'Xenosialitis': Past, Present, and Future Significance of the Non-human Sialic Acid Neu5Gc[END_REF] _______________ 122 Fig. 4. 11 MAA-I in complex with sialyllactose, PDB code 1DBN [START_REF] Imberty | An unusual carbohydrate binding site revealed by the structures of two Maackia amurensis lectins complexed with sialic acid-containing oligosaccharides[END_REF] ___________________________ 125 Fig. 4. 12 SNA in complex with N-Acetylgalactosamine, PDB code 3CA3 [START_REF] Maveyraud | Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra[END_REF]. __________________ 125 Fig. 4. 13 structure of VP1. PDB code 4POQ [START_REF] Bardor | Mechanism of uptake and incorporation of the nonhuman sialic acid N-glycolylneuraminic acid into human cells[END_REF]. __________________________________________ 126 Fig. 4. 14 3D structure of WGA [START_REF] Banda | Metabolism of vertebrate amino sugars with N-glycolyl groups: Mechanisms underlying gastrointestinal incorporation of the non-human sialic acid xeno-autoantigen N-glycolylneuraminic acid[END_REF] . ______________________________________________________ 126 Fig. 4. 15 A SDS PAGE analysis of MAAI. The protein sample was analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: MAAI (35 kDa) B GLYcoPROFILE in direct binding of MAAI interacting with labelled Neo6'SL, Neo3'SL, NeoMa and NeoF at 40 µg/mL. ________________________________________________________________________________________ 128 Fig. 4. 16 A SDS PAGE analysis of SNA. The protein sample was analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: SNA (33 kDa) B GLYcoPROFILE in direct binding of SNA interacting with labelled Neo6'SL, Neo3'SL and NeoMannose at 40 µg/mL. _______ 128 Fig. 4. 17 GLYcoPROFILE in direct binding representing the interaction between the lectin SNA and the labelled neoglycoprotein Neo6'SL at a starting concentration of 0.6 µM.The curve is obtained from four independent replicates. All measurements were performed in duplicates. ___________________ 130 Fig. 4. 18 GLYcoPROFILE in direct binding representing the interaction between the lectin MAAI and the labelled neoglycoprotein Neo3'SL at a starting concentration of 0.6 µM. The curve is obtained from four independent replicates. All measurements were performed in duplicates. ___________________ 130 Fig. 4. [START_REF] Bertok | Analysis of serum glycome by lectin microarrays for prostate cancer patients -a search for aberrant glycoforms[END_REF] Inhibition curves of SNA with the inhibitors Neo6'SL, Fetuin, Transferrin at the starting concentration of 10 µM and 6'SL at the starting concentration of 5mM. The labelled tracer Neo6'SL is at the fixed concentration of 20 µg/mL. The curve is obtained from three independent replicates. All measurements were performed in duplicates. _______________________________________________ 132 Fig. 4. [START_REF] Muthana | Glycan microarrays: Powerful tools for biomarker discovery[END_REF] Inhibition curves of SNA with the inhibitors Neo3'SL and Fetuin at the starting concentration of 10 µM and 3'SL at the starting concentration of 5mM. The labelled tracer Neo3'SL is at the fixed concentration of 20 µg/mL. The curve is obtained from three independent replicates. All measurements were performed in duplicates. _____________________________________________________________ 132 Fig. 4. 21 SNA and MAAI standard curves obtained with labelled Neo6'SL and Neo3'SL. _________ 135 Fig. 4. [START_REF] Suenaga | Monitoring influenza hemagglutinin and glycan interactions using surface plasmon resonance[END_REF] Interaction of MAAI and SNA specific lectins with labelled Fetuin at three different concentrations. The intensity of interaction of each lectin was studied before and after Fetuin digestion by total neuraminidase (NeuT) and α2-3 neuraminidase (Neuα2-3). Two independent assays (each with n = 3 replicates) were carried out. __________________________________________________________ 136 Fig. 4. 23 MALDI-TOF/MS. A Possible sialylated structures present on Fetuin. B Relative abundance of α2-6 and α2-6+α2-3sialic acid linkages present in sialylated glycans. _______________________ 137 Fig. 4. [START_REF] Reiding | High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification[END_REF] Interaction of MAAI and SNA specific lectins with labelled transferrin at three different concentrations. The intensity of interaction of each lectin was studied before and after transferrin digestion by total neuraminidase (NeuT) and α2-3 neuraminidase (Neuα2-3). Two independent assays (each with n = 3 replicates) were carried out. ________________________________________________ 138 Fig. 4. [START_REF] Community | Glyco@ALPS -Glyco 2030: A Roadmap for Glycoscience in Europe[END_REF] Interaction of MAAI and SNA specific lectins with labelled transferrin native and denatured at three different concentrations. Two independent assays (each with n = 3 replicates) were carried out. ________________________________________________________________________________________ 139 Fig. 4. 26 MALDI-TOF/MS A Possible sialylated structures present on Transferrin. B Relative abundance of α2-6 and α2-6+α2-3sialic acid linkages present in sialylated glycans. ____________ 140 Fig. 4.27 A Interaction of MAAI and SNA specific lectins with labelled porcine thyroglobulin at three different concentrations. B Interaction of MAAI and SNA specific lectins with labelled bovine thyroglobulin at three different concentrations. The intensity of interaction of each lectin was studied before and after thyroglobulin digestion by total neuraminidase (NeuT) and α2-3 neuraminidase (Neuα2-3). For each protein two independent assays (each with n = 3 replicates) were carried out. 141 Fig. 4. 28 MALDI-TOF/MS A Possible sialylated structures present on porcine thyroglobulin. B Relative abundance of α2-6 and α2-6+α2-3sialic acid linkages present in sialylated glycans. ____________ 142 4. 12 List of all lectins used through this thesis and corresponding specificities. ____________ has been tailored to serve the aim of the thesis that is building a lectin kit for the detection of the major undesirable glycan structures found mainly on biotherapeutic glycoproteins.

Table of tables

Table of Contents

The goal is to allow real-time monitoring and quality control of these biotherapeutics, but also other potential applications (vaccines quality control, diagnostics) .

Fig. 1. 2 synBIOcarb work packages

The research has been mostly focused on two glycans: the Galili antigen and the sialic acid.

Given the wide range of opportunities offered by this field, during the last three years my project has broadened. It has become interesting for us to imagine new areas other than the quality control, in which a rapid lectin kit could turn out to be a huge help in the detection of target glycans.

To help the reader navigate the thesis, here it is briefly described its structure:

Chapter 1 is a general introduction on the subject, especially focused on the study of glycans and lectin-glycan interactions and the importance of rapid quality control of biotherapeutics. Being my research inserted in the context of a company, the next step after the proof of concept is the redaction of all the documents required for the kits to be suitable for applications(user guide, specifications).

The good specifications and the precise definition of all the kits parameters are of fundamental importance at a research level to ensure stability and reliability to the potential users. 2. Synthèse des néoglycoprotéines. Les néoglycoprotéines sont une partie fondamentale de la construction du kit, car elles sont utilisées comme sondes standards pour établir les paramètres de référence pour l'utilisation des kits.

3. Etude des paramètres des kits : choix de la quantité de lectine/puits, choix du type de plaque, si adsorption ou covalente, la méthode de détection si en absorbance ou fluorescence, la limite de détection, la concentration inhibitrice à 50 % (IC50).

Ma recherche étant insérée dans le contexte d'une entreprise, l'étape suivante après la preuve de concept est la rédaction de tous les documents requis pour que les kits soient adaptés aux applications (guide d'utilisation, spécifications).

Les bonnes spécifications et la définition précise de tous les paramètres des kits sont d'une importance fondamentale au niveau de la recherche pour assurer la stabilité et la fiabilité aux utilisateurs potentiels.

Introduction

The evolution of genome sequencing, from the 1970 onwards, has opened up a completely new world in front of the human eyes: the possibility to understand the information hidden inside our cells, the genetic code. The central dogma of molecular biology, DNA copied into mRNA translated into proteins, finds its strength in the fact that DNA replication and translation are template-driven, meaning that proteins are primary genes products.

Considering that the human genome contains only 25000 genes, which is not much more than the one of Drosophila melanogaster (20000 genes), the hypothesis that came out was that human complexity lies in post-translational modifications of proteins [2]. One of them, and the most common one, is protein glycosylation. Hence, as it turned out, genome decoding does not explain all biological functions that our cells perform because it does not include a whole class of macromolecules: carbohydrates, also called glycans, or simply sugar chains. Glycans are not only energy-storage molecules, but they participate in many biological processes [3].

The glycome is defined as the entire pool of glycans, free or attached to other macromolecules, such as proteins or lipids, that is synthesized by our cells "under specific conditions of time, space, and environment" [4]. As the definition says, the glycome can change according to many factors and its synthesis does not depend on directly from the DNA sequence. It depends instead on enzymes, encoded by DNA, like glycosidases and glycosyltransferases that can attach carbohydrates in different order, organization, in a linear or branched configuration. The fact that it is not possible to predict the glycomes structure increases its complexity and makes its decodification very challenging. One of the keys to unlocking the so-called "glycocode" and deciphering the information that glycans come from natural inspiration from glycobiological interactions. Indeed, the main mechanisms driving plants and animals lives result from glycobiological interactions, that involve glycans specific recognition by glycan recognition molecules such as lectins.

Chapter 1 -Introduction and thesis objective 10

Lectins

The name "lectin" originates from the Latin word "legere" that means "to select". Lectins in fact are proteins with at least one non-catalytic domain, and of non-immune origin, which are able to specifically recognize, or select, and to reversibly bind carbohydrates. Lectins fall into the group of glycan-binding proteins, in which also sulphated glycosaminoglycan-binding proteins are placed. In 1919, the first lectin discovered was the Canavalia ensiformis or ConA or jack bean agglutinin, named from the leguminous plant it comes from and from the fact that it is able to clump red-blood cells. Years later, Landsteiner confirmed the idea that the agglutination was due to the interaction of the haemagglutinin with sugars in the blood, saying that "the actions of plant haemagglutinins resemble antibody reactions in all essentials" [6].

Nowadays we know that lectins/glycans interaction, unlike antibodies/antigens (Kd 10 -8 -10 -12 M), have a relatively low affinity ranging from millimolar for monosaccharides ligand to micromolar for oligosaccharides [7]. Lectins can be either a single carbohydrate-recognition domain (CRD) or an entire protein (containing more domains other than the CRD, e.g.

proteolytic domains) and are ubiquitous, meaning they are present in all living organisms.

They can be classified according to their monosaccharide specificity but more recently they are classified based on their CRD structure (Fig. 1.5) [4]. On one hand, many lectins are involved in immune responses, on the other hand, pathogens also often use their own lectins to attach to host cells during infection. Lectins also function in communication between cells in multicellular organisms, resulting in movement of molecules, cells, and information [4]. Lectins interact with their ligands mostly through weak interactions, mainly the formation of a hydrogen-bond network or, in the case of some positively or negatively charged carbohydrates (e.g. sialic acid) or modified carbohydrates (e.g. sulphated sugars), through electrostatic interactions [8]. Moreover, hydrophobic interactions ensure the interaction between the hydrophobic component of the glycan structure and the aromatic residues present in the CRD. Metal cations, such as Ca 2+ and Mn 2+ , can also be involved in the recognition as with Ca 2+ for C-type animal lectins. The reasons of the low affinity for the glycan-lectin interactions may be due to several variables: to the shallow binding pockets on the surface of the protein, to the limited changes in the conformations of the binding site after the ligand binding, the restricted binding surface available for the ligand, the inability of the ligand to exhibit multiple binding modes [3]. If the affinity is the strength of the interaction that occurs between a single protein and a single ligand, in the case of lectins it would be better to speak about avidity. The avidity compensates for the rather low affinity and it can be defined as the sum of all the strengths of all the multiple simultaneous interactions between the lectin and the interacting glycan, leading to an affinity enhancement [START_REF] Lee | Carbohydrate-Protein Interactions: Basis of Glycobiology[END_REF]. The importance of multivalent interactions in generating high affinity was demonstrated by Ashwell and co-workers, who showed that desialylated serum glycoproteins exposing several Gal residues could increase of 10000-fold the affinity towards rabbit hepatic lectin [START_REF] Grewal | The Ashwell-Morell receptor[END_REF]. So, for lectins to be able to cross-link glycan-containing structures, from which their agglutination ability come, they either are organized as oligomers of CRDs, either contain multiple binding sites on a single polypeptide chain, or again they are constituted by the association of polypeptides containing CRDs. Since their discovery, lectins have been used for all sorts of applications, spacing from lectin affinity chromatography, blood typing, histology, flow cytometry and blotting [START_REF] Hsu | Analyzing the dynamic bacterial glycome with a lectin microarray approach[END_REF]. With the advent of glycomics, they have been applied for lectin arrays, providing a fast, highthroughput screening platform that allows to monitor at the same time several lectin-glycan interactions. Lectin arrays were first introduced in 2005 [START_REF] Gemeiner | Lectinomics. II. A highway to biomedical/clinical diagnostics[END_REF] and the basic format consists of lectins immobilized on a surface that could be a plate or a glass slide, through adsorption, covalent or biochemical interaction. Methods for lectin immobilization could be carbene insertion, biotin-avidin bridge, coupling through primary amine on lysine of lectin with Nhydroxysuccinimydyl (NHS)-derivates esters, exploitation of self-assembled monolayers of thiols on gold-coated surfaces, and 3D hydrogel surfaces. None of this methods guarantees the complete control of the optimal lectin orientation [START_REF] Gupta | Lectin microarrays for glycomic analysis[END_REF]. The interacting sample is usually found in solution, and can be of various nature, from glycoproteins to cells, to vesicles (Fig.

1.6).

Fig. 1. 6 Lectin array design, adapted from [START_REF] Gupta | Lectin microarrays for glycomic analysis[END_REF].

The detection of the interaction can be achieved in three different ways: direct mode, indirect mode or competitive mode. In the direct mode the binding partner is labelled usually with a fluorophore, and the fluorescence is detected after the binding [START_REF] Gemeiner | Lectinomics. II. A highway to biomedical/clinical diagnostics[END_REF]. In the indirect mode, the partner is not labelled but it can be recognized by a fluorescent secondary antibody or the initial incubation with a biotinylated antibody is followed by incubation with fluorescent-labelled streptavidin. In this case, there is one step more before the fluorescence detection. In the competitive mode the binding partner could be either labelled or not labelled. If it is not labelled, another well-characterized glycan partner is labelled and the two molecules compete for the lectins on the array. In the GLYcoPROFILE ® in inhibition, the interaction is measured as percentage of inhibition of the binding of the labelled molecule to the lectin (Paragraph 1.7). If the binding partner is labelled, the competitor molecule is labelled as well but with a different dye. One example is the dual colour measurement where both samples compete for the lectin immobilized on the array, and there is different ratio of two dyes after the incubation time due to the different affinity of each binding partner for the lectin [START_REF] Pilobello | A ratiometric lectin microarray approach to analysis of the dynamic mammalian glycome[END_REF]. One drawback of lectin array are the washing steps that need to be performed to remove the unbound molecules. These steps are time-consuming and could be detrimental in the case of very weak lectin-glycan interactions. Hirabayashi et al. [START_REF] Kuno | Evanescent-field fluorescence-assisted lectin microarray: A new strategy for glycan profiling[END_REF] found a solution to this problem though evanescent-field fluorescence detection for weak interactions between lectins and carbohydrates in a liquid phase. This method does not require the washing step after a probing reaction. Another method, reverse phase dot blot lectin array is formed by immobilizing the samples containing glycoproteins on a surface, which is then analysed by addition of biotinylated lectins over the chip surface [START_REF] Angeloni | Glycoprofiling with micro-arrays of glycoconjugates and lectins[END_REF]. Chen et al. [START_REF] Chen | Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays[END_REF] designed a sandwich format of antibody microarray that capture multiple glycoproteins followed by detection with lectins. Lectin arrays find their application particularly in the following of the progression of pathologies such as cancer: Qiu et al. [18], showed the identification of potential plasma biomarkers for colorectal cancer, and Bertok at al. [START_REF] Bertok | Analysis of serum glycome by lectin microarrays for prostate cancer patients -a search for aberrant glycoforms[END_REF], showed the use of serum samples from healthy individuals, benign prostate hyperplasia patients, and prostate cancer patients.

Glycan arrays

Glycan arrays are another high-throughput screening technique to study lectin-glycan interactions but in this case, a variety of oligosaccharides, glycolipids, or glycoproteins are bound to solid supports and used to probe the carbohydrate-binding properties of proteins or cells. They can be printed in many different ways: chemically or enzymatically, without or with different types of linkers, but also the density of glycans on the surface can be adjusted according to the needs. Since they allow the screening of hundreds or thousands of samples at the same time, they are a valuable method for biomarker discovery, especially in the initial searching phase [START_REF] Muthana | Glycan microarrays: Powerful tools for biomarker discovery[END_REF]. Using an O-glycopeptide microarray, Wandall et al. demonstrated the presence of higher levels of antibodies against O-glycosylated mucin 1 in patients with breast, ovarian, and prostate cancer compared to that of healthy controls [START_REF] Wandall | Cancer biomarkers defined by autoantibody signatures to aberrant Oglycopeptide epitopes[END_REF]. Although these glycan arrays provide information about carbohydrate-interacting proteins, they do not allow us to directly examine changes in glycosylation. Among the studies about glycan-protein interactions with SPR, the glycan binding preferences of the hemagglutinin derived from influenza A viruses were explored in order to understand their potential ability for interspecies transmission [START_REF] Suenaga | Monitoring influenza hemagglutinin and glycan interactions using surface plasmon resonance[END_REF]. has been shown to be particularly effective for the resolution of protein glycoforms that can be difficult using conventional methods, among which the studies upon Carbohydrate-deficient Transferrin [START_REF] Prasad | Analysis of carbohydrate deficient transferrin by capillary zone electrophoresis[END_REF]. of N-glycans. The first step is the glycan release from the sample using PNGase F (Peptide -N-Glycosidase F). Afterwards, the glycans are purified using hydrophilic interaction liquid chromatography solid phase extraction. Then, the glycans are derivatized using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) and 1-hydroxy-benzotriazole (HOBt) as activators. EDC is meant for initial carboxylic acid activation and HOBt for catalysing the subsequent conversion to an ester. In Fig. 1.12 a, it is shown the reaction of α2-6 sialic acid that undergoes ethyl esterification and has a mass shift of +28 Da, while in Fig. 1.12 b, α2-3 sialic acid undergoes a lactonization, causing a mass shift of 618 Da [START_REF] Reiding | High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification[END_REF]. Then the sample are spotted for MALDI-TOF/MS analysis (Fig1.13 ). From the list in Fig 1 .14 A, it can be seen as well that of the five biotherapeutics, three are mAbs, Humira (Adanulinumab), Keytruda ( Pembrolizumab) and Opdivo (Nivolumab ). fusing rodent lymphocytes and myeloma cells. They are used for the treatment of a variety of disorders, including cancer, autoimmune diseases, cardiovascular disease, infections [START_REF] Dhara | Recombinant Antibody Production in CHO and NS0 Cells: Differences and Similarities[END_REF].

Glycosylation in biotherapeutics

Glycosylation is a fundamental post-translational modification, essential for protein folding, stability and molecular recognition, and it goes without saying that has also a strong impact on biotherapeutics. Glycosylation of a biotherapeutic protein is critical for product solubility, stability, pharmacokinetics and pharmacodynamics (PK/PD), bioactivity, and safety [START_REF] Zhang | The use of lectin microarray for assessing glycosylation of therapeutic proteins[END_REF]. . The crucial role of glycosylation is very well-studied also for mAbs: their functionality it is not only linked to the antigen binding domain (Fab) in their variable region, but also to the effector function mediated by the antibody Fc fragment (fragment crystallizable). The interaction of Fc with the Fcγ-receptors (FcγR) expressed on immune cell surfaces or the molecules of the complement triggers the ADCC (Antibody-Dependent Cell-mediated Cytotoxicity) and CDC (Complement-Dependent Cytotoxicity). The mechanism is not yet completely elucidated but it seems that sialylation has an anti-inflammatory effect, because it dramatically decreases mAb affinity for the Fcγ-receptor, inhibiting ADCC [START_REF] Raymond | Production of Highly Sialylated Monoclonal Antibodies[END_REF]. On the other side, the lack core fucosylation of IgG N-glycans, in which fucose is added α1,6 to the proteinadjacent GlcNAc of N-glycans, enhances ADCC via increased FcγR binding and signalling [START_REF] Cobb | The history of IgG glycosylation and where we are now[END_REF].

Production systems

Various production systems exist for the manufacturing of biotherapeutics. Indeed, recombinant DNA and hybridoma technology have a lot improved the production of customized recombinant proteins. Animal and human cells are the main platforms for the manufacturing, even though non-human mammalian cells are the most used at industrial scale [START_REF] Mabdesign | Immunowatch Edition n°3 -July 2021[END_REF]. This trend is mostly driven by the increased attention towards the biotherapeutics glycosylation state: non-human mammalian cells give to biotherapeutics a human-like glycosylation, with the advantage that are much easier to manipulate than human cells.

Human-like glycosylation makes them safer, in terms of being less immunogenic, and more • Non-human mammalian cells

Chinese hamster ovary cells or CHO: together with murine myeloma cells they are mammalian host gold-standard for the production mAbs and Fc-fusion proteins [START_REF] Beck | Glycofi's technology to control the glycosylation of recombinant therapeutic proteins[END_REF].

They account for the 70% of recombinant biotherapeutics production. The advantages are that they are less sensitive to contaminations by human viruses, making them safe for commercialization, they grow very fast in suspension and they support many geneexpressing systems, giving the possibility of upscaling the reaction in the bioreactors and increase the production. Although CHO cells possess many advantages for glycoprotein productions, they are unable to produce some types of human glycosylation, such as α-2,6-sialylation and α-1,3/4-fucosylation. Moreover, CHO cells produce glycans for humans that do not occur in human cells and are for this reason potentially immunogenic for patients: N-glycolylneuraminic acid (Neu5Gc) and Gal-α1,3-Gal (α-gal), even though these occurring at very low levels, <2% and<0.2%

respectively [START_REF] Lalonde | Therapeutic glycoprotein production in mammalian cells[END_REF].

Murine myeloma cells: NS0 and SP2/0 cells are used to produce mAbs. They exhibit small amounts of glycoforms, with additional α-1,3-Gal and N-glycolylneuraminic acid in considerably high levels, that result to be immunogenic in humans [START_REF] Beck | Glycofi's technology to control the glycosylation of recombinant therapeutic proteins[END_REF]. The case of Cetuximab is explanatory of the danger of this kind of residues (Chapter 3, Paragraph

3.2.1).

BHK: Baby Hamster kidney fibroblasts are mostly been used for the production of veterinary viral vaccines. Only two marketed recombinant glycoproteins are currently manufactured in these cells, Factor VIIa and Factor VIII, which are clotting factors [START_REF] Durocher | Expression systems for therapeutic glycoprotein production[END_REF].

• Human cell lines

Human embryonic kidney cells or HEK 293 and HT-1080 of fibrosarcoma origin are the most used human cell line to produce research-proteins for many years. Human cell-based expression systems produce recombinant proteins with glycosylation more similar to their natural counterpart [START_REF] Durocher | Expression systems for therapeutic glycoprotein production[END_REF].

PER.C6 cell line rFVIIIFc; Biogen, Cambridge, MA was created from human embryonic retinal cells, immortalized via transfection with the adenovirus E1 gene. This system was originally developed for the production of human adenovirus vectors for use in vaccine development and gene therapy. An advantage of PER.C6 is their ability to produce a high level of protein when used in the production of human IgG (Immunoglobulin) [START_REF] Dumont | Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives[END_REF].

• Hybridoma cells are the second most frequently used hosts for recombinant glycoprotein production [START_REF] Del Val | Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics[END_REF].

• Transgenic animals: in transgenic goat milk it is produced human anti-thrombin a, approved in Europe and the US in 2006 and 2009, respectively. The glycosylation profile changes significantly depending on the transgenic host used to express the proteins (goat, rabbit, chicken) [START_REF] Durocher | Expression systems for therapeutic glycoprotein production[END_REF].

• Insect cells infected with the viral vector baculovirus are mostly used for the development of virus-like particles and, as a consequence, vaccines. However, they lack of either galactosyltransferase and/or sialyltransferase activity, and even though they produce N-glycan precursors, these result in either high mannose or paucimannose residues that do not develop further into terminal galactose and/or sialic acid residues [START_REF] Dumont | Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives[END_REF].

• Plants expression systems are characterized for the absence of sialylation and the presence of potentially immunogenic α1,3-fucose and β1,2-xylose residues in plantderived glycoproteins glycoforms. There are currently a few plant-derived therapeutics approved in Europe for topical use in human and others are in clinical trials, including glucocerebrosidase manufactured in carrot cells [START_REF] Durocher | Expression systems for therapeutic glycoprotein production[END_REF].

• Yeast high mannose-type N-glycans contain from five to nine mannose residues and are found in yeasts [START_REF] Beck | Glycofi's technology to control the glycosylation of recombinant therapeutic proteins[END_REF] which may confer a short half-life and render proteins less efficacious and even immunogenic in humans

• Bacterial expression systems are convenient for the rapid cell growth and high yields. However, they are not able to glycosylate protein because they don't possess the required enzymes and cellular compartments. They are therefore generally used for production of non-glycosylated proteins, including some mAbs, hormones, cytokines and enzymes. Another problem is that mammalian proteins produced in bacteria often are not soluble, resulting in the formation of inclusion bodies and thus they need to be extracted from inclusion bodies and refolded [START_REF] Dumont | Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives[END_REF]. Hence, the choice of the production system influence the glycosylation pathway of the biotherapeutics. Being glycosylation non-template driven, the risk of glycan heterogeneity is high and it is connected not only to the production system, but also to the culture conditions.

Microheterogeneity is defined as the variation in the structure of the attached glycans, while macroheterogeneity is the variability of the glycosylation sites and glycans number [START_REF] Zhang | Glycan analysis of therapeutic glycoproteins[END_REF].

Genetic engineering is one way to introduce the required type of glycosylation. CHO cells were transfected with the gene for α2-6-sialyltransferase in order to increase the sialylation of various biologics, making them more human-like [START_REF] Lin | Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression[END_REF]. Another notable example of glycoengineering is human erythropoietin on which two additional N-linked glycosylation sites were introduced, allowing for a threefold increase in half-life and higher potency with a reduced frequency of administration in patients [START_REF] Elliott | Control of rHuEPO biological activity: The role of carbohydrate[END_REF]. In recent years, there have also been developments in the use of transgenic insect cells, engineered to mimic mammalian cell glycosylation of expressed proteins [START_REF] Kost | Baculovirus as versatile vectors for protein expression in insect and mammalian cells[END_REF].

Process optimization is the other approach to introduce the wanted glycan profile on biotherapeutics: media composition (glycosylation substrate feeding or media supplements)

and process optimization (pH, temperature, and osmolality) can be employed to increase sialic acid content [START_REF] Durocher | Expression systems for therapeutic glycoprotein production[END_REF]. One method that has been investigated is supplementation of the media with 25 mM glucose that enabled production of chimeric mAbs exhibiting greater homogeneity of the protein glycoforms [START_REF] Liu | The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the Nglycosylation profile of a monoclonal antibody[END_REF] to mitigate the toxic effects of ammonia generated by cellular respiration, the media can be supplemented with proline, threonine, and glycine resulting in increased sialic acid content of recombinantly expressed tissue plasminogen activator, used to treat stroke patients [START_REF] Roth | Recombinant Tissue Plasminogen Activator for the Treatment of Acute Ischemic Stroke[END_REF]. Metals such as iron have been added to cell culture media and were all shown to alter glycosylation levels of diverse proteins such as interferon-γ in CHO [START_REF] Clincke | Effect of iron sources on the glycosylation macroheterogeneity of human recombinant IFN-γ produced by CHO cells during batch processes[END_REF].

Biotherapeutics regulation

Having discussed the importance of glycosylation at a molecular level, now its impact on the marketing of biotherapeutics will be explored. Throughout the stages of drug development, all the data regarding the production process and its safety and efficacy are reviewed by regulatory agencies [START_REF] Del Val | Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics[END_REF]. Glycosylation is considered a critical quality attribute (CQA) that according to ICH Q8 [START_REF]EMEA/CHMP, 2009, ICH Topic Q 8 (R2) Pharmaceutical Development, Step 5: Note for Guidance on Pharmaceutical Development[END_REF] is a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. ICH is the International Council of Harmonization of Technical Requirements for Pharmaceuticals for Human Use, born in 1990, whose aim is to achieve an amity worldwide on the regulations to be applied in order to have safer, more effective and high quality medicines. Regulatory standards for biotechnology-derived pharmaceuticals are generally harmonized among the European Union, Japan and United

States. The ICH Q6B [45] is the one taking care of glycosylation, in the specific section 2.1.1 "Physicochemical properties". It is stated that the composition, physical properties and structure of a product have to be determined. It is admitted a degree of heterogeneity but this must be consistent with the one of the batches used in preclinical and clinical studies and it doesn't have to be detrimental for the safety and efficacy of the product.

Therefore, the degree of heterogeneity and the glycosylation profile of biotherapeutics has to be clearly defined during the research and clinical trials and has to be respected during the upscale of the process and the following production phases. Any alteration from the established glycosylation pattern has to be evaluated. The case of the company Genzyme is an example of the importance of the glycosylation profile of biotherapeutics. The company developed the drug Myozyme for the treatment of the Pompe disease, a glycogen storage disorder, made in relatively small 160-liter batches in the United States, that was only commercially available to children and infants. The biotechnology company in 2009 had asked the U.S. Food and Drug Administration for permission to make Myozyme in 2000-liter batches, in order to ease supply restraints and make the product available to adult patients, but the FDA delayed the approval of the medicine on large scale production for a difference in the glycans composition, causing a loss in the revenue of the company. The FDA determined that since the drug coming out of the 2000-liter batches was chemically slightly different than Myozyme, it needed to be approved as a separate medicine under a separate name, now marketed as Lumizyme [START_REF] Ratner | Genzyme's Lumizyme clears bioequivalence hurdles[END_REF].

The glycosylation control becomes even more important when it comes to biosimilars. A biosimilar is a biologic which is highly similar to the original medicine already approved, in terms of safety, purity and efficacy [START_REF] Community | Glyco@ALPS -Glyco 2030: A Roadmap for Glycoscience in Europe[END_REF]. It is the equivalent of a generic drug for small molecules and the first biosimilar that has been put on the market was infliximab biosimilar in 2013 [START_REF] Mabdesign | Immunowatch Edition n°3 -July 2021[END_REF]. The expiring of the patents in the next 20 years will contribute to the rise of biosimilars [START_REF] Community | Glyco@ALPS -Glyco 2030: A Roadmap for Glycoscience in Europe[END_REF]. Glycosylation is critical in the development of biosimilars because it has to be in accordance with the one of the original medicine, therefore quality control will play a vital role in the European biologics market.

Production of drugs involves a highly-controlled environment constrained by strict regulations and standards, and compared to chemical synthesis of small molecules, bioproduction of therapeutics includes many steps due to the complexity and the biological origins of these drugs. As such, cost of gods is higher for biologics than for small molecules [START_REF] Mabdesign | Immunowatch Edition n°3 -July 2021[END_REF]. The quality control is one of these steps to ensure that the end product has the desired properties. The widespread approach for drug quality control testing and approval is the so-called quality by testing (QbT). In QbT at the end of each batch, the product is tested for compliance with the desired quality. The QbT approach uncouples product end quality from the manufacturing process because doesn't require the knowledge that put in relationship the bioprocess inputs with the product quality outputs. On the contrary, the more recent idea is that quality must be built into the product. This is the main goal of the other approach, the quality by design (QbD) that means to build quality at every stage of process development [START_REF] Del Val | Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics[END_REF]. This concept was developed by Dr. Joseph M. Juran who believed that most quality problems are related to the way a product is designed in the first place. The QbD consists of the identification of CQA, product and process design, control for each step of the manufacturing process and process continuous improvement [START_REF] Yu | Understanding pharmaceutical quality by design[END_REF].

The current methods of quality control of biotherapeutics glycosylation make difficult to have an in process quality control as required by the QbD approach. The tools most commonly used for glycan characterization include HPLC and CE, followed MS. MALDI-TOF/MS analysis gives a reasonably good glycoform profile rapidly enough, although getting actual percentage results takes days. NMR imaging can also be used for glycans; but the drawback is that it requires reasonably pure glycan structure in relatively large quantity. When looking at the glycoproteins, it is important to determine glycosylation site occupancy as well as their overall intact protein profiles. For example, CE with sodium-dodecyl sulphate gel (CE-SDS) is readily used to determine the degree of occupancy at Asn 297 site for an IgG1 but for more complex glycoproteins, currently available methods are not yet optimal for quantitative purposes. Using peptide mapping, it can be seen whether a site is occupied, but that is difficult to quantify even using liquid chromatography-mass spectroscopy (LC-MS) [START_REF] Solá | Glycosylation of Therapeutic Proteins[END_REF].

Research aim

It would be ideal to have rapid tool allowing to examine more samples in-process with the aim of controlling and potentially optimising conditions in real time. The ideal tool would be affordable for the manufacturers, cost-effective, fast and easy-to use. The techniques described above give highly detailed carbohydrate structures but they present different drawbacks. First of all is the "human factor": they require knowledge, experience and training to be carried out correctly and to have reliable and reproducible results. Not all scientist have the skills to carry out experiments using a mass spectrometer. In addition those machines are very expensive and not all laboratories have one. Another factor is the accuracy: if carbohydrates need to be released for the analysis, it is very unlikely that they will be released at 100%, it is usually around 85%. Especially when it comes to O-linked glycans, because the methods of releasing are not as efficient as for N-linked glycans [START_REF] Solá | Glycosylation of Therapeutic Proteins[END_REF]. Another important point to consider is that the end product is a glycoprotein, so it would be in the best interest to look at the entire protein with the glycans still on it. Moreover, these techniques tend to be timeconsuming. The development of LEctPROFILE kits is meant to provide a convenient and complementary methodology to satisfy the requirements of the QbD process. First of all, many lectins show promiscuous binding: one of the goal for the development of the kits is the purification of highly-specific lectins for the recognition of unwanted glycan structures on biotherapeutics. It can be carried out by non-specialized researcher: it is not needed a printing machine to spot the lectins, because it is in a 96-well plate format. Everything can be done manually with the basic tools that every laboratory has and without a specific training for the users. There is no need to remove glycans but the glycoprotein can stay in its natural condition.

The glycan-containing sample has to be labelled prior to the analysis only if the direct-binding mode is chosen (Fig. 1.16 A). To avoid this additional step and above all to avoid adding unwanted variability due to the labelling efficiency, a secondary antibody can be used. Even better, the assay can be performed in a inhibition mode(Fig. 1.16 B): the sample competes with a previously labelled neoglycoprotein that has the same specificity of the sample for the lectin immobilized on the plate. Another advantage is that is a high-throughput analysis, and the plate can be designed according to the user's needs, customizing the plate according to the number of samples. It is also a very fast method that in 2 hours give the desired results, using for the detection a plate reader that is found in the majority of lab facilities. Results

analysis is also taken into consideration during the development: the user will only need to insert the raw data and the output will be given thanks to a predesigned data sheet. Fig. 2. 1 A structure of the neoRSLVI in complex with aMetilFucose (spheres), all binding sites result occupied, adapted from [START_REF] Arnaud | Membrane deformation by neolectins with engineered glycolipid binding sites[END_REF]. B Cartoon representation of a Janus lectin, able to cross-link biological surfaces thanks to its double specificity for fucosylated glycans on one side and sialylated ones on the other side, adapted from [START_REF] Ribeiro | Tailor-made Janus lectin with dual avidity assembles glycoconjugate multilayers and crosslinks protocells[END_REF].

Another approach that allows to study protein-glycan interactions is from the glycan's point of view. In nature, the affinity is increased when glycans are presented linked to other structures, such as glycolipids or glycoproteins. In The glycoside cluster effect occurs when multivalency is achieved on both sides, the one of the protein and the one of the glycan [START_REF] Lee | Carbohydrate-Protein Interactions: Basis of Glycobiology[END_REF]. However, multivalent glycans in nature are structurally complex and thus not suitable to study physiological mechanisms. Therefore, another way to increase the avidity and consequently the affinity, is to chemically synthetise multivalent glycans choosing the type of scaffold, playing with their size, the density of ligand, the orientation and the flexibility. The general idea is to have a multivalent glycoconjugate that has one glycan specificity displayed in a way that results in a strong multivalency. • Glycoclusters usually contain a limited number of linked glycoconjugates of which the orientation and position are synthetically controlled [8].

.

• Glycodendrimers are macromolecules completely built around a core unit and completely branched throughout their structures. Also in this case, the valency and the terminal functional groups are well-controlled [3].

• Glycopolymers are wholly synthetic multivalent or polyvalent cluster glycosides [3].

For example, the polymer polyacrylates were used to incorporate 5-N-acetyl neuraminic acid in order to reveal the maximal inhibition of the influenza virus receptorbinding activity [START_REF] Mochalova | Synthetic polymeric inhibitors of influenza virus receptor-binding activity suppress virus replication[END_REF].

• Glyconanoparticles are made of magnetic materials tethered with specific glycans.

The most used are made of iron oxide and gold for their nontoxic and biodegradable properties [8]. Reynolds et al. [START_REF] Reynolds | Multivalent gold glycoclusters: High affinity molecular recognition by bacterial lectin PA-IL[END_REF] showed that gold nanoparticles exhibiting Gal ligands in a multivalent fashion had a 3000-fold increase in the affinity of the lectin PA-IL, from Pseudomonas aeruginosa, over the monovalent ligand.

• Neoglycoproteins defined as a glycoproteins produced by the covalent attachment of mono-, oligosaccharides, or dendrimers to proteins that may or may not already contain naturally occurring, covalently bound carbohydrate [START_REF] Stowell | Neoglycoproteins the preparation and application of synthetic glycoproteins[END_REF]. The main difference with a glycoprotein is that of course, the linkage with the glycans is not natural. In addition the occupancy and glycan density are controlled, as well as the homogeneity of glycosylation.

Neoglycoproteins

Applications

Extensive research has shown that neoglycoproteins are suitable for many practical applications other than the study of biological interactions. Applications for imaging include human serum albumin modified with 99mTc-galactose for radiolabelling. It is exploited in order to estimate the hepatocyte mass and function by imaging the expression of the asialoglycoprotein receptor in the liver [START_REF] Srinivas | 3.23 Neoglycoproteins[END_REF].

Neoglycoproteins have been also used successfully as drug carriers in order to selectively release drugs inside cells. Methotrexate, which acts as a metabolic inhibitor within the cells, is a chemotherapic drug for the treatment of leukemia. It was synthetized a fucosyl serum albumin conjugate to methotrexate neoglycoprotein which was more cytotoxic than free methotrexate, indicating that methotrexate bound to the neoglycoprotein is more actively internalized than the free drug [START_REF] Srinivas | 3.23 Neoglycoproteins[END_REF].

Pathogenic infections are often mediated by an initial glycan-protein interaction, as it happens for example, for the recognition of sialic acid on human cells by the influenza hemagglutinin.

Since neoglycoproteins can present multiple glycans epitopes, their potential immunogenic nature have been used to design vaccines against viruses, bacteria, parasites. One case is the one of the gram negative bacterium Neisseria meningitidis responsible of the epidemic meningitis in Sub-Saharan Africa. Human Serum Albumin conjugates of synthetic fragments of bacterium capsular polysaccharide of this bacterium were synthesized and it was showed that even small as monosaccharide fragments was recognized by polyclonal Meningococcus A antiserum [START_REF] Berkin | Towards a synthetic glycoconjugate vaccine against Neisseria meningitidis A[END_REF].

Chapter 2 -Neoglycoproteins 35

Synthesis

The first method for preparing neoglycoproteins was developed in 1929 by Goebel and Avery, who attached a variety of mono-and oligo-saccharides to ovalbumin and horse serumglobulin [58].

A number of criteria must be considered in developing the protocol for the chemical synthesis of neoglycoproteins:

• Reproducibility is one of the main factors, therefore the conditions of preparation of glycans need to be stable and applicable to a great variety of glycans. It is preferable if the reaction is rapid and works under mild conditions. The modification should be specific, preferably for one functional group. and stable to physiological conditions.

• The protein chosen as a scaffold, it should maintain its native structure and its biological activity, again underlining the importance of keeping mild conditions for the reaction. If the modified protein needs to be used in vivo, the linkage should be resistant to metabolic and enzymic degradation. The modified protein should be easy to purify from the reaction mixture, and the modification must be analysed and quantified.

• The linkage between carbohydrate and protein should not introduce long-chain hydrocarbons, aromatic rings, or ionic species, as such groups as these may interfere with the activity of the protein, or interact non-specifically in biological systems [START_REF] Stowell | Neoglycoproteins the preparation and application of synthetic glycoproteins[END_REF].

There are many ways to synthetise neoglycoproteins, for example the the conjugation to cysteine with terminal thiol, conjugation to aspartic or glutamic acid through their carboxylic group [1]. The conjugation to lysine with terminal primary amine is one of the most used. The most common way to do this is through reductive amination. In this type of reaction, the protein is solubilized in borate buffer at pH around 8 or 9 with a large excess of glycans. The reducing end of the target glycan forms a Schiff's base and react with the lysins ε-amino group on the protein. Since Schiff's base is not a stable product, by adding cyanoborohydride (NaBH3CN) in solution, the equilibrium of the reaction is pushed towards the formation of the product [START_REF] Roy | Improved procedures for the conjugation of oligosaccharides to protein by reductive amination[END_REF].

Another more versatile strategy, is to use a linker for the modification of lysins on the protein scaffold. The linker can be of different length and it can present different active groups: isothiocyanate, 2-imino-2-methoxyethyl -1-thioglycoside, carbodiimide-activated carboxylic acid. All these groups are activated in order react with primary amine, while, on the other side, the glycan moiety remains intact [START_REF] Ogura | Exploring the glycan interaction in vivo: Future prospects of neo-glycoproteins for diagnostics[END_REF].

In this thesis it is explored only the synthesis of neoglycoprotein by click-chemistry after modification of the lysine amino acid with a propargyl or azido linker.

Synthesis by click chemistry

The CuAAC click-chemistry reaction (copper-catalysed azide-alkyne cycloaddition), was discovered in 2002 by two independent groups [START_REF] Tornøe | Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides[END_REF] [62], and, since then, it has been improved and now it has become an excellent tool in organic synthesis. This reaction occurs between two molecular entities that are functionalized by azido and alkyne groups, giving as product the 1,4-disubstituted-1,2,3-triazole molecules (Fig. 2.4 A). It is an advantageous compound because it is chemically inert against oxidation, reduction and hydrolysis under acidic or basic conditions. The advantage of the addition of catalytic amount of copper is that the reaction goes much faster, taking few hours, and can be performed at room temperature.

Additionally, this reaction is very convenient because it does not require any products separation since it has a very high regioselectivity being the 1,4-disubstituted isomer the only product formed (Fig. 2.4, A, B).

Results and discussion

In the recent years, the synthesis of neoglycoproteins has been a focal point for For the synthesis of neoglycoproteins used for this thesis, the scaffold used is the protein Bovine Serum Albumin (BSA). It is a protein of 66.5 kDa and consists of three homologous domains, conferring a heart-shaped molecule. It is soluble in neutral and alkaline medium and it is big enough to allow a heavy sugar substitution [START_REF] Srinivas | 3.23 Neoglycoproteins[END_REF]. It is cheap, enabling to produce large batches of neoglycoproteins and it is not glycosylated so there is no interference in the glycans of interest-lectin interactions.

The neoglycoproteins synthesis is carried out following two main steps:

1. The first step is the functionalization of BSA with Propargyl-N-hydroxysuccinimidyl ester (PNHS) obtaining BSA-alkyne (Fig. 2.5 A). In fact, PNHS react with primary amines(-NH2) of lysine. This step was optimized by Dr. Blanka Didak who obtained a ratio of 35-40 alkyne/BSA, which is very good, considering that on BSA there are around 60 lysins, leaving 20 lysins for additional modifications such as labelling with biotin.

The "reverse" protocol to obtain BSA-azide was optimized during the course of my thesis thanks to Dr. Benoît Roubinet. This functionalization is carried out again via the primary amine groups (-NH2) of BSA reacting with the N-hydroxysuccinimide ester (NHS) of the reagent 2-azidoacetic acid NHS ester (Fig. 2.5 B). In this case, a ratio of Fig. 2. 5 A Reaction between the primary ammines of the BSA and PNHS ester, followed by the click-chemistry reaction (CuAAC) between BSA-alkyne and the glycan functionalized with an azide group. B Reaction between the primary ammines of the BSA and the NHS, followed by the click-chemistry reaction (CuAAC) between BSAazide and the glycan functionalized with an alkyne group.

Synthesis of BSA-alkyne and BSA-azide

We performed the synthesis of BSA containing alkyne groups and BSA containing azide groups, in order to have the right scaffolds to build the desired neoglycoproteins for the LEctPROFILE kits. For the synthesis of BSA-alkyne, the chemical reaction happens between the primary amines NH2 of BSA lysins and PNHS (Fig. 2.5 A). For the synthesis of BSA-azide, the chemical reaction happens between the primary amines NH2 of BSA lysins and NHS (Fig. The choice of BPA is to see if there is a cross-reaction of the neoglycoprotein with lectins specific for β-Gal. As expected, the NeoGalili showed no interactions (fluorescence intensity < 0) with UEA-I and WGA, the first one specific for fucose and the second one for Neu5Ac and GlcNac. From the chart, it can be seen that by far the highest NeoGalili interaction is with the lectin GSLIB4 and after with MOAβT and the full MOA. Overall, these results indicate that the synthesis via click chemistry of the NeoGalili neoglycoprotein was successful and therefore this neoglycoprotein makes an optimal probe for the development of the Galili antigen LEctPROFILE kit.

B)

Synthesis of Neo6'SL and Neo3'SL neoglycoproteins

In order to synthetize the neoglycoproteins Neo6'SL and Neo3'SL, needed as probes for the presence of α2-3/ α2-6 linkages within the LEctPROFILE kit, we used the BSA functionalized with the alkyne group to react with the azide group on the thrisaccharide 6'sialylactose-N3 and 3'-sialylactose-N3 (Fig. 2.12).

Conclusion

In this Chapter, it the synthesis of five neoglycoproteins of interest, by coppercatalysed azide-alkyne cycloaddition, is described. The neoglycoproteins produced will serve as standard probes for testing the performances of the three LEctPROFILE. The protocol developed for the synthesis of these molecules resulted in good yield, of 5 mg/synthesis, and high reliability batch to batch, key parameters to have reliable reagents for the kits development. The neoglycoprotein were also characterised through SDS-PAGE, MALDI-TOF/MS analysis and GLYcoPROFILE, allowing to confirm a high level of purity, quantify the ratio of sugar/BSA and study their specificity to each neoglycoprotein. Overall, these results build a solid ground for the next steps in the development of the kits.

Paper 1-Homo-and Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins

Abstract

The Galα1-3Gal epitope, also called Galili antigen, is found in mammals except from humans, apes and Old World Monkeys that lost its encoding gene (GGTA1) for the enzyme α1,3galactosyltransferase (α1,3GT) during evolution. As a consequence, 1% of the circulating antibodies in humans are anti-Gal antibodies. This is an obstacle for the production of biotherapeutics in nonhuman mammalian cell lines, such as murine myeloma cells. The case of the Cetuximab monoclonal antibody, which is produced in these type of cells, was a striking case of hypersensitive reaction among patients due to the Galili antigen. For the same reason, anti-Gal antibodies are an obstacle for the transplantation of organs from pig into humans. To date, there is no a fast, high-throughput screening able to detect the presence of the Galili antigen on biotherapeutics and transplanted tissues and organs. We want to address this gap by building a lectin array highly specific and easy to use, in order to detect this antigen on biotherapeutics and biological samples. In this chapter, a review about the Galili antigen and an overview on the potential application of the Galili antigen

The Galili antigen

The enzyme α1,3-galactosyltransferase (α1,3GT), encoded by the gene GGTA1, synthesizes the α1,3-galactose epitopes (Galα1-3Galβ1-4GlcNAc-R) (Fig. 3.1A), which can be found at the external part of glycoproteins and glycolipids. α1,3GT uses N-acetyllactosamine residues as the sugar acceptor and UDP-Gal as the sugar donor (Fig3. 1B). This reaction takes place in the Golgi apparatus of non-primate mammals, prosimians and New World monkeys (South American monkeys). This enzyme is not found in humans, apes and Old World monkeys (African monkeys) that in contrast produce antibodies (Abs) against the Galα1-3Galβ1-4GlcNAc-R epitope, to which was given the name of Galili antigen from the scientist who discovered it [START_REF] Galili | Anti-Gal: An abundant human natural antibody of multiple pathogeneses and clinical benefits[END_REF]. It can also be called α-Gal epitope or xenotransplantation epitope for reasons that will be explained further on in this chapter (Paragraph 3.2.2). The gene GGTA1 was inactivated in ancestral Old World monkeys and apes around 20-28 million years ago. One hypothesis on the disappearance is that it was due to an epidemic caused by some infectious agents spread in the Old World and expressing the Galili antigen. The pathogen exerted a selective pressure for these lineages to develop anti-Gal antibodies and to suppress the GGTA1 gene in order to, respectively, get rid of the Galili structure to survive the pathogen and to avoid autoimmune reactions. The other hypothesis is that a pathogen in the Old World was using the Galili antigen to enter the cells and it caused a selective pressure for primates to inactivate the GGTA1 gene. Consequently, the Galili structure started to be recognized as an antigen and antibodies against it started to be produced [67]. Now, it is calculated that there are around 1% of circulating anti-Gal IgG in immunocompetent individuals, produced in response to intestinal bacteria, many of which express this carbohydrate antigen [START_REF] Galili | One Percent of Human Circulating B Lymphocytes Are Capable of Producing the Natural Anti-Gal Antibody[END_REF]. Antibodies can bind either only the disaccharide but also the pentasaccharide as long as it contains the Galα1-3Gal epitope [START_REF] Kirkeby | Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B4 isolectin to αgalactosyl carbohydrate antigens in the surface phase[END_REF]. [START_REF] Biolabs | Glycoproteomics Technical Guide[END_REF]. B, the cetuximab mechanism: it binds to the EGFR and prevents its dimerization, blocking all its pro-tumour functions and promoting apoptosis, adapted from [START_REF] Patil | Cetuximab and biomarkers in non-small-cell lung carcinoma[END_REF].

Chapter 3 -

Hypersensitivity is defined as the allergic immune reactions, stimulated by soluble antigens, that can have different degrees of intensity and, in the case of anaphylaxis, it could be fatal. In the early Chapter 3 -The Galili Antigen 60 2000s, some patients started to show immediate onset of anaphylaxis after the administration of intravenous Cetuximab. Further studies pointed in the direction of anti-Gal IgE Abs and found that individuals with anti-Gal IgE ≥0.35 IU/mL were more likely to develop a reaction against Cetuximab.

As seen in Paragraph 3.1, all humans have IgG antibodies specific for the disaccharide Galα1-3Gal and Cetuximab is produced in murine myeloma cells which can synthesise this glycan motif. Of the total α-gal in Cetuximab, most of it is located on the Fab domain [START_REF] Steinke | The alpha-gal story: Lessons learned from connecting the dots[END_REF]. Anti-Gal-producing B cells occasionally undergo isotypes switch to produce IgE antibodies, which cause allergic reactions by binding to α-gal epitopes [START_REF] Galili | The α-gal epitope (Galα1-3Gal1-4GlcNAc-R) in xenotransplantation[END_REF].

The investigation of this phenomena started considering the geographical area where the hypersensitivity was more common and it turned out it was in the South-eastern regions of the United States, mainly in North Carolina, Arkansas,Missouri, Virginia, and Tennessee. In the same regions there were some cases of people, having spent much time outdoors, developing gastrointestinal symptoms, skin rash and urticaria around 3 to 6 hours after red meat consumption. After careful analysis, it was stated that also in this case the allergy was triggered by anti-Gal IgE Abs.

What was left to be understood was the reason why so many cases were concentrated in the same area, what was the link between Cetuximab anaphylaxis and red meat allergy and what is the mechanism behind the isotype switch from IgG to IgE. Most patients of the very same regions with red meat hypersensitivity reported having recently been bitten by the Lone Star tick (Amblyomma americanum). Isotype switch to anti-Gal IgE was observed in several individuals following a bite from this tick. In Europe and Australia, the isotype switch was connected to different tick species than in the United States. In Europe, Ixodes ricinus was implicated, while in Australia the relevant tick was Ixodes holocyclus. There are three theories to explain the isotope switch: the response could be caused by some component of tick saliva, by some residual glycans from a previous blood meal or by another organism commensal of the tick [START_REF] Steinke | The alpha-gal story: Lessons learned from connecting the dots[END_REF].

The big appearance in North America was due to the increase of deer's population on the territory since these animals are a major carrier of lone star ticks. In turn, the increasing deer population was linked to several reasons, among which the enactment of leash laws for dogs and a decrease in the number of hunters. This phenomenon got the name of α-gal syndrome [START_REF] Steinke | The alpha-gal story: Lessons learned from connecting the dots[END_REF] (Fig. 3. 3). Therefore, the conclusion was that the Galili contained in the Cetuximab administered to those patients was enough to trigger a strong immune response in individuals having already a significant anti-Gal IgE titre. Thus, the Galili antigen LEctPROFILE kit could be used as a screening method to detect as early as possible (research stage) the level of Galili antigen in biotherapeutics and make sure that it does not overcome the threshold established by the Regulatory Agencies (Chapter 1, Paragraph 1.6.4).

Chapter 3 -The Galili Antigen

In organ transplantation

Transplantation is the implant of a living tissue or an organ from a donor into a recipient. It mostly consists of allotransplantation, when an organ is transplanted from a person, living or deceased depending on the organ donated, to another one. Since 1980, it has been a revolutionary medical practice but it still presents many setbacks which are really hard to overcome. First of all, patients must take immunosuppressive drugs that do not always prevent rejection. Second, transplantation is a treatment, not a cure, and the implanted organs last a determined period of time (on average around 10 years [START_REF] Rana | [END_REF]) after which they should be replaced. And here pops up the third and main problem: the shortage of organs coming from compatible donors. According to the US Health Resources and Services Administration, currently, there are more than 100000 people waiting for an organ and every nine minutes a person is added to the list. To solve this critical issue, it has been thought that organs coming from other mammals could be used: pig organs have attracted attention for their size which is really similar to humans. Mammal organs, that are not humans, used for transplantation, are defined as "xenograft" from which the process takes the name "xenotransplantation".

There is anyway an immunologic barrier between pigs and humans and one of the immunogenic epitopes is the Galili antigen that is produced in pigs and for which humans have circulating antibodies (Paragraph 3.1). The Galili antigen for this reason is also called xenotransplantation epitope and it is to be expected that transplantation of pig organs into humans results in the binding of anti-Gal Abs to the Galili antigen on the xenograft cells with their consequent destruction.

Immunosuppressive drugs are not sufficient to avoid the organ rejection, unless used at a dose that Chapter 3 -The Galili Antigen 62 completely inhibits the immune system, preventing the recipient from being able to respond to eventual infections [START_REF] Galili | The α-gal epitope (Galα1-3Gal1-4GlcNAc-R) in xenotransplantation[END_REF].

In the early 2022, the first xenograft transplant was performed at the University of Maryland School of Medicine (Baltimore, MD, USA), on the patient David Bennett, a 57-year-old man with advanced heart failure and a type of arrhythmia called ventricular fibrillation, who was declared ineligible to receive a human heart. The genetically modified pig heart was provided by Revivicor, a regenerative medicine company based in Blacksburg, Virginia (USA). They are known for the production of the so-called "Galsafe pigs" that are α1,3GT-deficient [START_REF] Phelps | Production of α1,3-galactosyltransferase-deficient pigs[END_REF]. Other than the Galili antigen as carbohydrate knockouts, the gene edits included the Sda blood group antigen, and N-glycolylneuraminic acid.

Seven more genetic modifications were added: 6 human genes were inserted that are two antiinflammatory genes, two genes that promote normal blood coagulation and prevent blood vessel damage, and two other regulatory proteins that help downsize the antibody response plus a modification for a growth hormone to control the size of the heart and reduce the chance the organ would grow once implanted [START_REF] Griffith | Genetically Modified Porcine-to-Human Cardiac Xenotransplantation[END_REF].

Unfortunately, after two months from the transplant, the patient died from a porcine virus. Of course, the Galili antigen is not the only source of rejection or death, nevertheless, the fast screening of knock-out pigs could help the detection of the unwanted glycan and add a small brick in the improvement of these organs. The LEctPROFILE kit could be an interesting option for detection of the Galili antigen in knock-out animals.

In vaccines and other clinical settings

Since humans have natural anti-Gal antibodies and the Galili antigen is quite easy to chemically synthesise, it could be potentially applied to increase the immunogenicity of viral vaccines. The immunogenicity of influenza vaccine is not optimal, leading to the main problem of the low uptake of the vaccine by antigen presenting cells (APC). This happens because the quantity of hemagglutinin A (HA), the viral envelope glycoprotein included in the annual vaccines, is very low (15 μg HA of each vaccinating strain) [START_REF] Macher | The Galα1,3Galβ1,4GlcNAc-R (α-Gal) epitope: A carbohydrate of unique evolution and clinical relevance[END_REF]. Moreover, the APCs at the vaccination site uptake the vaccine by random endocytosis because they do not recognize any glycoprotein marker.

The mechanism of action of viral vaccines is shown in Fig. 3.5: the viral antigen is recognized by the APCs that could be macrophages, dendritic cells and Langerhans cells of the skin. Dendritic cells further process the viral glyco/protein into peptides and present them by major histocompatibility complex (MHC) activating the cell-mediated immune response. MHCII presents the antigen to CD4+Tcells. CD4+T cells differentiate into CD4+T helper cells that help activate CD8+T cells that release granzyme, perforins and interferon γ, and B cells that produce antibodies. Eventually, memory T cells and memory B cells are produced [START_REF] Sokolova | The potential of nanoparticles for the immunization against viral infections[END_REF]. If the viral antigen injected with the vaccine is not a sufficient amount, it will not be enough to activate a good adaptive immune response and the efficacy of the vaccine will be lowered. The HA of influenza has 6-8 N-linked glycans [START_REF] Roberts | Role of Conserved Glycosylation Sites in Maturation and Transport of Influenza A Virus Hemagglutinin[END_REF] whereas major glycoprotein in the envelope of HIV is the gp120 which has 24 N-linked glycosylation sites. Studies were carried out to see how the immunogenicity of vaccines against these pathologies could be improved by carrying alpha-gal epitopes on their multiple carbohydrate chains. Flu virus is usually produced in embryonated chicken eggs. Chickens lack α-gal epitopes, and since Sia is removed from viral glycoproteins by viral neuraminidase, the carbohydrate chains on HA terminate with N-Acetyllactosamine (Galβ1-4GlcNAc-R). Thus, α-gal epitopes could be added to all carbohydrate chains by recombinant α1,3GT. α1,3GT knockout mice immunised with inactivated flu virus vaccine expressing α-gal epitopes presented higher anti-flu virus antibody and T cell response in comparison to knockout mice immunised with the original inactivated flu virus lacking α-gal epitopes [START_REF] Abdel-Motal | Immunogenicity of Influenza Virus Vaccine Is Increased by Anti-Gal-Mediated Targeting to Antigen-Presenting Cells[END_REF]. The gp120 carbohydrate chains, produced in CHO cells, have the terminal structure Sia-Galβ1-4GlcNAc-R. They were converted to α-gal epitopes by incubation of the glycoprotein with a mixture of neuraminidases for removal of Sia plus α1,3GT and UDP-Gal. Immunizations of knockout mice with the gp120 expressing α-gal epitopes resulted in higher anti-gp120 antibody response and a similar higher T cell response, in comparison to the immune response in knockout mice receiving similar immunizations with gp120 lacking α-gal epitopes. This principle could be applied to different types of vaccines including the ones for cancer [START_REF] Abdel-Motal | Increased Immunogenicity of Human Immunodeficiency Virus gp120 Engineered To Express Galα1-3Galβ1-4GlcNAc-R Epitopes[END_REF].

Since anti-Gal Abs are the most abundant natural antibodies in humans, there are other clinical settings in which the Galili antigen/anti-Gal interaction could be potentially exploited : one example is the injection into patients with solid tumours of glycolipids with carbohydrate chains capped with Galili epitopes (Fig. 3.6). They would act as Tumor Associated Antigen (TAA) recruiting APC to the site and starting the immune response against cancer cells. In clinical studies, patients with advanced-stage cancer survived more than expected after the injection and no patients developed clinical signs of toxicity or autoimmunity [START_REF] Whalen | Cancer immunotherapy by intratumoral injection of α-gal glycolipids[END_REF]. The same principle can be applied also for accelerating the healing of wounds, burns, regeneration of ischaemic myocardium and of injured nerves; α-gal nanoparticles applied to the injury site will bind anti-Gal and may induce rapid macrophage migration and activation for the secretion of vascular endothelial growth factor but further studies need to be conducted on mammalian animal models Chapter 3 -The Galili Antigen 65 [START_REF] Galili | Anti-Gal: An abundant human natural antibody of multiple pathogeneses and clinical benefits[END_REF]. Taking these studies into account, the Galili antigen LEctPROFILE kit could be a fast screening tool for the presence and relative quantification of Galili antigen during all phases of the vaccine and α-gal glycolipids and nanoparticles production. is detected with blood group A or H oligosaccharides [START_REF] Abdel-Motal | Increased Immunogenicity of Human Immunodeficiency Virus gp120 Engineered To Express Galα1-3Galβ1-4GlcNAc-R Epitopes[END_REF]. It also binds to linear oligosaccharides such as Galα1-3Gal1-4Glc on glycosphingolipid, isoGb3 and the Galili antigen Galα1-3Gal1-4GlcNAc [START_REF] Gemeiner | Lectinomics. II. A highway to biomedical/clinical diagnostics[END_REF]. The overall protein sequence is around 290 amino acids. The entire structure consists of a N-terminal β-trefoil domain and a C-terminal proteolytic domain [START_REF] Juillot | Uptake of Marasmius oreades agglutinin disrupts integrin-dependent cell adhesion[END_REF]. Three carbohydrate-binding sites are located on the trefoil domain but they have different degrees of affinities for the ligand, with the α and β sites appearing to have an higher affinity than the γ (Fig. 3. 7). The proteolytic domain doesn't bear binding sites but is important for protein homodimerization [START_REF] Grahn | Crystal Structure of the Marasmius Oreades Mushroom Lectin in Complex with a Xenotransplantation Epitope[END_REF].

In our case, only the N-terminal β trefoil domain (156 amino acids) was cloned, expressed in E. coli and purified by affinity chromatography to perform the experiments described in this thesis. We named it MOAβT, which means MOA beta trefoil.

GSLI-B4

Griffonia simplicifolia lectin-1 is a carbohydrate-binding protein present in the seeds of the African leguminous shrub. It is a mixture of five tetrameric isolectins that vary in their content of A and B subunits. It is highly specific for α-Galactose and the xenotransplantation antigen was often studied by using this lectin in the course of the years [START_REF] Kirkeby | Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B4 isolectin to αgalactosyl carbohydrate antigens in the surface phase[END_REF]. We have decided to include it in the Galili antigen LEctPROFILE kit study because it is instructive to compare it with MOAβT. Structural data suggest that the GSLI-B4 isolectin has a very restricted binding site that does not appear to easily accommodate glycans beyond the monosaccharide unit [START_REF] Tempel | The xenograft antigen in complex with GS-1-B 4 lectin: crystallization and preliminary X-ray analysis[END_REF]. This was verified by the X-ray crystallographic structure of the B4 isolectin complexed with Galα1-3Gal in which it is shown that only the non-reducing galactosyl group makes contact with the lectin (Fig. 3.8) [START_REF] Winter | The mushroom Marasmius oreades lectin is a blood group type B agglutinin that recognizes the Galα1,3Gal and Galα1,3Gal,β1,4GlcNAc porcine xenotransplantation epitopes with high affinity[END_REF]. Moreover, Kirkeby and Moe, using an enzyme-linked lectin assay, showed that galactosyl disaccharides linked in α1-2, α1-3, and α1-4-galactobiosyl groups as well as the linear Galili antigen, linked to human serum albumin were very similar in their binding affinity to GSLI-B4 [START_REF] Lee | mAbs Glycosylation profile and biological activity of Remicade® compared with Flixabi® and Remsima® Glycosylation profile and biological activity of Remicade Ò compared with Flixabi Ò and Remsima Ò[END_REF]. Results and discussion

MOAβT Purification

The β-trefoil domain was cloned and purified during my secondment at the CERMAV (Centre de Recherches sur les Macromolécules Végétales) in Grenoble, in January 2020. The purification protocol and lectin characterization are described in the paper at the end of this chapter [START_REF] Notova | Extending Janus lectins architecture: characterization and application to protocells[END_REF]. At GLYcoDiag, we optimized the purification protocol for industrial scale purification. The purification procedure is property of GLYcoDiag and for this reason it will not be described in detail

GSLI-B4 Functionality

This lectin was not produced at GLYcoDiag but it was purchased from Vector. We checked the functionality of the lectin and its selectivity through GLYcoPROFILE in direct binding. The bar chart in Fig. 3.10, shows that the specificity of GSLI-B4 for NeoGalili is higher than thyroglobulin, in accordance to literature: Kirkeby and Moe obtained higher affinity when thyroglobulin was denatured, proving that the accessibility of the galactosyl residues needs to be taken into account [START_REF] Lee | mAbs Glycosylation profile and biological activity of Remicade® compared with Flixabi® and Remsima® Glycosylation profile and biological activity of Remicade Ò compared with Flixabi Ò and Remsima Ò[END_REF].

Parameters of the Galili antigen LEctPROFILE kit

To optimize the kit for comprehensive glycans analysis, a number of parameters were studied. First of all, since the lectin MOAβT was new in GLYcoDiag's catalogue of lectins, the optimal quantity of lectin/well was evaluated. Moreover, the type of detection, meaning the measurement of the interaction lectin-glycans either in fluorescence or absorbance, was studied. Once completed these two steps, the neoglycoprotein NeoGalili was chosen as a model probe for the characterization of the sensitivity of the kit and studies upon the IC50 determination. Except from the choice of the type of plate and the quantity of lectin/well, the same process was applied to the lectin GSLI-B4.

Furthermore, a set of glycoproteins and neoglycoproteins was tested on the kit to prove the specificity of the two lectins and for further studies on the IC50. To validate the assay for potential applications, therapeutic monoclonal antibodies were tested on the kit.

Determination of MOAβT quantity/well

In order to establish the optimum detection signal, the determination of the right quantity of lectin/well is a step of paramount importance. At the same time, we chose the most suitable plate for the by testing the lectin on adsorption plates and on covalent plates. The lectin was immobilized on covalent a adsorption plate at decreasing concentrations starting from 4 µg/well. To test the optimal interaction only based on the lectin quantity/well, not dependent from the neoglycoprotein concentration, the NeoGalili was added in each well at the same concentration of 1.15 µM. The detection of the signal was measured in absorbance units for the covalent plate, and in absorbance and fluorescence units for the adsorption plates (Fig. 3.11 A, B). As it can be seen in Fig 3 .11 A, in both the adsorption and covalent plate the Absorbance at 450 nm stays at a constant value until the quantity of lectin decreases to 0.5 µg. The main difference between the covalent and the adsorption plate is that the covalent plate seems to show a more intense signal (Abs 400 nm>1.5 versus Abs 400 nm= 0.6 ) and therefore probably a better sensitivity. In Fig 3 .11

B, we see that the intensity of the signal in fluorescence on adsorption plate it's strong enough to think of a good interaction between MOAβT and NeoGalili; there is a slight decrease of the intensity following the decrease of the quantity lectin/well that however are not directly proportional. This makes us think of an experimental error more than a real difference connected to the quantity of lectin/well which is confirmed by the repetition of the experiment that gave the same results (data not shown) and by the higher standard deviation reported at lower concentrations (e.g. at 0.5 µg, the standard deviation=1000 fluorescence units). Therefore, the quantity chosen in any condition is 1 µg/well because it is the right quantity to achieve a good signal while more would give the same result with a higher waste of lectin and less would compromise the quality of the signal. It needs to be pointed out that we chose to follow up with experiments on the covalent plate because of the strong signal that might have resulted in a very sensitive LectPROFILE kit.

To confirm that the right quantity was 1 µg/well, and show that the high intensity of the signal on the covalent plate was not dependent on the NeoGalili high concentration, we performed an experiment with MOAβT at decrescent concentrations, and NeoGalili at the lower concentration of 0.3 µM. We can see in Fig 3 .12 A, that again, no matter we decreased the NeoGalili concentration, the absorbance remains high and at a plateau up until around 0,5 µg/well, with a big drop at lower quantities of lectin. To see this event magnified, we decided to keep the same concentration of

NeoGalili but to apply it on decreasing concentrations of lectin going from 1 µg/well to 0.1 µg/well. The last proof of the fact that the optimal quantity of lectin is 1µg/well was obtained making two experimental curves in direct binding of NeoGalili interaction with MOAβT at 1 µg/well and 0.5 µg/well. This time, the lectin was imobilized at a fixed quantity/well and the neoglycoprotein was added in serial dilutions starting from 0.85 µM. The results of the direct binding of NeoGalili neoglycoprotein on different quantities of MOAβT/well are shown in Fig. 3.13: the data obtained fit saturation binding curves obtained using a non-linear regression model from which the BC50 and Bmax were calculated. The BC50 represents half of the maximum ligand concentration at which there is interaction with the lectin. This value can also be called Kd and reflects a surface concentration of the binding sites because the area in the microtiter plate is constant. The Bmax is the plateau value of adsorption and reflects the maximum number of the lectin binding sites [START_REF] Kirkeby | Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B4 isolectin to αgalactosyl carbohydrate antigens in the surface phase[END_REF].

These values are shown in the Table 3.2. The Kd is 10 times lower when the lectin is coated at 1 µg/well, meaning an higher sensitivity. For both curves the number of binding sites calculated are around 2, in accordance with the crystallographic studies that reveal three binding sites/monomer, each one with different ligand occupancy and with ELLA studies that calculates 4 binding sites using the full MOA including the dimerization domain

Direct binding in absorbance on covalent plate

To continue the development of the Galili antigen LEctPROFILE kit, it was chosen the covalent immobilization of MOAβT in the microtiter plate at 1 µg/well. More experiments in direct binding were performed to confirm the value for the BC50 and to calculate the detection limit of the kit, meaning the absorbance units below which the interaction between the lectin and the neoglycoprotein is considered non-specific. The data shown in Fig 3 .14 represent a direct binding curve made with MOAβT immobilized at 1µg/well followed by the application of the labelled NeoGalili at 1.15 µM and serially diluted. Again, the value calculate for the BC50 with the non-linear regression model it is confirmed to be in the nanomolar range even though in this case it is ten times higher (40 nM) than the one calculated for the previous experiment (2 nM) at the same quantity of lectin/well (1 µg/mL, Fig 3.13). The detection limit of the kit was settled at 0.1 absorbance units considering that the significative value of interaction of the wells lectin-PBS is above 0.1 absorbance units. We can see from the experimental curve that also the detection limit is very low and it can be included in the range going from 0.001-0.01 µM.

Inhibition in absorbance on covalent plate

The inhibition experiments are designed to understand the ability of a compound to inhibit the binding between MOAβT and the NeoGalili neoglycoprotein. The inhibition assay proves to be particularly advantageous because the compounds of interest do not need to be labelled, saving time and reagents, and they can directly be put in relation with the already characterised neoglycoprotein. The NeoGalili is well-characterized and it is used as a reference inhibitor so that, using the Galili antingen LEctPROFILE kit, a relative quantity of Galili antigen for an unknown compound can be estimated in relation to the NeoGalili. In Fig 3 .15 the labelled NeoGalili functions as a tracer applied to the lectin plate at a fixed concentration of 0.5 µg/mL. The compounds of choice, named inhibitors, compete with the tracer for the binding to the lectin. The data fit a non-linear regression model from which the respective IC50 were calculated. As model compounds, we used the glycoproteins porcine thyroglobulin and the disaccharide melibiose (Galα1-6Glc). They were used respectively at a starting concentration of 12 µM for the NeoGalili and thyroglobulin and at 50 mM for the melibiose and then serially diluted. It is evident the affinity of the porcine thyroglobulin for the lectin is higher than the NeoGalili affinity for the lectin, in fact the IC50 of Porcine Thyroglobulin is almost ten times less than the one of the NeoGalili neoglycoprotein. This can be due either to the avidity effect caused by a bigger quantity of NeoGalili on the thyroglobulin or a better arrangement of the sugar moieties on this glycoprotein. Hence, MOAβT has a strong selectivity for the Galα1-3Gal linkage and it is clear that melibiose has a poorly inhibitory effect on MOAβT unless it is used at very high concentration (100 mM). No inhibition is obtained with the galactose monosaccharide (not shown). The low affinity of MOAβT for the monosaccharide has a structural reason: the binding sites are found on the protein surface and are extended and shallow, and this is the main feature that make this lectin more suitable for the study on the Galili antigen than the lectin GSLI-B4 (Paragraph 3.6).

MOAβT studies on adsorption plate with fluorescence detection

At the same time, further studies were carried on using the adsorption plate in fluorescence. The choice to continue also in this direction was due to different reasons:

1. The other lectin considered for this kit, GSLI-B4, gives good results in fluorescence and it was interesting to see the comparison with MOAβT.

2. There are lectins that cross-react with the extravidin-peroxidase used in the absorbance assay, so in the case the use of the kit needs to be extended to those lectins, fluorescence would be the only possible choice.

3. The use of extravidin-peroxidase adds one step more to the experiment protocol, which does not take up much time when it comes only to one test but thinking it on an industrial scale is for sure time-consuming.

4. The use of the substrate for extravidin-peroxidase makes the kit development more expensive.

5. The covalent binding of the lectin to the plate cannot be easily controlled.

The lectin MOAβT was compared to the full MOA and to GSLI-B4 in order to have a clear overview on the potential use of these lectins for the kit.

Direct binding in fluorescence on absorption plate

We performed the GLYcoPROFILE in direct binding of MOAβT on adsorption plate in fluorescence in order to define the kits characteristics for this specific type of plate and this method of detection. As shown Fig. 3.16 A, knowing that the fluorescence detection is slightly less sensitive than the absorbance, the starting concentration of the NeoGalili neoglycoprotein was set at a double concentration of 2.3 µM if compared to the experiment in Fig. 3.14. Since in this case we do not see saturation in the binding of MOAβT to the NeoGalili neoglycoprotein, a simple linear regression analysis was used to calculate the BC50. [START_REF] Kirkeby | Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B4 isolectin to αgalactosyl carbohydrate antigens in the surface phase[END_REF]. The BC50 calculated for MOAβT interaction with NeoGalili neoglycoprotein is in the micromolar range (1µM) while the one calculated in absorbance on covalent plate is in the nanomolar range (40 nM). A different sensitivity was expected because the signal has a higher amplification in absorbance thanks to the use of the colorimetric reaction. The detection limit of the kit, set at 500 fluorescence units, for this reason is affected too, being around 0.3 µM, which would not be suited for lowconcentration samples. In order to see if this is due to the fact that MOAβT has less avidity for the neoglycoprotein because of the lack of the dimerization domain, experiments in direct binding with the full MOA were performed.

Comparison between MOAβT and rMOA

The GLYcoPROFILE in direct binding of the full MOA was performed in order to compare it with the one of MOAβT (Fig. 3. [START_REF] Angeloni | Glycoprofiling with micro-arrays of glycoconjugates and lectins[END_REF]). In the course of the thesis I have attempted to purify the natural MOA directly from the fairy ring mushrooms but with poor results, so we performed the experiments with the commercial lectin. In order for the experiments to be comparable, the same parameters as

Chapter 3 -The Galili Antigen

75

in the experiment in Fig. 3.16 were kept for the curve shown in Fig. 3.17. No matter the higher signal (max fluorescence intensity around 10000 fluorescence units), also in this case the BC50 is in the millimolar range (1.2 µM) and it is difficult to calculate the detection limit for 500 fluorescence units because below 0.5µM the signal drops. Overall, the lack of dimerization domain does not seem to affect the binding performance of MOAβT, as it is also highlighted in the paper at the end of this chapter (Notova et al.,).

GSLI-B4 direct binding in fluorescence on adsorption plate

The same experiment in direct binding that were performed for MOAβT and full were carried out for GSLI-B4. The use of the adsorption plate in fluorescence was chosen due to previous studies and for the advantages of this detection method over the absorbance one, already described in Paragraph 3.7.4. As shown in Fig. 3.18, GSLI-B4 presents a very good interaction with the NeoGalili reflected in the high fluorescence intensity measured (>10000 fluorescence units). Again, in this case we do not see saturation in the binding of GSLI-B4 to the NeoGalili neoglycoprotein, hence a simple linear regression was used to estimate the BC50. The BC50 is in the micromolar range (0.9µM) as for MOA and MOAβT but the detection limit is lower, around 0.1 µM, showing a better sensitivity compared to the MOAβT in fluorescence on adsorption plate, and to full MOA.

GSLI-B4 inhibition in fluorescence on adsorption plate

The inhibition experiments were designed to understand the ability of a compound to inhibit the binding between GSLI-B4 and the NeoGalili neoglycoprotein and it follows the same principle explained for MOAβT in Paragraph 3.7.3, this time in fluorescence on adsorption plate. In the experiment in Fig. 3.19 the labelled NeoGalili functions as a tracer applied to the lectin plate at the fixed concentration of 35 µg/mL. In this case, as model compounds for inhibition, we used the glycoproteins porcine and bovine thyroglobulin, the NeoGalili neoglycoprtein, the disaccharide Melibiose (Galα1-6Glc) and the monosaccharide α-galactose. They were used respectively at a starting concentration of 12 µM for the NeoGalili and thyroglobulin and at 100 mM for melibiose and galactose and then serially diluted. The IC50 could not be calculated for galactose and melibiose because of the signal saturation. It is observed saturation also with NeoGalili, so in order to have a more precise calculation of the IC50, the experiment need to be repeated. Even though we performed the inhibition assay on two different plates and with two different detection methods for GSLI-B4 and MOAβT, it is evident that the first lectin, as described literature [START_REF] Winter | The mushroom Marasmius oreades lectin is a blood group type B agglutinin that recognizes the Galα1,3Gal and Galα1,3Gal,β1,4GlcNAc porcine xenotransplantation epitopes with high affinity[END_REF] [69], shows a far less selective binding, interacting not only with the monosaccharide but also being inhibited to saturation by the melibiose that barely inhibits MOAβT. The explanation is once again in the binding site structure: for GSLI-B4 it is a deep pocket, less large and less prone to accommodate large glycans. µg/mL and with the non-labelled inhibitors applied in serial dilution, from a starting concentration of Porcine Thyroglobulin=12µM, NeoGalili=12 µM, Melibiose=100 mM, α-galactose= 100 mM. The IC50 for α-galactose and Melibiose could not be calculated because of the saturation of the signal.

Chapter 3 -

Monoclonal antibodies: a proof of concept

In the Paragraph 3.2.1 we have seen how the presence of the wrong sugar moiety, the Galili antigen on Cetuximab, could affect patient's safety. To validate the kit we have performed the GLYcoPROFILE® of Cetuximab and two other mAbs, Infliximab and Rituximab. Their characteristics are summarized in the Table 3.3. These mAbs were chosen because they are extensively used worldwide for diseases treatment, and their glycosylation is well documented [START_REF] Biolabs | Glycoproteomics Technical Guide[END_REF] [START_REF] Lee | mAbs Glycosylation profile and biological activity of Remicade® compared with Flixabi® and Remsima® Glycosylation profile and biological activity of Remicade Ò compared with Flixabi Ò and Remsima Ò[END_REF]. In Fig. 3.20 and 3.21, the panel of chosen lectins is based on the known glycan structures that can be find on the mAbs. A list of all lectins mentioned throughout this thesis and their specificity is enclosed in Annex 2 at the end of Chapter 4. The experiment was performed with native mAbs and digested with total neuraminidase (NeuT) and total neuraminidase+α-galactosidase, as negative control. The availability of the sample was limited, therefore the concentration of mAbs used for the experiment could not exceed the 40 μg/mL. This is reflected in the Infliximab GLYcoPROFILE in Fig. 3.20A

where the signal for all lectins is below the detection limit so it cannot be considered as significant.

The same happens for the Rituximab in Fig. 3.20B for which the most abundant glycan species are known to be G0F, G1F, G2F but the only glycan moiety that is detected is the core fucose interacting with the lectin PSA. For the Cetuximab in Fig. 3.21, despite the low quantity of sample available, we can see a GLYcoPROFILE consistent with published data obtained with LC/MS [START_REF] Biolabs | Glycoproteomics Technical Guide[END_REF]. One hypothesis for this difference between the three mAbs, that goes beyond the sample concentration, is that the mAbs Infliximab and Rituximab only contain glycans on the Fc domain while the Cetuximab presents the N-glycans also on the Fab region (Fig 3 .2). Those are more exposed so it is possible that they are more accessible to the interaction with lectins, whereas the Fc glycans are positioned inside a pocket formed by the two CH2 domain of the antibody (Fig. 4.7, Chapter 4) so they are difficult to be detected. In order to have a quantification of the glycan moieties, we have transformed the fluorescence intensity into neoglycoprotein equivalents (Table 3.4). It means that for each lectin included in this experiment a direct binding curve was performed with a corresponding neoglycoprotein known to have high affinity for the lectin. For each curve a linear regression equation was calculated (Annex I) and from each y values corresponding to the fluorescence intensity of the native antibody interaction with the lectin, a x value was calculated that corresponds to the concentration of the glycan moiety on the mAb, equivalent to the concentration of neoglycoprotein.

Name Commercial name

Molecular weight

Mechanism

Lectins

Conclusion

In this Chapter, we provided a complete characterization of the kit in relation with the lectins GSLI-B4 and MOAβT. The fact that GSL1B4 shows affinity not only for the Galili antigen but also for different α-galactosylated carbohydrates, monosaccharides and disaccharides in particular, could be seen as a drawback in using the lectin as a reliable marker for the xenotransplantation antigen. On another point of view, used jointly with MOAβT that has a strong selectivity for the Galili antigen,

Paper 2 -Extending Janus lectins architecture: characterization and application to protocells

This paper is currently on BioRxiv and it was submitted to Computational and StructuralBiotechnology Journal.

Chapter 4 -Sialic acid

Chapter 4 -Sialic acid 109

Abstract

Sialic acids are a family with a wide structural diversity, found mostly at the outer end of the glycan chains. They can be divided in two big subgroups, the N-acetyl neuraminic acid (Neu5Ac)

and N-glycolyl neuraminic acid (Neu5Gc). While the latter is not found in humans, the former is of paramount importance in humans. In fact it can be linked to the glycan chain with α2-3 or α2-6 linkage, and thanks to its location and variety of structures it is in charge of mediating many physiological and pathological functions like cell-cell communication, immune response, intra membrane transport, bacterial and viral infections.

Alteration of sialylation could lead to pathological states and it is well-known as a hallmark of cancer.

Moreover, the changing of the ratio α2-3/α2-6 linkage is involved in pathogens tropism, in antiinflammatory activity of antibodies, and in recombinant biotherapeutic glycoproteins are different depending on the system of production.

In this chapter, a brief review of sialic acid biochemistry and role in biological systems is provided with a major focus on the α2-3/α2-6 linkage and the significance of their recognition by specific lectins. Here, a LEctPROFILE kit able to discriminate between the two linkages is developed, providing a rapid semi-quantificative tool with industrial and research and development applications.

Biochemical and functional overview

The family of nine-carbon backbone acid-amino sugars can be called Sialic acid (Sia) or Neuraminic acid (Fig. 4.1). Both names are retained because they were given separately by two different scientists around the same period. Gunnar Blix isolated it from salivary mucins in 1936 and named his substance "sialic acid" after the Greek word for saliva (σíαλoν). Ernst Klenk isolated it from brain glycolipids in 1941 and named it "neuraminic acid" from neurons in the brain [4]. In nature, around 50 derivatives of sialic acid are known, due to the fact that its amino group is substituted either by an acetyl or glycoloyl residue, and the hydroxyl groups may be methylated or esterified with acetyl, lactyl, sulfate or phosphate group (Fig. 4.1). Non-substituted sialic acid does not occur in nature [START_REF] Schauer | Sialic acids and their role as biological masks[END_REF]. Sias are abundant on various glycoproteins and glycolipids and are mainly found at the outermost non-reducing end of glycan chains, linked to other sugars such as galactose with α2-3-or α2-6glycosidic bonds or through poly-sialic acid repeats in α2-8-linkage. They are found on all types of cells and on secreted proteins, in vertebrates and higher invertebrates and the sum of all sialoglycans on a cell is named "sialome". Their location, ubiquitous distribution, and peculiar structural features give sialic acids the role of mediators of many biological functions that are different depending on the cell type they are located on. In this thesis, more attention will be given to the functions connected to the objective of the study, which is the building of kits discriminating the α2-3 from the α2-6 linkage and Neu5Ac from Neu5Gc. They will be described more in detail later on in the corresponding paragraphs.

Because Sias are negatively charged, they play a role in the transport of positively charged compounds via Ca 2+ channels. They are involved in conformational changes of glycoproteins which are important for their organization on cell membranes, and they contribute to the resistance of serum glycoproteins to proteolytic degradation [START_REF]Serum sialylation changes in cancer[END_REF]. In fact, when initially sialylated molecules age, they progressively lose their sialic acid moieties. Asialoglycoproteins are recognized by galactose-specific lectins on hepatocytes, called asialoglycoprotein receptors (ASGPR) that bind them, resulting in their update and degradation (Fig. 4.2) [START_REF] Maverakis | Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: A critical review[END_REF]. This is the main reason why Sias and their receptors give many opportunities for therapeutic development. An example of drugs based on Sia metabolism are Relenza and Tamiflu, competitive inhibitors of influenza A and B sialidases, another one is Rivipansel, a Sia-mimetic drug that inhibits selectins and has shown promising results in clinical trials. Pathogens can also display the so-called "molecular mimicry", which means that they express Sias to evade the host immune response (Fig.

4.

3) [START_REF] Varki | Sialic acids in human health and disease[END_REF].

Sialylation often results altered in presence of diseases. This is again connected to the fact that sialylation is involved in the regulation of many intrinsic receptors. Selectins recognize malignant tumour cells causing their interaction with platelets, leukocytes and endothelium, facilitating the motility and metastasis. Tumour Sias also bind to factor H to limit the complement activation and modulate cell attachment to the matrix, promoting invasion and metastasis [4].

N-acetyl neuraminic acid

N-acetyl neuraminic acid (Neu5Ac or NANA) is the most common sialic acid derivative found in mammals and it occurs when the C 5 amino group is substituted with an acetyl group (Fig. Golgi and eventually transported to the cell surface or secreted [START_REF] Gu | Improvement of interferon-γ sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine[END_REF].

4.4).

Neu5Ac in forensic sciences

Human Transferrin is the iron transport serum glycoprotein and it consists of a polypeptide chain with two binding sites for iron and two N-linked glycan chains. It can exist in different glycoforms, related to the number of Sia antennae (n-antennary) that it contains (Fig. 4.6), which can go from zero to four. The major human transferrin glycoform in physiological conditions consists of four sialic acid residues (tetrasialo) and it is about 80% of the total. [START_REF] Porpiglia | A new sample treatment for asialo-Tf determination with capillary electrophoresis: an added value to the analysis of CDT[END_REF]. It is known that a regular consumption of 50-80 g of alcohol per day for at least one week is associated with an increase of and typically of asialo-transferrin (no glycosylation) and disialo-transferrin. This increase has been adopted in clinical and forensic medicine as a biomarker of alcohol abuse, with the name of carbohydrate deficient Transferrin (CDT). Its measurement is little affected by other conditions and it is apparently independent from liver comorbidities [START_REF] Stibler | Carbohydrate-deficient transferrin in serum: A new marker of potentially harmful alcohol consumption reviewed[END_REF]. ST6GAL2 expression is mainly restricted to the embryonic and adult brain but its functions are currently unknown [4].

3). The viral hemagglutinin of influenza infects cells binding to cell-surface Sia and fusing with

the host cell membrane. The virion is released inside the cells, where it exploits the host machine to replicate and assemble new virions that eventually are released from the host cell surface through the action of a viral neuraminidase. The hemagglutinin directs species and tissue tropism: human influenza virus for example binds preferentially to sialic acid residues in α2,6 linkage, whereas avian influenza recognizes the α2,3-linkage of sialic acids found in the intestinal epithelium of birds, where it resides [START_REF] Imberty | Microbial recognition of human cell surface glycoconjugates[END_REF]. The possible jump from birds to humans has drawn interest for the dangers that it could bring, as it could cause an epidemic outburst. This could happen if the hemagglutinin component of the virus underwent specific mutations, switching to preferentially binding α2-6-linked sialic acids, which are enriched on the airway epithelium of humans. It occurred in 1918 when the influenza pandemic, very well-known as Spanish flu, killed at least 20 million people. What is believed is that there was no direct jump from birds to humans, but most likely pigs acted as a 'mixing vessel' as they have both α2-3-and α2-6-linked sialic acids on the epithelium.

Glycans bearing α2-3 sialic acid residues have also been implicated in bacterial pathogenesis.

Helicobacter pylorii is a human and primate-specific pathogen found in the gastric mucus layer or attached to the gastric epithelium. Infection by this bacterium, which affects about half the world population, results in chronic active gastritis and is a risk factor for the development of peptic ulcer disease, gastric adenocarcinoma, and gastric lymphoma. The wild-type bacteria binds to Nacetyllactosamine-based gangliosides with terminal α2-3-linked Neu5Ac, while gangliosides with terminal Neu5Gc, Neu5Acα2-6, or Neu5Acα2-8, Neu5Acα2-3 are not recognized [START_REF] Roche | Helicobacter pylori and Complex Gangliosides[END_REF].

The study of α2-6-and α2-3-sialylation is a promising biomarker for cancer diagnosis since increasing levels of α2-3 linkage point towards tumour malignancy. Prostate cancer (PCa) is one example. The current golden standard method for PCa screening is the blood quantification of the Prostate Specific Antigen (PSA), a glycoprotein produced by normal, as well as malignant, cells of the prostate gland. The higher a man's PSA level, the more likely it is that he has PCa (PSA > 10 ng/mL). However, this kind of test is not accurate, in fact men with levels of PSA that are in a range considered normal or intermediate were found to have PCa. Moreover, high levels of PSA could also be connected to benign prostate hypertrophy due to excessive exercise or linked to urinary tract infection or assumption of some medications [START_REF] Bertok | Analysis of serum glycome by lectin microarrays for prostate cancer patients -a search for aberrant glycoforms[END_REF]. Only about 25% of people who undergo prostate biopsy due to an elevated PSA level are diagnosed with PCa. Therefore, a more accurate test is needed. The levels of α2-3-linked sialic acids on PSA could improve PCa diagnosis since it illustrated great potential in discriminating malignant from benign conditions. During the years there have been different studies in this direction: electrochemical label-free glycoprofiling showed that Maackia amurensis agglutinin recognizing α2-3-terminal sialic acids binding to serum PSA was significantly higher for PCa samples than for healthy controls [START_REF] Pihikova | Sweet characterisation of prostate specific antigen using electrochemical lectinbased immunosensor assay and MALDI TOF/TOF analysis: Focus on sialic acid[END_REF]. In another study, the evaluation of α2-3sialylated PSA in serum with a lectin immunoaffinity column revealed that α2-3-sialic acid on PSA exhibited high performance in discriminating between high-risk prostate cancer patients and the benign prostate hyperplasia individuals [START_REF] Llop | Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes[END_REF].

In biotherapeutics

It has been estimated that in 2018 Chinese hamster ovary cells (CHO) had been used for the production of 84% of FDA-approved biotherapeutics [START_REF] Coulet | Metabolic Profiling of CHO Cells during the Production of Biotherapeutics[END_REF]. They are, in fact, advantageous for many reasons. They are less susceptible to infections by human viruses, making them safer for commercial purposes. They are easy to manipulate since they duplicate quickly both in adhesion and in suspension, and for this reason are suitable for large-scale industrial suspension culture.

Moreover, the biotherapeutics produced by CHO have a human-like glycosylation, thus making the products more compatible with the human hosts. Despite all these upsides, CHO cells are not able to produce some types of human glycosylation such as α2,6-sialylation, as they lack the ST6 sialyltransferase gene [START_REF] Lalonde | Therapeutic glycoprotein production in mammalian cells[END_REF]. As described in paragraph 4.1, sialylation helps to keep glycoproteins in the bloodstream, preventing the recognition by receptors on liver hepatocytes. Increased protein sialylation may decrease the frequency of injection or the amount of therapeutic protein used in a single dose. Moreover, the fact that both linkages are present in humans' glycoproteins has an impact on their activity inside the body (Paragraph 4.2.2). Hence, many strategies have been put in place to increase sialylation and recreate a glycosylation profile beneficial for the host that could also generate savings for the industry. One of these approaches is the supplementation of CHO culture media with different precursors of the sialic acid pathway to improve the sialylation of interferon-γ [START_REF] Gu | Improvement of interferon-γ sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine[END_REF]. Another strategy is the transient expression of sialylation enzymes into CHO cells, as in the case of ST6Gal1 to increase the sialylation of Trastuzumab F243A mutant antibody [START_REF] Raymond | Production of α2,6sialylated IgG1 in CHO cells[END_REF].

Finally, other expression systems have been exploited, among which human cell lines HEK293 cells and the HT-1080, respectively from human embryo kidney [START_REF] Graham | Characteristics of a human cell line transformed by DNA from human adenovirus type 5[END_REF] and fibrosarcoma origin [START_REF] Rasheed | Characterization of a newly derived human sarcoma cell line (HT-1080)[END_REF]. Only few biotherapeutics produced in these cell lines have been approved so far. Another one, the Nicotiana benthamiana plant expression system was genetically modified on a combination of stably transformed plants with transient expression modules. They are intended to carry the human-type sialylation pathway and to generate mono-and disialylated structures with α2-3 and α2-6 linkage [START_REF] Kallolimath | Engineering of complex protein sialylation in plants[END_REF].

For all the cases mentioned above, the α2,3/α2,6 LEctPROFILE kit could be a tool to detect the right clones in the early phases of production of these engineered strains, accelerating drug development and production, reducing manufacturing costs and improving drug efficiency. CH2 domains of the antibody where they interact with internal amino acid residues through hydrogen and CH-p bonds. As a consequence of this embedment, the Fc N-glycans are mostly limited to di-antennary complex types with partial galactosylation and low sialylation. Fc-sialylation in circulating human IgGs is generally believed to be mainly of α2-6 type [START_REF] Gu | Improvement of interferon-γ sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine[END_REF]. The most common glycan on circulating human IgGs is a core fucosylated complex structure with one galactose (G1F), followed by fucosylated complex glycans with 0 and 2 galactoses (G0F and G2F). There can be glycans also in the Fab regions but they are not conserved (Fig. 4.9).

Fig. 4. 9 Different glycan structures that can be found on IgG. G= galactose, F= fucose, S= sialic acid [START_REF] Raymond | Production of α2,6sialylated IgG1 in CHO cells[END_REF] .

Studies over the past 30 years have provided important information regarding how glycosylation affects Fc-mediated effector functions. Antibody glycosylation was found to be changing in numerous diseases; one that was particularly studied is the autoimmune rheumatoid arthritis, in which are present immune complexes consisting only of immunoglobulin (most commonly IgG), implying that the IgG is both the 'antibody' and the 'antigen'. Autoantigenic reactivity was localised to the constantregion domains of lgG where there is a decrease of the level of outer-arm galactosylation of the complex N-linked glycans. As a consequence, there is a decrease of Neu5Acα(2-6)Gal segments and an increase in IgG molecules bearing oligosaccharides terminating in N-acetylglucosamine.

Immune complexes formed through G0 IgG can bind and activate mannan-binding lectin and a pseudo-complement cascade [START_REF] Parekh | Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG[END_REF]. Amongst patients with the most severe rheumatoid arthritis, articular amelioration occurred during pregnancy, linked to a six-fold reduction in the actual concentration of IgG lacking galactose, with consequent remission of symptoms during gestation [111].

Studies on intravenous immunoglobulin therapy (IVIG) for rheumatoid arthritis show that the antiinflammatory activity of purified IgG from pooled human plasma can be attributed to the presence of terminal sialic acid. In fact, removal of the terminal sialic acid of IVIG, in animal models, abrogates the anti-inflammatory activity. On the contrary, enrichment of the sialylated fraction of IVIG enhances this activity. This study was conducted only on the Fc portion, and it was the first time when it was demonstrated the independence of the Fc from the antigen-binding domain. Through sequential mass spectrometric based glycan analysis and treatment with either α2-3 or α2-3/ α 2-6 sialidase, it was determined that the linkage type present on IVIG is the 2-6 linkage to the penultimate galactose. No α2-3 linkages were detected on intact IVIG and no attenuation of the antiinflammatory activity was observed following α2-3 SA treatment [START_REF] Anthony | A RECOMBINANT IgG Fc THAT RECAPITULATES THE ANTI-INFLAMMATORY ACTIVITY OF IVIG[END_REF]. In another paper by the same author [113] it is theorized that the addition of sialic acid moieties has structural implications and consequently the effector function of the IgG is affected. In fact, the glycans at N-297 keep the Fc

region in an open conformation to promote the binding to the FcγR. The addition of sialic acid would reduce this binding and drive the IgG to bind to the lectin receptors SIGN-R1 or DC-SIGN thereby attenuating autoantibody-initiated inflammation. However, this mechanism of sialylated IgG/Fc to bind DC-SIGN was later discussed in other studied and not confirmed [START_REF] Cobb | The history of IgG glycosylation and where we are now[END_REF] and it remains under debate. Whether α2-6-sialylated IgG/Fc actively inhibits inflammation remains an interesting topic and still there is much to investigate. The effect probably depends upon the context of the disease and metabolic factors.

It appears to be a certain variability in Fc glycans across individuals. At the Croatian company Genos, under the leadership of Gordon Luc, studies were carried out through the hydrophilic interaction liquid chromatography (HILIC) method, performed on a large scale of patients affected by different pathologies. The biggest differences in glycosylation were found at different stages of life and among individuals coming from different parts of the world; the HILIC method, does not takes into account the linkage of sialic acid though [START_REF] Cobb | The history of IgG glycosylation and where we are now[END_REF]. Therefore, insight in the sialylation pattern through the α2-3/α2-6 LEctPROFILE kit may provide valuable clues on how sialylation affects IgGs effector functions in different pathologies and individuals.

N-glycolyl neuraminic acid

It is a glycoform of Sia occurring when there is the addition of a single oxygen atom to the Nacetyl group of Neu5Ac at the C5 amino group, resulting in the glycolyl group (Fig. 4.10 B).

It is found in most mammals, with exceptions for taxa that have lost the capacity to synthesize to add Neu5Gc to newly synthesised glycoconjugates. These two nucleotides seem to be used interchangeably by the Golgi CMP-Sia transporter and by the mammalian sialyltransferases, which transfers Sia residues to cell surfaces. Moreover, Neu5Ac or Neu5Gc released from glycoconjugates during lysosomal degradation processes can also be exported back into the cytosolic compartment by a specific transporter. Again, there appears to be no major difference in their conversion by CMP-Sia synthase [START_REF] Bardor | Mechanism of uptake and incorporation of the nonhuman sialic acid N-glycolylneuraminic acid into human cells[END_REF].

Although found in mammals, humans are incapable of synthesising Neu5Gc due to an inactivating single-exon deletion mutation in the CMAH gene [START_REF] Banda | Metabolism of vertebrate amino sugars with N-glycolyl groups: Mechanisms underlying gastrointestinal incorporation of the non-human sialic acid xeno-autoantigen N-glycolylneuraminic acid[END_REF].The reason why this mutation occurred in the first place it's still a matter of discussion. Many pathogens use Sias to bind and infect, and some specifically target Neu5Gc so one explanation is the loss of Neu5Gc to survive a lethal infectious pathogen. Another possibility that does not exclude the first one, it is called the "cryptic female choice" which is the selective fertility of Neu5Gc-deficient females with Neu5Gc-deficient males, leading to positive selection of this genotype [START_REF] Dhar | From 'Serum Sickness' to 'Xenosialitis': Past, Present, and Future Significance of the Non-human Sialic Acid Neu5Gc[END_REF]. in salivary mucin, but this has resulted to be poor and without any connection to the findings in endothelial, epithelia, cancers, or foetuses [START_REF] Tangvoranuntakul | Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid[END_REF]. Afterwards, Banda et al. proposed a mechanism to elucidate this accumulation using a Neu5Gc-deficient mouse with a human-like defect in Cmah. They followed the ingested free Neu5Gc by fluorescent tagging of free sialic acids with 1,2-diamino-4,5methylenedioxybenzene dihydrochloride (DMB) for HPLC, and the Neu5Gc-glycoproteins by Neu5Gc IgY. The former showed rapid absorption into the circulation and urinary excretion, the latter was absorbed into the small intestinal wall, appeared in circulation at a steady-state level for several hours, and was incorporated into multiple peripheral tissue glycoproteins and glycolipids. This proved that Neu5Gc can be metabolically incorporated from food [START_REF] Banda | Metabolism of vertebrate amino sugars with N-glycolyl groups: Mechanisms underlying gastrointestinal incorporation of the non-human sialic acid xeno-autoantigen N-glycolylneuraminic acid[END_REF]. This is the first example where a non-human dietary molecule becomes incorporated onto human cell surfaces, also being epidemiologically linked to the increased cancer risk [START_REF] Hedlund | Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression[END_REF]. Hence, Neu5Gc presence has consequences on health and it can be specifically linked with development of anti-Neu5Gc antibodies and diseases.

In cancer

It is described that certain types of cancer, including colon carcinomas, retinoblastomas, breast cancers, and melanomas, show an increase of Neu5Gc and Neu5Gc antibodies [START_REF] Hedlund | Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression[END_REF].

The first to describe the presence of anti-Neu5Gc antibodies in humans were Hanganutziu and Deicher, from which now they are called HD-antibodies. They observed a "serum sickness" derived from the development of these Abs after administration of therapeutic animal antiserum to humans.

Later, it was demonstrated that they are directed against the Neu5Gc antigen contained in gangliosides. They were later detected in some cancer-patients' human serum, without the patients having ever received animal serum, and afterwards it had been shown that also healthy individuals can have high levels of anti-Neu5Gc antibodies [START_REF] Hedlund | Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression[END_REF]. The frequency of cancer patients possessing HD-Abs is 80% of hepatocellular carcinomas and 25-28% in colon and gastric cancer cases. In general, HD-antibodies from patients with melanoma and hepatic cancers are identified as IgG and

IgM [START_REF] Malykh | N-Glycolylneuraminic acid in human tumours[END_REF]. The appearance of HD-Abs in tumours has been connected to the chronic inflammatory state of the cancer tissue. Inflammation is the biological response of body tissues after an injury and consists in the recruitment of tissue mast cells and the migration of leukocytes from the blood vessels to the interested site. The idea would be that Neu5Gc is a potential link between diet and human cancer. Red meat and milk-derived products triggers the accumulation of Neu5Gc with consequential "xenosialitis" an inflammation due to reaction against a xeno-sialic acid that is now part of "self" and HD-Abs production. The inflammation causes the recruitment of innate immunity growth factors, cytokines, and angiogenic agents and the Neu5Gc the recruitment of antibodies to the inflammation site. This causes a chronic situation in which the immune response is not sufficient to shut down the inflammation, but instead stimulates tumour growth and survival [START_REF] Prehn | The Autoimmune Nature of Cancer[END_REF]. The inflammation status favours angiogenesis and cell extravasation, originating the metastatic tumours.

This hypothesis matches with the decreased cancer risk associated with veganism and the rarity of carcinomas in the chimpanzees who normally express Neu5Gc and thus do not make antibodies against it. In any case, an increase in the Neu5Gc titre correlated well with the chance to develop cancer and analysis of tissues through the NeuAc/NeuGc LEctPROFILE kit could be of diagnostic significance to give an early indication of the inflammatory status.

Xenosialitis is connected also to the formation of atherosclerotic plaques. Studies have shown that

Neu5Gc can be detected in the endothelium and the sub-endotelium of the atherosclerotic plaques.

In vitro experiments with human endothelial cells fed with Neu5Gc and subsequently exposed to serum containing anti-Neu5Gc glycan antibodies showed the presence of IgG and complement deposition which increase endothelial activation, cytokine production, all events associated with early atherogenesis. These effects were inhibited by Neu5Gc-alpha-methyl glycoside, a specific competitor to anti-Neu5Gc antibodies [START_REF] Dhar | From 'Serum Sickness' to 'Xenosialitis': Past, Present, and Future Significance of the Non-human Sialic Acid Neu5Gc[END_REF].

In biotherapeutics

The use of systems for the expression of high levels of protein, such as bacterial, yeast and insect cells for production of biotherapeutic glycoproteins, are limited to small proteins without extensive post-translational modification. Besides CHO, another widely-used expression system is mouse myeloma cells. The drawback of these cells is that they have very high levels of Neu5Gc and patients receiving products coming from them might enhance their own intrinsic anti-Neu5Gc antibody production.

A more serious possibility is the formation of antigen-antibody complexes, which could secondarily trigger an immune reaction against the biotherapeutic agent. Some limitations of mammalian cell culture systems have been overcome by the expression of recombinant proteins in transgenic animals such as human recombinant antithrombin (rhAT) into the milk of transgenic goats. The study of Zhou et al. compared the carbohydrate structures in rhAT from several transgenic goat lines and clones. rhAT carbohydrates differ from human plasma AT with increasing levels of Neu5Ac and this carbohydrate heterogeneity was found also in different transgenic goat lines [START_REF] Zhou | Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats[END_REF].

For this reason it is of paramount importance to profile the glycosylation in the early stages of production, in order to select the right clones from the beginning of the process and maintain a glycosylation homogeneity. 4Glc), but it is not inhibited by either 2-6-sialyllactose or free NeuAc [START_REF] Imberty | An unusual carbohydrate binding site revealed by the structures of two Maackia amurensis lectins complexed with sialic acid-containing oligosaccharides[END_REF]. Studies show that the 50% of inhibition is reached at a concentration of 0.5 mM of the trisaccharide ɑ2-3-sialyllactose and lactose was found to inhibit at 5 mM [START_REF] Knibbs | Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis. Comparison with other sialic acid-specific lectins[END_REF]. 4.5.5 SSL Scylla serrata lectin takes its name from the homonymous marine crab, and it's a C-type lectin specific for Neu5Gc. Crustaceans cannot synthesise sialic acids, but most probably they make sialic acid binding lectins as a defence against bacteria sialoconjugates. The molecular weight of pure SSL is about 55 kDa and under reducing conditions it resolves into two subunits of 30 kDa and 25 kDa. NeuGc inhibits hemagglutination activity of the purified lectin at a concentration of 0.6 mM

[127]. To date, there are no crystal structures of this protein and the number of binding sites is unknown.

The purification of this lectin was carried out starting from the natural source, the crab Scylla serrata.

The yield of the purification was too low to perform any experiment and it was not also possible to detect a clear band on the SDS-PAGE, even though signs of lectin activity were detected in the extract.

Results and discussion

MAAI Purification

At GLYcoDiag we established purification protocol for the industrial production of MAAI based on Cummings [START_REF] Wang | The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked alpha-2,3 to penultimate galactose residues[END_REF], starting from the seeds of the leguminous tree Maackia amurensis. The purification procedure is property of GLYcoDiag and for this reason it will not be described in detail in this thesis. The protocol was successful in the purification of around 10 mg/lectin per 100 g of seeds. The GLYcoPROFILE in direct binding in The kit was optimized to discriminate the α2-3/α2-6 glycosidic linkages. In this specific case, the choice of the type of plate and the method of detection were immediately directed to the fluorescence detection on adsorption plate, based on previous studies conducted at GLYcoDiag before my arrival (data not shown). Based on previous studies performed at GLYcoDiag (data not shown), the quantity of lectin/well chosen for SNA is 2.5 µg/well and for MAAI is 1 µg/well.

Direct binding

We performed the GLYcoPROFILE in direct experiments to test the specificity of the lectins MAAI and SNA and their selectivity towards the corresponding Sia linkages and to test the sensitivity of the kit, determining the limit of detection. The determination of the detection limit is useful to establish the minimum concentration of unknown sample necessary to have a robust interaction with the lectin on the plate. The neoglycoproteins Neo3'SL and Neo6'SL were chosen as a model probes.

In Fig. 4.17, it is represented the direct binding curve of the lectin SNA interaction with Neo6'SL at starting concentration of 0.6 µM and then serially diluted on the plate. The data obtained fit a nonlinear regression model, from which the BC50 was calculated. The BC50, as for the Galili antigen LEctPROFILE kit, represents half of the maximum ligand concentration at which there is interaction with the lectin and for SNA interaction with Neo3'SL resulted to be 0.2 µM. Then, we set the detection limit at 500 fluorescence units, below which, the signal can be considered as background noise. The concentration of Neo6'SL giving a signal below 500 fluorescence units in the chart in Fig. 4.17 is 0.01 µM. 4. 17 GLYcoPROFILE in direct binding representing the interaction between the lectin SNA and the labelled neoglycoprotein Neo6'SL at a starting concentration of 0.6 µM.The curve is obtained from four independent replicates. All measurements were performed in duplicates.

The direct binding curve was performed as well for MAAI interacting with Neo3'SL at a starting concentration of 0.6 µM and then serially diluted on the plate. In Fig 4 .18 is shown the chart obtained: the data fit a non-linear regression model and the calculated BC50 is also in this case 0.2 µM. The detection limit is below 0.01 µM, concentration of Neo3'SL. What emerges from this direct binding experiment is that the sensitivity of the kit is very good and it could allow to detect Sia linkages at concentrations up to 0.01 µM.

Inhibition

The inhibition experiments are designed to understand the ability of a compound to compete for the binding between the lectin and the specific neoglycoprotein. The labelled neoglycoproteins are the "tracer" applied to the lectin plate at a fixed concentration. The inhibitors are the non-labelled compounds applied to the plate in serial dilutions. For each tested inhibitor it was calculated the IC50 (half-maximal inhibitory concentration), which is the half concentration of inhibitor necessary to displace the interaction between the lectin and the tracer. After the determination of the IC50, the Relative Inhibitory Potency (RIP) was calculated (Table 4.1) for each inhibitor. This was done with the purpose of standardizing all values and to have reference values to which a target unknown sample can be compared to. For the RIP, the obtained IC50 values were normalized to the IC50 value of nonconjugated trisaccharide 6'SL for SNA and 3'SL for MAAI. Thus, stronger binding to SNA or MAAI results in a lower IC50 value and a higher RIP. RIP was calculated according to the formula:

𝑅𝐼𝑃 = 𝐼𝐶50(𝑠𝑖𝑚𝑝𝑙𝑒 𝑡𝑟𝑖𝑠𝑎𝑐𝑐ℎ𝑎𝑟𝑖𝑑𝑒) 𝐼𝐶50 (𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟)
The GLYcoPROFILE in inhibition is carried out using as tracer the labelled neoglycoproteins Neo6'SL and Neo3'SL, respectively, for SNA and MAAI. The choice of the concentration was determined according to the direct binding curve: the concentration that gives the 50% of interaction between the lectin and the neoglycoprotein (BC50) is chosen as the concentration of tracer for the inhibition experiment and for both lectins it was fixed at 20 µg/mL (Fig. 4.17 The values of IC50 and RIP obtained for the two lectins are reported in Table 4.1. The IC50 values obtained for the two lectins with the respective trisaccharide are in accordance with previous studies [124] [123]. It is interesting to notice that the RIP for fetuin interacting with SNA is higher than for fetuin interacting with MAAI, that could mean this glycoprotein presents more α2-6 linkages than α2-3 which would be in accordance with literature [START_REF] Guttman | Site-Specific Mapping of Sialic Acid Linkage Isomers by Ion Mobility Spectrometry[END_REF].

SNA MAAI

Compound

Validation of the kit: a comparison study

Taking into account the applications reviewed at the beginning of this chapter, it is of great interest the quantification of α2-3-and α2-6sialyllactose in a rapid and simple way. The goal of this study is to do it through two relevant and complementary methods, the MALDI-TOF-MS and the GLYcoPROFILE®. The purpose of the collaboration with Juvissan Aguedo of the Slovak Academy of Sciences was to calculate the ratio of α2-6-and α2-3glycosidic linkages contained in reference commercial glycoproteins through the two techniques and compare the results obtained.

GLYcoPROFILE® and MALDI-TOF-MS of reference glycoproteins

We used four reference native glycoproteins listed in the Table 4.2. The choice of these particular glycoproteins is due to their interesting applications, for example transferrin, which has gained attention in the forensic field (Paragraph 4.2.1), and to the fact that their glycosylation was previously described. However, in literature is missing the exact quantification of the Sia linkages on these glycoproteins, a part from Fetuin for which the percentage of α2-3-and α2-6sialyllactose has been described [START_REF] Narhi | The effect of carbohydrate on the structure and stability of erythropoietin[END_REF], hence, for this reason, it can be considered as a control. The GLYcoPROFILE experiments were carried out in direct binding. As negative control, digestion with α2-3 neuraminidase (Neuα2-3) and total neuraminidase (NeuT) was carried out in order to confirm that the results were in good agreement with the expected specificities of the lectins.

Name

Neuraminidase is the common name for the Acetyl-neuraminyl hydrolase also known as Sialidase.

α2-3 Neuraminidase (Neuα2-3) is a highly specific exoglycosidase that catalyses the hydrolysis of α2-3 linked N-acetylneuraminic acid residues from glycoproteins and oligosaccharides. The α2-3,6,8

Neuraminidase (NeuT) catalyses the hydrolysis of α2-3, α2-6, and α2-8 linked N-acetylneuraminic acid residues from glycoproteins and oligosaccharides. What we expected to see is a drop of fluorescence intensity for the lectins SNA and MAAI interaction with samples treated with NeuT and a drop only in the fluorescence intensity interaction of MAAI with the samples treated with Neuα2-3.

The ratio α2-3/ α2-6 was calculated based on the equivalents of neoglycoprotein determined for each glycoprotein, for the highest concentration tested. To do so, we performed a direct binding curve for SNA and MAAI with the corresponding neoglycoproteins Neo6'SL and Neo3'SL. For each curve a linear regression equation was calculated (Fig.

Fetuin

The use of bovine fetuin is widespread as a model glycoprotein for the study of the biosynthesis, structure, functions and lately also for the carbohydrate characterization. N-linked and O-linked oligosaccharide on Bovine Fetuin have been extensively described, and to date it is known that the N-linked account for the 80% of Fetuin oligosaccharide while the O-linked for the 20% [START_REF] Green | The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy[END_REF].

Sialic acid linkage isomers contained into fetuin were recently studied by Guttman and Lee by Ion Mobility Spectrometry in addition to conventional glycoproteomic platforms with the main purpose of differentiating sialic acid linkages on both N-and O-linked glycopeptides [START_REF] Guttman | Site-Specific Mapping of Sialic Acid Linkage Isomers by Ion Mobility Spectrometry[END_REF]. They state that with their technique, the overall ratios of sialic acid linkage isomers observed on Fetuin is 38.4% for α2-3 and 59.4% for α2-6, with a 2.2% attributed to sialylation at the GlcNAc. These results are consistent with previous quantitative measurements of α2-6:α2-3 sialylation ratio of Fetuin that range from 62:38 up to 49:51. The oscillation of the percentage could be possibly due to differences in the methods used for glycan isolation, the sample preparations and the data collection.

Observing the GLYcoPROFILE in Fig. 4.22, the experiment gives coherent and reliable results. In fact, as expected, a decrease in the signal for the interaction SNA/NeuT+Fet and MAAI/NeuT+Fet.

The digestion of the glycoprotein with α2-3 neuraminidase worked too: being MAAI the lectin that recognize the α2-3 linkage, there is a decrease only in the interaction MAAI/Neuα2-3+Fetuin but not for SNA/Neuα2-3+Fetuin. The ratio α2-6:α2-3 sialylation, obtained as equivalents of neoglycoprotein, is 56:44: this observation is in line with the previously cited studies [START_REF] Guttman | Site-Specific Mapping of Sialic Acid Linkage Isomers by Ion Mobility Spectrometry[END_REF]. To confirm the level of a2-3-or a2-6-terminally sialylated structures, the background level of interaction obtained after neuT digestion was substracted from the results obtained for the interaction of each lectin with the native glycoprotein at the maximum concentration, following the method of Damiani et a [START_REF] Damiani | Stable expression of a human-like sialylated recombinant thyrotropin in a Chinese hamster ovary cell line expressing α2,6-sialyltransferase[END_REF].

The calculated ratio α2-6:α2-3, which is 54:46, is in agreement with literature and the calculation based on the neoglycoprotein equivalents. However, the results reported for fetuin by MALDI-TOF-MS are not in agreement with the GLYcoPROFILE neither with literature. According to MS data, the most abundant sialylated glycan in Fetuin is a non-core fucosylated bi-antennary di-sialylated glycan with full α2-6-sialylation (Fig.

A)

.The difference of ratio α2-6:α2-3 (Table 4.4) is too drastic (97:3 versus 56:44) to attribute the diverging results to the techniques used so an hypothesis might be that the lack of α2-3 sialylation is due to the fact that only the fetuin N-glycans were analysed in this MALDI-TOF-MS experiments, in fact, the sample preparation includes only the digestion with PNGase F.

Transferrin

The studies that focus on human transferrin sialylation have as their main object the discrimination of transferrin glycoforms, classified by the number of sialic acids branches found on the two N-glycosylation sites of this glycoprotein. We wanted to have a more complete view on this matter, searching for the type of Sia linkages found on human transferrin and the ratio α2-6:α2-3.

The GLYcoPROFILE in Fig. 4.24 implies that Transferrin only contains the ɑ2-6 linkage, given that the only interaction that we see is with the lectin SNA. This is not in accordance with the information found on Glyconnect by Expasy, neither with the MALDI-TOF-MS results and could result from a limited accessibility of the ɑ2-3 linkage by the lectin. To know if this hypothesis of the accessibility was correct, we performed a GLYcoPROFILE of transferrin both in native and in denaturing conditions. As shown in Fig. 4.25, the fluorescence units value of MAAI incubated with denatured transferrin are 10 folds higher than values obtained after lectin incubation with native transferrin. For both lectins we can see an increase of fluorescence intensity when transferrin is in denaturing conditions, but, the difference is that for SNA the increment is proportional to the fluorescence values of the native transferrin at the three different concentrations, while for MAAI the native transferrin gives a signal close to zero, proving that there is a problem of accessibility. The ratio α2-6:α2-3 calculated on the neoglycoprotein equivalents goes from 99:1 for native transferrin to 56:44 for the denatured (Table 4.6). According to the MALDI-TOF MS, the most abundant species is a non-core fucosylated tri-antennary tri-sialylated glycan with two α2-6-linkages and one α2-3-linkage (Fig 4.26). The ratio α2-6:α2-3 (Table 4.7) is comparable to the one obtained in GLYcoPROFILE, being 65:35, in this case the small difference can be attributed to the assay. According to the MALDI-TOF-MS, Bovine Thyroglobulin presents a fucosylated bi-antennary disialylated glycan with fully α2-6-sialylation and Porcine Thyroglobulin presents a fucosylated biantennary mono-sialylated glycan with one α2-6-sialylation (Fig. 4.28). Only α2-6 glycosidic linkage is reported (Table 4.9) as opposite as the GLYcoPROFILE in in which also α2-3 is detected.

SNA

Concentratio

Equivalent of neoglycoprotein

Ratio α2-

Glycoprotein

Linkage

Relative abundance % α2-6 sialyllactose 100 α2-3 sialyllactose 0 α2-6 + α2-3 linkage 0 Table 4. 9 Summary of glycosidic linkages relative abundances of both porcine and bovine thyroglobulin calculated according to MALDI-TOF/MS with linkages derivatization.

The two methods agree on the fact that the α2-6 linkage is the one always in major quantity; sialylated glycans containing α2-6-linkages are more abundant in fetuin, porcine and bovine thyroglobulin whereas glycans with both linkages α2-6and α2-3were found in lower levels, except in transferrin, which presents higher abundance of glycans containing α2-6and α2-3sialylation.

However, the two methods are not completely in accordance because with the GLYcoPROFILE we saw the presence of α2-3 linkages also in fetuin and in both thyroglobulins. In the case of transferrin, we see that the two methods give a very similar ratio α2-6:α2-3, but, in the case of the GLYcoPROFILE we needed to perform the denaturation of transferrin to gain accessibility to the α2-3 linkages: Further analyses need to be performed to clarify why there is such a big difference in the detection of the α2-3 linkages. One idea could be to introduce in the study another glycoprotein well characterized and of a lower molecular weight than the thyroglobulins, which can be for example the alpha(1)-Acid glycoprotein (AGP), with a molecular weight of 41-43 kDa and heavily glycosylated (45%), which is also already described [START_REF] Guttman | Site-Specific Mapping of Sialic Acid Linkage Isomers by Ion Mobility Spectrometry[END_REF].

This work wants to show the first step towards a complementary and high throughput method for discrimination of α2-6and α2-3-sialylation to be used in biomarker discovery and in quality control of biotherapeutics proteins. The advantage of MALDI-MS method for derivatized glycans is the possibility to get additional structural information regarding the exact position of sialic acid linkage and when both linkages α2-6and α2-3are present on the glycan. The advantage of the GLYcoPROFILE is to have a very fast analysis because the results are obtained in a couple of hours and moreover the glycans don't need to be removed from the glycoproteins so we can obtain additional information on their accessibility. Generally, for cells GLYcoPROFILE, the results are not expressed in terms of fluorescence intensity but rather as percentage of bound cells, which is more comparable between experiments, while the fluorescence can move according to the quality and the concentration of cell during the labelling.

The percentage is obtained thanks to a reference linear regression equation calculated from the standard curve in Fig 4 .31A.

For the experiment in Fig. 4.31B, the panel of lectins was chosen based on previous results (data not shown) and on literature (for a detailed description of all lectins used in this thesis refer to AnnexI I). According to literature, both CHO grown in monolayer (adherent) and in suspension have a similar range of glycan structures. High mannose structures, complex N-glycans (mixture of bi-, tri-, tetraantennary structures) fucosylated at the core and bearing LacNAc extensions, capped with sialic acid residues. Sia is predominant in the Neu5Ac form, the NeuGc in small quantities can be attributed to the presence of foetal bovine serum in the culture medium and to the fact that CHO can synthesise a small amount of CMP-NeuGc. Not a significant number of hybrid structures was observed [START_REF] North | Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals Nglycans of a novel size and complexity[END_REF].

The results are in agreement with what it was expected in terms of glycan structures: the lectins ConA, GNA and PSA highlight the presence of high-mannose and core-fucosylated structures, WFA and DSA respectively GalNac and GlcNac, AIA could underline the presence of Lac residues while PHA-L PHA-E the presence of complex N-glycans. It is striking the interaction with MAAI and the absence of signal for SNA, linked to the well-known lack of ST6 and thus α2-6-sialylation in CHO cells. 4.10.

conditions and the purified IgGs of the sample 3 and 4 are at a very low concentration (Table 4.10)

that might affect the detection of IgG sialic acid linkages. However, the healthy individuals show a very similar profile, letting us think that the changes could be attributed to the disease.

4.12 Neu5Ac/Neu5Gc LEctPROFILE kit 4.12.1 HPyL Purification

The lectin from HPyV9 was expressed and purified at GLYcoDiag following the protocol described by Khan et al. [START_REF] Khan | Crystallographic and Glycan Microarray Analysis of Human Polyomavirus 9 VP1 Identifies N-Glycolyl Neuraminic Acid as a Receptor Candidate[END_REF]. The purification protocol for industrial scale purification was optimised at GLYcoDiag and it is property of GLYcoDiag, for this reason it will not be described in detail in this thesis. In

Direct binding

The GLYcoPROFILE in direct binding was used to test the specificity of the lectins WGA and HpyL towards the corresponding neoglycoproteins and to test the sensitivity of the kit, determining the limit of detection. The neoglycoproteins NeoNeuAc and NeoNeuGc were chosen as a model probes. The preliminary results in Fig. 4.39 A and B show the direct binding curve of WGA with NeoNeuAc and HPyL with NeoNeuGc. The data obtained fit the non-linear regression model from which the BC50 is calculated, in both cases it is around 1µM. The limit of detection was set at 500 fluorescence units. For WGA, the limit is around 0.03 µM. For HPyL the sensitivity is low, being the detection limit ten times higher than the one of WGA, around 0.3 µM. In the future, further experiment need to be performed to have a complete view of this kit.

5.2.Biotinylation of neoglycoproteins

The labelling of NeoGPs was carried out by amide coupling with the EZ-link sulfo-NHS-LCbiotin (10 equivalents) in PBS 10 mM at pH 7.4 for 30 min at room temperature. The excess of biotin reagent was removed by dialysis process. The number of biotin coupled on the neoglycoprotein was determined according to the standard HABA (4'-hydroxyazobenzene-2-carboxylic acid)/ avidin quantification method. This value is related to the biotin amount of the sample. The same protocol was applied to the biotinylation of cells culture medium and cell culture supernatant.

Determination of protein concentration with Bradford Protein Assay

The Brilliant Blue G250 (Sigma). The staining was removed from the gel by heating in water until bands appeared visible or after staining with a MeOH/AcOH solution for 1 h. In order to reveal the glycoproteins present in a sample, the glycoproteins band can been revealed by using the Schiff reagent PAS (Periodic Acid Schiff stain, Sigma) protocol. Scan records of the gels were taken.

GLYcoPROFILE®

The covalent and adsorption 96-microtiter plates for GLYcoPROFILEs study were purchased from Biomat. Streptavidin-DTAF and extravidine-peroxydase solution, the revealing solution used in GLYcoPROFILEs were respectively obtained from Jackson Imuno Research and Sigma Aldrich. The OPD tablet was purchased from Sigma Aldrich. All absorbance and fluorescence measurements were recorded on a FluoStar Omega (BMG LabTech). The full recombinant MOA was purchased from Sobioda, French distributor for the Japanese lectins Fujifilm WAKO. GSLI-B4 was purchased from Vector. WGA was purchased from Medicago.

GLYcoDiag's GLYcoPROFILE is a lectin-array based technology. Lectins were immobilized overnight at 4°C on a 96-well microtiter plate. The quantity of each lectin was determined empirically in order to get the optimum binding. For covalent plates, surfaces are functionalized with carboxylic groups that can react covalently through amidification with the free amino groups of lectins using the EDC activating reagent. After lectins incubation overnight, the plates were washed with PBS/Tween 20 0,05% and saturated with PBS/BSA 0.5%.

GLYcoPROFILE in direct binding: after 30 minutes incubation time with PBS/BSA 0.5%, the plates were washed with PBS. Then, the labelled glycoprotein/neoglycoprotein or the monoclonal antibodies were applied to the plate in serial dilutions and they were left for 30 minutes at room temperature. After this 30 min of incubation, the plates were washed with PBS, and streptavidin-DTAF for fluorescent plates or ExtrAvidin-peroxidase for absorbance plate were added. They were incubated 30 minutes at room temperature, sheltered from light. In the case of monoclonal antibodies, before adding the streptavidin-DTAF, a biotinylated secondary antibody was added to the plate and it was incubated for 30 minutes at room temperature. GLYcoPROFILE in inhibition: the protocol is the same with few differences. After the saturation step with PBS/BSA 0.5% and the washing step, the non-labelled inhibitors were added to the plate in serial dilution while the labelled tracers (a biotinylated neoglycoprotein) were added to the plate at a fixed concentration, determined empirically. The plates were incubated for two hours at room temperature. The fluorescence or absorbance signal then were expressed in % of inhibition.

5.6.2B16

B16 murine melanoma cells were grown in medium DMEM supplemented with heat inactivated foetal bovine 10%, L-Glutamine 200 mM and mix of antibiotics 1X. They were kept in the incubator at 37°C with 5% of CO2. Before reaching the confluence, the cells were harvested using Tripsin/EDTA and after harvesting, cells were washed in PBS pH 7.4 without cations, collected by centrifugation and resuspended in PBS pH 7.4 without cations. The cells were counted using the Trypan blue 0.4% from Sigma. To perform a cell count, the Malassez counting chamber was used.

After counting, all cells were diluted to 2x10 6 cells/mL and labelled with CFSE at 40 μM for 15 min at 37 °C under gentle agitation sheltered from the light. After washing with PBS without cations, cells were incubated on the previously prepared LEctPROFILE plate at a concentration of 2x10 5 cells/well.

After washing, the fluorescence intensity was measured using the microplate reader. Data analysis was performed as previously described (5.7.1.).

Enzimatic digestion

α2-3 Neuraminidase Sfrom Streptococcus pneumoniae (P0743) and α2-3,6,8

Neuraminidase (P0720) from Clostridium perfingens were purchased from New England Biolabs.

The α-1-(3,6) Galactosidase from green coffee beans was purchased from Sigma.

For the α2-3 Neuraminidase digestion, the glycoproteins or the monoclonal antibodies were incubated with the enzyme in a ratio of 8 units/µg of glycoconjugate. The reaction mix was incubated in a buffer at pH 6 (supplied with the enzyme) at 37°C for 1 hour. After the incubation time, the reaction was stopped on ice and the pH was reset to 7.4 with PBS. The samples were dialyzed against PBS pH 7.4 prior to use in GLYcoPROFILE. For the α2-3,6,8 Neuraminidase digestion, the glycoproteins or the monoclonal antibodies were incubated with the enzyme in a ratio of 2,5 units of enzyme/µg of glycoconjugate. The reaction mix was incubated in a buffer at pH 6 (supplied with the enzyme) at 37°C overnight. After the incubation time, the reaction was stopped and the pH was reset to 7.4 with PBS. The samples were dialyzed against PBS pH 7.4 prior to use in GLYcoPROFILE.

After digestion with α2-3,6,8 Neuraminidase, the monoclonal antibodies were incubated with the α-1- (3,6) Galactosidase in a ratio of 14 units/µg of glycoconjugate. The reaction mix was incubated in PBS pH 7.4 at 37°C for 24 hours and afterwards the reaction was stopped on ice and the pH was reset to 7.4 with PBS. The samples were dialyzed against PBS pH 7.4 prior to use in GLYcoPROFILE.

Serum IgG Purification

The blood samples were provided by Etablissement français du sang (EFS) for healthy donors, and supplied by the department of infectious and tropical diseases of the "Centre hospitalier régional d'Orléans (CHRO)" for sick donors.

The blood was clotted by leaving it undisturbed at room temperature for 15-30 minutes. The clot was removed by centrifugation of the samples at 2,000 x g for 10 minutes at 4°C and filtered through 0.45 µM. The samples were then purified on Protein A after dilution by 3 of sample in the equilibration buffer (0.02M sodium phosphate, pH 7.0). Pure antibodies were eluted with 0.1M glycine-HCl buffer (pH 2.7) and immediately neutralized with a TrisHCl buffer (1M, pH 9.0). The purified antibodies were controlled for their purity by Bradford and SDS-PAGE before their use in GLYcoPROFILE studies.

The antibodies were kept at -20°C.

Final conclusion and perspectives

The aim of the synBIOcarb network is to study the glycobiological interactions in order to design targeted drug-delivery systems and diagnostics tools. This ambitious goal requires a high- The development of the Galili antigen LEctPROFILE kit was the following: first, we studied the optimal quantity of lectin MOAβT/well to be immobilized on the microtiter plate and the best type of plate. We chose the covalent plate with EDC mediated amination linkage and an absorbance detection because it was the one giving the most robust signal. Then, we tested MOAβT also with fluorescence detection: the sensitivity of the kit is lower in fluorescence but it is much more exploitable. The fluorescence detection removes the step of the addition of the substrate for the extravidin-peroxidase that makes the kit cheaper and the experiment faster. In addition, if MOAβT is included in a wider kit containing more lectins, the fluorescence is advantageous because some lectins cross-react with the peroxidase. The lectin GSLIB4 was also tested in fluorescence, having high specificity for the NeoGalili neoglycoprotein and a very good sensitivity. The two lectins MOAβT and GSLIB4 can be considered complementary tools on the same kit: MOAβT has a strong selectivity for the Galili antigen but it has low affinity for the monosaccharide and disaccharides whereas GSLIB4 has higher affinity for monosaccharides and disaccharides also linked in 1,4 and 1,6.

The development of the α2-3/α2-6 LEctPROFILE was carried out in fluorescence using the α2-3 sialic acid-linkage specific lectin MAAI and α2-6 sialic acid-linkage specific SNA. The kit performs well in direct binding mode, where the quantity of the glycosidic α2-3 and α2-6 linkages can be expressed as equivalents of neoglycoprotein or as ratio α2-3/α2-6 and in inhibition mode, where the results can be expressed as relative inhibition potency (RIP). Sialic acid was also the focus of another study in collaboration with Dr. S. Gouin et al. "Polymers of a transition-state sialyl cation strongly Inhibit Bacterial Sialidases", at the end of Chapter 4 . Here, we evaluated the IC50 of potential sialidase multivalent inhibitors, thanks to sialic acid néoglycoproteins.

Altogether, considering the work and results presented in this thesis, the objective of the development of kits was met. However, several short-term and long-term perspectives should be considered for the future work. The purification of more lectins that may expand the screening of a wider variety of glycan motifs, such as core fucose, which is another important feature for the functionality of mAbs. Together with this, other than the neoglycoproteins already available at GLYcoDiag, the synthesis of neoglycoproteins containing more complex glycan motifs can be considered. In addition, it would be of great interest to test the kit with a wider pool of macromolecules. For example, more mAbs, IgG from healthy individuals and pathological individuals at different stages in order to monitor the glycosylation changes of the disease. It would be also

LEctPROFILE kits: towards quality control and new potential applications

Glycosylation is one of the most important post-translational modifications of proteins: it affects folding, stability, molecular recognition and functionality. It also influences the properties of biotherapeutics, medicinal products manufactured in living systems like mammalian cells, bacteria, yeast. It has been shown that lack of sialylation in biotherapeutics can affect the serum half-life of erythropoietin and accelerate its clearance from the body. It can also modify the monoclonal antibodies effector functions. The presence of sugar moieties like Galα1,3Gal (Galili antigen) and glycolyl neuraminic acid (Neu5Gc), can make a medicine unsafe, triggering an immune response from the human's body.

Biotherapeutics glycosylation strictly depends on the expression system and on the manufacturing process: the use of non-human mammalian host expression system like Chinese hamster ovary cells and murine myeloma cells is very convenient because it gives the drugs a human-like glycosylation pattern, but it has its downsides. CHO only sialylate in the α2,3 linkage affecting the functionality of the biotherapeutic molecules and murine cells can add the immunogenic epitopes Galili antigen and Neu5Gc. When it comes to glycan analysis in industrial production, the main used techniques like mass spectrometry and HPLC are very accurate but they can be time-consuming and require a high-level of knowledge and expertise. The main idea has been to develop a tool that makes easy to the industrial manufacturer the quality control of their biotherapeutics at the earliest possible stage of research, then each stage of the development and production: a lectin array based on the GLYcoDiag's GLYcoPROFILE® technology. Among the main features of the LEctPROFILE kits there is the high-specificity for the glycans of interest: it is a tool that helps to discriminate the sialic acid linkages and to distinguish the unwanted glycans Galili and Neu5Gc. This is achieved thanks to the purification of lectins with high-selectivity for the target glycan moiety. In addition, it has to be fast, high-throughput and it has to allow non-specialized researchers to be able to perform the analysis. In order to develop this kind of assay, we have synthetised neoglycoproteins that work as specific probes for the study of the kits performances. To do so we have established a protocol for batch production of five main neoglycoproteins of interest, and we have demonstrated their purity, activity and specificity towards the target lectins. They allow to understand the lectins selectivity, the detection limit of the kits and the IC50 of different reference molecules. During the course of the thesis, the idea of application of the kits has broadened: the same principle of a fast and easy-to use screening can be applied to other fields than the quality control of biotherapeutics. For example the Galili antigen is an obstacle for the transplantation of organs from pig into humans. To date, there is no a fast, high-throughput screening able to detect their presence on transplanted tissues and organs. This gap can be addressed by building a lectin array in order to detect this antigen on biotherapeutics and biological samples. Another example is the alteration of sialylation that could lead to pathological states and it is wellknown as a hallmark of cancer. Moreover, the changing of the ratio α2,3/α2,6 linkage is involved in pathogens tropism and It is also described that certain types of cancer, including colon carcinomas, retinoblastomas, breast cancers, and

43 Fig. 2 . 14 A 47 Chapter 3 -

 43214473 Fig. 2. 9 SDS-PAGE of NeoGalili synthesis. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA, Line 3: BSA-alkyne, Line 4: BSA-azide, Line5: NeoGalili neoglycoprotein. ______________________________________________ 41 Fig. 2. 10 MALD-TOF spectrum of NeoGalili neoglycoprotein. __________________________________ 41 Fig. 2. 11 GLYcoPROFILE in direct binding of the NeoGalili neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with six different lectins. The data shown are the average of experiments performed in triplicate. _________________________ 42 Fig. 2. 12 CuAAC reaction between BSA-alkyne and 6'-sialylactose-N3 or 3'-sialylactose-N3. ______ 43 Fig. 2. 13 SDS-PAGE of Neo6'SL and Neo3'SL synthesis. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA-alkyne, Line 3: Neo6'SL, Line 4: Neo3'SL. _________________________________________________________________ 43 Fig. 2. 14 A MALD-TOF spectrum of Neo6'SL B MALD-TOF spectrum of Neo3'SL. _______________ 44 Fig. 2. 15 A GLYcoPROFILE in direct binding of the Neo6'SL neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with four different lectins. B GLYcoPROFILE in direct binding of the Neo3'SL neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with four different lectins. The data shown are the average of experiments performed in triplicate. __________________ 44 Fig. 2. 16 CuAAC reaction between BSA-azide and propargylNeu5Ac or propargylNeu5Gc. _______ 45 Fig. 2. 17 SDS-PAGE of NeoNuAc and NeoNeuGc synthesis. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA, Line 3: BSAazide, Line 4: NeoNeuAc, Line 4: NeoNeuGc. ________________________________________________ 46 Fig. 2. 18 A MALD-TOF spectrum of NeoNeuAc B MALD-TOF spectrum of NeoNeuGc. __________ 46 Fig. 2. 19 A GLYcoPROFILE in direct binding of the NeoNeuAc neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with seven different lectins. B GLYcoPROFILE in direct binding of the NeoNeuGc neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with seven different lectins. The data shown are the average of experiments performed in triplicate. __________________ 47

Fig. 3 . 20 A

 320 Fig. 3. 10 GLYcoPROFILE in direct binding of GSLI-B4 interacting with labelled Porcine Thyroglobulin, Bovine Thyroglobulin, Invertase, NeoGalili, NeoL, NeoF. ______________________________________ Fig.3. 11 A Comparison of the binding of MOAβT at decreasing quantity/well to the NeoGalili neoglycoprotein at a fixed concentration (1.15 µM) on covalent plate (black) and adsorption plate (pink).The two experiments can be compared because the signal in both cases is measured in absorbance. B Binding of MOAβT at decreasing quantity/well to the NeoGalili neoglycoprotein at a fixed concentration (1.15 µM) on adsorption plate, this time the interaction is reported as fluorescence intensity. _________________________________________________________________________________ Fig.3. 12 A MOAβT decreasing quantity/well (4 µg/well to 0.12 µg/well) plotted against the absorbance at 450 nm resulting from the lectin interaction with the NeoGalili neoglycoprotein at a fixed concentration (0.3 µM) on covalent plate. B MOAβT decreasing quantity/well (1 µg/well to 0.1 µg/well) plotted against the absorbance at 450 nm resulting from the lectin interaction with the NeoGalili neoglycoprotein at a fixed concentration (0.3 µM) on covalent plate. ___________________ Fig.3.[START_REF] Gupta | Lectin microarrays for glycomic analysis[END_REF] Comparison of MOAβT binding to NeoGalili neoglycoprotein when MOAβT is at a fixed quantity/well of either 1 µg/well or 0.5 µg/well with NeoGalili added in serial dilutions. _____________ Fig.3. 14 Direct binding curve of MOAβT immobilized on covalent plate at the fixed quantity of 1 µg/well with NeoGalili neoglycoprotein applied to the plate at 1.15 µM and serially diluted. _________ Fig.3. 15 Inhibition experiment performed with MOAβT at 1 µg/well interacting with labelled NeoGalili at the fixed concentration of 0.5 µg/mL and with the non-labelled inhibitors applied in serial dilution, from a starting concentration of Porcine Thyroglobulin=12µM, NeoGalili=12 µM, Melibiose=50 mM. Fig.3. 16 Direct binding curve of MOAβT coated on adsorption plate at the fixed quantity of 1 µg/well with NeoGalili neoglycoprotein applied to the plate at 2.3 µM and serially diluted. _________________ Fig.3. 17 Direct binding curve of MOA coated on adsorption plate at the fixed quantity of 1 µg/well with NeoGalili neoglycoprotein applied to the plate at 2.3 µM and serially diluted. _________________ Fig.3. 18 Direct binding curve of GSLI-B4 coated on adsorption plate at 1µg/well interacting with NeoGalili neoglycoprotein applied to the plate at 2.3 µM and serially diluted. _____________________Fig. 3. 19 Inhibition experiment performed with GSLI-B4 interacting with labelled NeoGalili at the fixed concentration of 35 µg/mL and with the non-labelled inhibitors applied in serial dilution, from a starting concentration of Porcine Thyroglobulin=12µM, NeoGalili=12 µM, Melibiose=100 mM, α-galactose= 100 mM. The IC50 for α-galactose and Melibiose could not be calculated because of the saturation of the signal. ________________________________________________________________________________ Fig. 3. 20 A Infliximab GLYcoPROFILE in direct binding with a panel of lectins. B Rituximab GLYcoPROFILE in direct binding with a panel of lectins. _______________________________________ Fig. 3. 21 Cetuximab GLYcoPROFILE in direct binding with a panel of lectins. ___________________ Fig. 3. 22 MOAβT standard curve. __________________________________________________________ Fig. 3. 23 GSLI-B4 standard curve. __________________________________________________________ Fig. 3. 24 HPyL standard curve. ____________________________________________________________ Fig. 3. 25 SNA standard curve. _____________________________________________________________ Fig. 3. 26 MAAI standard curve. ____________________________________________________________ Fig. 3. 27 PSA standard curve. _____________________________________________________________

Fig. 4 . 29 A

 429 Fig. 4. 29 A Possible sialylated structures present on bovine thyroglobulin. B Relative abundance of α2-6 and α2-6+α2-3-sialic acid linkages present in sialylated glycans. __________________________ 143 Fig. 4. 30 Intracellular reaction of the non-fluorescent CFDA-SE cells-permeant molecule which diffuses into the cells and it is convertend into the fluorescent CFSE by intracellular esterases. ___________ 145 Fig. 4. 31 A CHO standard curve with linear regression equation. B GLYcoPROFILE of CHO k1 adherent cells. The data are mean values of three replicates. The wells "without lectins" correspond to the empty wells where only cells are deposed. The percentage of bound cells is calculated based on the standard curve, considering that 2x10 5 cells/well are deposed. _____________________________ 146 Fig. 4. 32 A. GLYcoPROFILE of CHO supernantant at three different concentrations on a panel of lectins. The supernatant is labelled at a concentration of 1 mg/mL and then serially diluted to 0.25 mg/mL B. GLYcoPROFILE of CHO medium at three different concentrations on a panel of lectins. The medium is labelled at a concentration of 1 mg/mL and then serially diluted to 0.25 mg/mL C Illustrative bar chart of the subtraction of cell supernatant GLYcoPROFILE from which the values of fluorescence intensity of the cell medium GLYcoPROFILE are removed. Below 500 fluorescence units the signal is considered background noise. The subtraction was made only for the highest concentration of 1 mg/mL. ________________________________________________________________________________________ 147 Fig. 4. 33 A B16 standard curve with linear regression equation. B GLYcoPROFILE of B16 melanoma cells. The data are mean values of three replicates. The wells "without lectins" correspond to the empty wells where only cells are deposed. The percentage of bound cells is calculated based on the standard curve, considering that 2x10 5 cells/well are deposed. _________________________________________ 148 Fig. 4. 35 SDS-PAGE on 14% polyacrylamide gel of IgG after elution from affinity column with protein A. Line 1: Protein marker Line 2 sample 1 Line 2 sample 2 Line 3 sample 4 Line 5 sample 4. _____ 149 Fig. 4. 36 GLYcoPROFILE® of purified IgG from Sample 1, 2, 3 and 4 performed in direct binding with the lectins SNA and MAAI. ________________________________________________________________ 150 Fig. 4. 37 A SDS PAGE analysis of HPyL. The protein sample was analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: HPyL (35 kDa). B GLYcoPROFILE in direct binding of HPyL interacting with labelled NeNeuGc, NeoNeuAc, and NeoFucose at2.3 µM. ____________________________________________________________________ 151 Fig. 4. 38 GLYcoPROFILE in direct binding of WGA interacting with labelled NeoNeuAc, NeoNeuGc and NeoFucose at 0.6 µM. ________________________________________________________________ 152 Fig. 4. 39 A GLYcoPROFILE in direct binding representing the interaction between the lectin WGA and the labelled neoglycoprotein NeoNeu5Ac at a starting concentration of 2 µM.The curve is obtained from three independent replicates. All measurements were performed in duplicates. B GLYcoPROFILE in direct binding representing the interaction between the lectin HPyL and the labelled neoglycoprotein NeoNeu5Gc at a starting concentration of 2 µM.The curve is obtained from three independent replicates. All measurements were performed in duplicates ___________________________________ 153 Fig. 4. 40 Inhibition curves of WGA with the inhibitors NeNeuAc and Transferrin at the starting concentration of 10 µM. The labelled tracer NeoNeuGc is at the fixed concentration of 50 µg/mL. All measurements were performed in duplicates. _______________________________________________ 153

Chapter 1 - 1 Chapter 1 -Chapter 1 -Introduction and thesis objective 2 1. 1

 111121 Introduction and thesis objective ..................................................................................... 1.1 Project description and thesis structure ............................................................................................ 1.1.1 Description du projet et structure de la thèse ................................................................................ 1.2 Introduction ....................................................................................................................................... 1.3 Lectins ............................................................................................................................................. 1.4 Techniques to study glycobiological interactions ............................................................................ 1.4.1 Lectin arrays ............................................................................................................................. 1.4.2 Glycan arrays ........................................................................................................................... 1.4.3 SPR .......................................................................................................................................... 1.4.4 ITC............................................................................................................................................ 1.5 Other techniques to study glycans .................................................................................................. 1.5.1 HPLC ........................................................................................................................................ 1.5.2 Capillary electrophoresis .......................................................................................................... 1.5.3 Mass spectrometry ................................................................................................................... 1.6 Biotherapeutic glycoproteins ........................................................................................................... 1.6.1 History ...................................................................................................................................... 1.6.2 Glycosylation in biotherapeutics .............................................................................................. 1.6.3 Production systems .................................................................................................................. 1.6.4 Biotherapeutics regulation ....................................................................................................... 1.7 Research aim .................................................................................................................................. 1.7.1 Objectif de la recherche ........................................................................................................... Chapter 2 -Neoglycoproteins ........................................................................................................... Abstract ................................................................................................................................................. Introduction and thesis objective Project description and thesis structure Synthetic Glycobiology is a rapidly growing field aiming at engineering and redesigning glycans, glycan-active enzymes and glycan-binding proteins for fundamental research, biomedicine and biotechnology. My PhD thesis has been carried out in the framework of the synBIOcarb (https://synbiocarb.science/) Innovative Training Network (ITN) part of the European Union's Horizon 2020 research and innovation programme, under the Marie Skłodowska Curie grant agreement no. 814029. SynBIOcarb started in 2019 and it is a consortium of European academic and industrial beneficiaries where 15 PhD students, called Early Stage Researchers (ESR), have been trained. In the network there are other partners, with the purpose to contribute to the consortium, giving the students the adequate support required and the opportunity to carry out secondments. In the map in Fig. 1.1, are highlighted the European countries that take part in the synBIOcarb ITN and a list of the beneficiaries and partners of the network; in red are highlighted our main collaborators. SynBIOcarb is a multidisciplinary network and each one of the participant has high expertise either in chemistry, biochemistry, molecular biology and protein engineering, structural biology, cell biology, bioanalytical tools. The final common goal is the development and exploitation of synthetic glycobiology for diagnostics and targeted drug delivery through the building of collaborations.

Fig. 1 .Chapter 1 -Introduction and thesis objective 3

 113 Fig. 1. 1 synBIOcarb network. On the left, highlighted in yellow all countries where the beneficiaries and partners are located. On the right, the logos of all beneficiaries and partners involved in the network.

Chapter 1 -Introduction and thesis objective 4 Chapter 2 ,Chapter 2 Chapter 3 Chapter 5 1 . 2 .

 14223512 3 and 4 contain the main results and are designed for the reader to be read independently: each one has its own abstract, introduction, results and discussion and conclusion. is the prelude to Chapter 3 and 4 because it contains the results regarding the neoglycoprotein synthesis which, together with lectins, are the main tools for the development of the kits. At the end of this chapter, it is attached the paper in collaboration with Dr. Goyard and Prof. Renaudet of University of Grenoble-Alpes: "Homoand Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins". is entirely dedicated to the Galili antigen. At the end of this chapter, it is attached the paper in collaboration with Dr. Simona Notova of Cermav in Grenoble: "Extending Janus lectins architecture: characterization and application to protocells". Chapter 4 is entirely dedicated to sialic acid. At the end of this chapter, it is attached the paper in collaboration with Dr. Gouin of University of Nantes: "Polymers of a transition-state sialyl cation strongly Inhibit Bacterial Sialidases". is dedicated to materials and methods and it is in common for all experiments because the techniques used are the same for all the three results chapters. Chapter 6 is a short summary of what are the thesis main accomplishments and the future perspectives. The experimental work can be divided into four big chunks that put together have led to reach the final objective of building the lectin kits. The workflow is represented in Fig. 1.3. The four steps are: Purification of highly specific lectins. This part of the work intended to target above all the lectins that are specific towards the glycans of interest. The lectins are either recombinant or from natural sources. Concerning recombinant lectins, the first one that was purified is MOAβT and this was done in collaboration with Dr. Simona Notova of the synBIOcarb network, during my secondment at the Cermav-CNRS in Grenoble in January 2020. The other recombinant one is HPyL specific for Neu5Gc, purified at GLYcoDiag. Regarding the natural lectins, the lectin MAAI was purified by me, and the lectin SNA has been purified at GLYcoDiag before my arrival. Synthesis of neoglycoproteins. Neoglycoproteins are a fundamental part of the construction of the kit, because they are used as standard probes to set the reference parameters for the kits use.

Chapter 1 -Introduction and thesis objective 5 3. 4 .

 154 Study of the kits' parameters: choice of the quantity lectin/well, choice of the type of plate, if adsorption or covalent, the method of detection if in absorbance or fluorescence, the detection limit, half-maximum inhibitory concentration (IC50). Proof of concept, meaning testing the kit with relevant molecules, one part of this work was done in collaboration with Juvissa Aguedo , another PhD student of the synBIOcarb network.

Fig. 1 . 3

 13 Fig. 1. 3 Thesis workflow.

Fig. 1 . 4

 14 Fig. 1. 4 The non-template glycosylation drastically increases biological functional diversity, adapted from [5].

Fig. 1 . 5

 15 Fig. 1. 5 Classification of lectin based on their CRDs and their presence across the kingdoms of life adapted from [4]

Chapter 1 -Introduction and thesis objective 12 1. 4

 1124 Techniques to study glycobiological interactions 1.4.1 Lectin arrays

Fig. 1 . 7

 17 Fig. 1. 7 Glycan array principle, adapted from[START_REF] Muthana | Glycan microarrays: Powerful tools for biomarker discovery[END_REF]

Fig. 1 . 8

 18 Fig. 1. 8 SPR principle adapted from https://bit.ly/2Es3k2s.

Fig. 1 . 9 Chapter 1 -Introduction and thesis objective 16 1. 5

 191165 Fig. 1. 9 ITC principle adapted from https://bit.ly/2rP1OAL

Fig. 1 .

 1 Fig. 1. 10 Principle of HPLC, adapted from https://bit.ly/3UWsiMH

Fig. 1 .

 1 Fig. 1. 11 Schematic representation of CE system, adapted from https://bit.ly/3SHSR64

Fig. 1 .

 1 Fig. 1. 12 Derivatization of sialic acid linkages.Adapted from the poster "Rapid and high-throughput methods for discrimination of sialic acids linkages in glycoproteins" J. Aguedo and F. Vena.

Fig. 1 .

 1 Fig. 1. 13 MALDI-TOF/MS for the discrimination of α2-3 and α2-6 sialic acid linkages workflow. Adapted from the poster "Rapid and high-throughput methods for discrimination of sialic acids linkages in glycoproteins" J. Aguedo and F. Vena.

Fig. 1 .Chapter 1 -

 11 Fig. 1. 14 A 2020 best-selling drugs, adapted from [25]. B Biotherapeutics global revenues from 2005 to 2006 adapted from [26].

Chapter 1 -

 1 Introduction and thesis objective 21 efficient, with longer half-life in the blood. There are different types of cells currently used for the manufacturing:

  The Swiss company GlycoVaxyn, now part of GSK, has designed a bioconjugation platform which enables the in vivo synthesis of protein-polysaccharide complexes in E. coli, via recombinant DNA technology. All these systems have their advantages and disadvantages, and above all, they present very different glycosylation pathways (Fig 1.15).

Fig. 1 .

 1 Fig. 1. 15 Different glycosylation according to the kind of expression system.

Chapter 1 -

 1 Introduction and thesis objective[START_REF] Reiding | High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification[END_REF]

Fig. 1 . 16 AChapter 2 -Neoglycoproteins 31 2. 1

 1162311 Fig. 1. 16 A GLYcoPROFILE in direct binding. B GLYcoPROFILE in inhibition.

Fig. 2 . 2 ,Chapter 2 -Neoglycoproteins 32 b)

 22232 are represented the four different mechanisms of interaction of multivalent partners [8] [53]: a) The chelate binding. The n binding sites of a homomultimeric lectin can interact with m ligands from a multi-glycosylated molecule on the cell surface. The receptor clustering. It happens when monovalent lectins or ligands are anchored to the cell membrane. For example, in the presence of a multivalent ligand, receptors can diffuse through the lipid bilayer and become clustered by the multivalent ligand. c) The subsite binding. Some proteins in addition to the first binding site possess a second one that can be occupied by a multivalent glycan with different affinity compared to the first binding site. d) Statistical reassociation. A monovalent lectin could improve the affinity interacting with multivalent glycans present in close proximity of the binding site and in high concentration.

Fig. 2 . 2

 22 Fig. 2. 2Mechanisms of interaction of multivalent partners, adapted from[8]

Chapter 2 -Neoglycoproteins 33 2. 2

 332 Different types of multivalent synthetic glycoconjugates Different types of multivalent glycoconjugates have been designed with different valencies and topologies (Fig 2.3).

Fig. 2 . 3

 23 Fig. 2. 3Types of multivalent synthetic glycoconjuagtes, adapted from[8]

Fig. 2 . 4 A

 24 Fig. 2. 4 A CuAAC via 1,3-dipolar cycloaddition of azides and alkynes reaction resulting in 1,2,3-triazoles, adapted from [63]. B CuAAC reaction mechanism (adapted from, http://bitly.ws/uSiZ)

  GLYcoDiag's research and development. The previous Glycodiag's PhD student, Dr. Blanka Didak, mainly worked on neoglycoproteins as strong multivalent ligands for C-type lectins (especially DC-SIGN and Langerin), by preparing a large variety of multimeric mannosylated neoglycoproteins and neoglycoclusters. During the course of my thesis, the neoglycoproteins synthesis was optimised with the purpose of having the right probes for the development of the LEctPROFILE kits. The neoglycoproteins are, in this case, addressed towards an application; they work as model macromolecules that, thanks to their high avidity and strong specificity for the target lectin, help to design the assay in order to have all the information for the analysis of an unknown sample. Thus, in this section are presented the results that are the prerequisites for the implementation of the Galili antigen LEctPROFILE kit, the α2-6/α2-3 LEctPROFILE kit and the Neu5Ac/Neu5Gc LEctPROFILE kit.

Chapter 2 -Neoglycoproteins 38 20- 25

 3825 azide groups/BSA is expected. The reaction product is purified on Sephadex G-25 and the ratio is verified by MALDI-TOF/MS. This protocols are optimized to obtain around 100 mg of functionalized protein.2. The second step is the actual CuAAC reaction (Fig.2.5 A, Bright part) between the BSA-alkyne or BSA-azide prepared previously and the alkyne or azide group of a simple or complex glycan. The number of glycans conjugated on BSA is determined by MALDI-TOF/MS analysis. Further steps for characterization of these glycoconjugates are SDS-PAGE, Bradford for protein quantification. After neoglycoprotein biotinylation, their specificity is evaluated through GLYcoPROFILE in direct binding. This protocol is optimized to obtain around 5 mg of neoglycoprotein.

Fig 2 . 6

 26 shows the SDS-PAGE that was carried out at the end of the reactions, after the products purification by sephadex G-25. The most interesting aspect of this gel is that the band at line 3 representing the BSA-alkyne, and the band at line 4 representing the BSAazide, clearly show an increase in the molecular weight compared the simple BSA at line 2.

Chapter 2 -Neoglycoproteins 39

 39 This is the first confirmation that the reaction worked. The MALD-TOF spectra in Fig 2.7 A, B and C are the final proof of the success of the synthesis. On the top, the spectrum of BSA confirms the molecular weight of around 66 kDa of the pure protein. On the bottom left, in the spectrum of the BSA-alcyne we can see a single peak indicating the purity of the protein and the increased molecular weight of 70674 Da compared to the BSA 66 kDa. On the right, in the spectrum of the BSA-azide we can see a single peak indicating the purity of the protein and the increased molecular weight of 68206 Da. The molecular weight calculated in the MALD-TOF spectra for the two proteins is also in accordance with what we see on the SDS-PAGE (Fig 2.6). The calculated number of alkyne/BSA calculated from the MALD-TOF analysis is 37 and the calculated number of azide/BSA is 21, as expected.

Fig. 2 . 6

 26 Fig. 2. 6 SDS-PAGE of BSA functionalization with alkyne and azide groups. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA, Line 3: BSA-alkyne, Line 4: BSA-azide.

Fig. 2 . 7 A

 27 Fig. 2. 7 A MALD-TOF/MS spectrum of BSA B MALD-TOF/MS spectrum of BSA-alkyne C MALD-TOF spectrum of BSA-azide.

Fig. 2 . 8

 28 Fig. 2. 8 CuAAC reaction between BSA-azide and Galα1-3Gal-N-Acetyl-Propargyl.

Fig. 2 . 9

 29 Fig. 2. 9 SDS-PAGE of NeoGalili synthesis. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA, Line 3: BSA-alkyne, Line 4: BSA-azide, Line5: NeoGalili neoglycoprotein.

Fig. 2 .

 2 Fig. 2. 10 MALD-TOF spectrum of NeoGalili neoglycoprotein.

Fig. 2 .

 2 Fig. 2. 11 GLYcoPROFILE in direct binding of the NeoGalili neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with six different lectins. The data shown are the average of experiments performed in triplicate.

Fig. 2 .

 2 Fig. 2. 12 CuAAC reaction between BSA-alkyne and 6'-sialylactose-N3 or 3'-sialylactose-N3. After purification of the reaction product, the SDS-PAGE presented in Fig 2.13 confirmed that the reaction was successful, with a clear molecular weight shift of the Neo3'SL and Neo6'SL towards 70 kDa.

Fig. 2 .

 2 Fig. 2. 13 SDS-PAGE of Neo6'SL and Neo3'SL synthesis. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA-alkyne, Line 3: Neo6'SL, Line 4: Neo3'SL.

Fig. 2 .

 2 Fig. 2. 14 A MALD-TOF spectrum of Neo6'SL B MALD-TOF spectrum of Neo3'SL.

Fig. 2 . 15 A

 215 Fig. 2. 15 A GLYcoPROFILE in direct binding of the Neo6'SL neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with four different lectins. B GLYcoPROFILE in direct binding of the Neo3'SL neoglycoprotein labelled. The neoglycoprotein at three different concentrations was

Fig. 2 .

 2 Fig. 2. 16 CuAAC reaction between BSA-azide and propargylNeu5Ac or propargylNeu5Gc.

Fig. 2 .

 2 Fig. 2. 17 SDS-PAGE of NeoNuAc and NeoNeuGc synthesis. The protein samples were analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: BSA, Line 3: BSA-azide, Line 4: NeoNeuAc, Line 4: NeoNeuGc.

Fig. 2 .

 2 Fig. 2. 18 A MALD-TOF spectrum of NeoNeuAc B MALD-TOF spectrum of NeoNeuGc.

Fig. 2 . 19 A

 219 Fig. 2. 19 A GLYcoPROFILE in direct binding of the NeoNeuAc neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with seven different lectins. B GLYcoPROFILE in direct binding of the NeoNeuGc neoglycoprotein labelled. The neoglycoprotein at three different concentrations was added to the plate coated with seven different lectins. The data shown are the average of experiments performed in triplicate.

  LEctPROFILE kit are described. The results about the study of the kit are presented as well as a proof of concept using the above-cited mAbs Cetuximab and two other therapeutic mAbs. The Galiliantigen specific lectins MOAβT and GSLIB4 are carefully characterized and studied for the development of the kit. At the end of the chapter is enclosed the paper "Extending Janus lectins architecture: characterization and application to protocells" by Notova et al., in which the purification and characterization of MOAβT is described.RésuméL'épitope Galα1-3Gal, également appelé antigène Galili, est expriméchez tous les mammifères à l'exception des humains, des singes et des singes de l'Ancien Monde qui ont perdu le gène codant (GGTA1) pour l'enzyme α1,3-galactosyltransférase (α1,3GT) au cours de l'évolution.En conséquence, 1% des anticorps circulants chez l'homme sont des anticorps anti-Galili. Cette caractéristique crée un obstacle pour la production de biothérapeutiques dans des lignées cellulaires de mammifères non humains, telles que les cellules de myélome murin. Le cas de l'anticorps monoclonal Cetuximab, qui est produit dans ce type de cellules, a été un cas frappant de réaction d'hypersensibilité chez les patients dû à l'antigène Galili. Pour la même raison, les anticorps anti-Gal sont un obstacle à la transplantation d'organes de porc chez l'homme. Il n'existe pas à ce jour de criblage rapide à haut débit capable de détecter la présence de l'antigène Galili sur les biothérapies et les tissus et organes destinés à être greffés. Nous voulons combler cette lacune en développant une puce à lectine hautement spécifique et simple d'utilisation, afin de détecter cet antigène sur des échantillons biothérapeutiques et biologiques. Dans ce chapitre, un examen de l'antigène Galili et un aperçu des applications potentielles du kit LectPROFILE d'antigène Galili sont décrits. Les résultats de l'étude du kit sont présentés ainsi qu'une preuve de concept utilisant le Chapter 3 -The Galili Antigen 58 mAbs cetuximab cité ci-dessus et deux autres mAbs thérapeutiques. Les lectines spécifiques de l'antigène Galili MOAβT et GSLIB4 ont été particulièrement caractérisées et étudiées pour le développement du kit. L'article « Extending Janus lectins architecture: characterization and application to protocells» de Notova et al., dans lequel la purification et la caractérisation de MOAβT est décrite, est joint dans son intégralité, à la fin du chapitre.

Fig. 3 . 1 A

 31 Fig. 3. 1 A, the Galili antigen trisaccharide. B, the reaction catalysed by the α1,3GT, adapted from [67].

Fig. 3 . 2 A

 32 Fig. 3. 2 A, Cetuximab structure. It contains two glycosylation sites in the Asn 299 on the Fc portion and two at Asn88 in the Fab portion, adapted from[START_REF] Biolabs | Glycoproteomics Technical Guide[END_REF]. B, the cetuximab mechanism: it binds to the EGFR and prevents its dimerization, blocking all its pro-tumour functions and promoting apoptosis, adapted from[START_REF] Patil | Cetuximab and biomarkers in non-small-cell lung carcinoma[END_REF].

Fig. 3 . 3

 33 Fig. 3. 3 Schematic representation of the distribution of hypersensitivity reactions to Cetuximab and red meat in the southeast region of North America and its connection to the increasing deer population carrying ticks and tick bites, adapted from [72]].

Fig. 3 . 4

 34 Fig. 3. 4 Galsafe engineered pigs used for xenotraspantation, adapted from [77].

Fig. 3 . 5

 35 Fig. 3. 5 Schematic representation of the viral vaccine mechanism of action, adapted from [79].

Fig. 3 . 6

 36 Fig. 3. 6 Rrepresentation of the α-Gal glycolipids and how they are recognized by anti-gal Abs when injected in tumour tissues, adapted from [66].

3. 3

 3 The Marasmius oreades agglutinin MOA (Marasmius Oreades agglutinin) is a lectin from the edible fairy ring mushroom. MOA is specific for oligosaccharides with terminal non-reducing Galα1-3Gal linked galactose, while disaccharides linked in α1,2, α1,4, and α1,6 show no binding. It agglutinates group B blood cells which display the branched trisaccharide epitope Galα(1,3)-[Fucα(1,2)]Galβ-epitopes but no binding

Fig. 3 . 7

 37 Fig. 3. 7 MOA β-trefoil portion crystal structure in complex with the Galili antigen. This figure was created with Pymol, adapted from the crystal structure of Grahn et al, ([85] PDB: 2IHO).

Fig. 3 . 8

 38 Fig. 3. 8 Crystal structure of GSLI-B4 in complex with the xenograft antigen. PDB code 1HQL, adapted from [86].

Chapter 3 -

 3 The Galili Antigen 67 in this thesis. All lectins manufactured at GLYcoDiag are carefully quantified through the Bradford protein assay: the yield of MOAβT reached for the industrial batch production is of around 15 mg of protein/ culture litre. We checked the lectin purity through SDS-PAGE: in Fig.3.9A it is shown the lectin at the expected molecular weight of around 17 kDa. No impurities were detected. We tested also the lectin activity and selectivity for the target sugar through GLYcoPROFILE in direct binding.The bar chart in Fig.3.9B shows the selectivity of the protein for the thyroglobulin glycoproteins known to bear the Galili antigen, and for the NeoGalili neoglycoprotein.

Fig. 3 . 9 A

 39 Fig. 3. 9 A SDS-PAGE analysis of MOAβT The protein sample was analyzed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: MOAβT (17,2 kDa). B GLYcoPROFILE in direct binding of MOAβT interacting with labelled Porcine Thyroglobulin, Bovine Thyroglobulin, Invertase, NeoGalili and NeoβGal, NeoF.

Chapter 3 -The Galili Antigen 68 PFig. 3 .

 3683 Fig. 3. 10 GLYcoPROFILE in direct binding of GSLI-B4 interacting with labelled Porcine Thyroglobulin, Bovine Thyroglobulin, Invertase, NeoGalili, NeoL, NeoF.

Fig. 3 . 11 A

 311 Fig. 3. 11 A Comparison of the binding of MOAβT at decreasing quantity/well to the NeoGalili neoglycoprotein at a fixed concentration (1.15 µM) on covalent plate (black) and adsorption plate (pink). The two experiments can be compared because the signal in both cases is measured in absorbance. B Binding of MOAβT at decreasing quantity/well to the NeoGalili neoglycoprotein at a fixed concentration (1.15 µM) on adsorption plate, this time the interaction is reported as fluorescence intensity.

Fig 3 .

 3 Fig 3.12 B clearly shows a drop of the signal directly proportional to the lectin quantity.

Fig. 3 . 12 A

 312 Fig.3. 12 A MOAβT decreasing quantity/well (4 µg/well to 0.12 µg/well) plotted against the absorbance at 450 nm resulting from the lectin interaction with the NeoGalili neoglycoprotein at a fixed concentration (0.3 µM) on covalent plate. B MOAβT decreasing quantity/well (1 µg/well to 0.1 µg/well) plotted against the absorbance at 450 nm resulting from the lectin interaction with the NeoGalili neoglycoprotein at a fixed concentration (0.3 µM) on covalent plate.

Fig. 3 .

 3 Fig. 3. 13 Comparison of MOAβT binding to NeoGalili neoglycoprotein when MOAβT is at a fixed quantity/well of either 1 µg/well or 0.5 µg/well with NeoGalili added in serial dilutions.

Fig. 3 .

 3 Fig. 3. 14 Direct binding curve of MOAβT immobilized on covalent plate at the fixed quantity of 1 µg/well with NeoGalili neoglycoprotein applied to the plate at 1.15 µM and serially diluted.

Fig. 3 .

 3 Fig.3.[START_REF] Kuno | Evanescent-field fluorescence-assisted lectin microarray: A new strategy for glycan profiling[END_REF] Inhibition experiment performed with MOAβT at 1 µg/well interacting with labelled NeoGalili at the fixed concentration of 0.5 µg/mL and with the non-labelled inhibitors applied in serial dilution, from a starting concentration of Porcine Thyroglobulin=12µM, NeoGalili=12 µM, Melibiose=50 mM.

Fig. 3 .

 3 Fig. 3. 16 Direct binding curve of MOAβT coated on adsorption plate at the fixed quantity of 1 µg/well with NeoGalili neoglycoprotein applied to the plate at 2.3 µM and serially diluted.

Fig. 3 .

 3 Fig.3. 17 Direct binding curve of MOA coated on adsorption plate at the fixed quantity of 1 µg/well with NeoGalili neoglycoprotein applied to the plate at 2.3 µM and serially diluted.

Fig. 3 .

 3 Fig. 3. 18 Direct binding curve of GSLI-B4 coated on adsorption plate at 1µg/well interacting with NeoGalili neoglycoprotein applied to the plate at 2.3 µM and serially diluted.

Fig. 3 .

 3 Fig. 3. 19 Inhibition experiment performed with GSLI-B4 interacting with labelled NeoGalili at the fixed concentration of 35

Fig. 3 . 20 AFig. 3 .

 3203 Fig. 3. 20 A Infliximab GLYcoPROFILE in direct binding with a panel of lectins. B Rituximab GLYcoPROFILE in direct binding with a panel of lectins.

Chapter 3 -The Galili Antigen 81 WhenFig. 3 .Fig. 3 .Fig. 3 .Fig. 3 .Fig. 3 .Fig. 3 .

 381333333 Fig. 3. 22 MOAβT standard curve.

AChapter 4 -Sialic acid 110 Résumé

 4110 comparison with MALDI-TOF-MS results is showed to prove the reliability of the kit. The sialylation of IgGs from healthy patients and patients with pathologies is explored, and CHO and melanoma cells are also tested on the kit. At the end of the chapter, preliminary results about the development of Neu5Ac/Neu5Gc LEctPROFILE kit are reported.Les acides sialiques sont une famille de sucres avec une grande diversité structurelle, que l'on trouve principalement à l'extrémité externe des chaînes de glycanes. Ils peuvent être divisés en deux grands sous-groupes, l'acide N-acétyl neuraminique (Neu5Ac) et l'acide N-glycolyl neuraminique (Neu5Gc), ce dernier n'étant pas expriméchez l'homme. Le premier, au contraire, est d'une importance primordiale chez l'homme, il peut être lié à la chaîne glycannique avec une liaison α2-3 ou α2-6, et grâce à sa localisation et à la variété de ses structures, il est impliqué dans la médiation de nombreux aspects physiologiques tels que la communication cellule-cellule, la réponse immunitaire, le transport intramembranaire, les infections bactériennes et virales. L'altération de la sialylation pourrait conduire à des états pathologiques et elle est bien connue comme une caractéristique du cancer. De plus, la modification du rapport de liaison α2-3/α2-6 est impliquée dans le tropisme des agents pathogènes, dans l'activité pro-ou anti-inflammatoire des anticorps et présente un impact dans les fonctions effectrices des glycoprotéines recombinantes puisque la sialylation est différente selon le système de production. Dans ce chapitre, après un bref résuméde la biochimie dès l'acides sialiques et de leur rôle dans les systèmes biologiques, l'accent est ensuite placé sur la liaison α2-3/α2-6 et l'importance de leur reconnaissance par des lectines spécifiques. Nous verrons le développement I d'un kit LEctPROFILE destiné à discriminer les liaisons α2-3 et α2-6afin de fournir une semi-quantification de chacune et d'avoir un outil rapide susceptible de répondre à des applications potentielles industrielles et de recherche et développement. Une comparaison avec les résultats MALDI-TOF-MS prouve la fiabilité du kit. La sialylation des IgG de patients sains et de patients atteints de pathologies est explorée ; les surnageants de culture des cellules CHO et de mélanome sont également testées. A la fin du chapitre, les résultats préliminaires du développement du kit LEctPROFILE Neu5Ac/Neu5Gc sont décrits.

Fig. 4 . 1

 41 Fig. 4. 1 Structure of sialic acid, adapted from [90].

Fig. 4 . 2

 42 Fig.4. 2 Asialoglycoprotein receptors. ASGP receptors in the liver do not recognize desialylated proteins, targeting them for uptake and degradation, adapted from[START_REF] Maverakis | Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: A critical review[END_REF]

Fig. 4 . 3

 43 Fig. 4. 3 Roles of sialic acids. They can act as ligands for intrinsic receptors as well as extrinsic receptors, adapted from [92].

Fig. 4 .

 4 Fig. 4. 4 N-Acetyl neuraminic acid. The red circle highlights the acetyl functional group on C5

Fig. 4 . 5

 45 Fig. 4. 5 Neu5Ac acid metabolism in mammalian cells, adapted from [93].

Fig. 4 . 6

 46 Fig. 4. 6 Human Transferrin glycoforms, adapted from[START_REF] Porpiglia | A new sample treatment for asialo-Tf determination with capillary electrophoresis: an added value to the analysis of CDT[END_REF]

Fig. 4 . 7

 47 Fig. 4. 7 Sialome organization, adapted from [4].

4. 3 . 3

 33 In antibodies Immunoglobulins or IgGs are the most common type of antibodies (Ab) found in blood circulation, representing 75% of serum antibodies in humans. The four IgGs subclasses, IgG1, 2, 3 and 4 are named based on their abundance in serum. IgGs are large globular proteins of around 150 KDa. They are part of the humoral immunity and they control infections of body tissues binding to viruses, bacteria and fungi. They are represented with the classical Y shape, where the bottom part is made of the Fc domain, and the upper part is the Fab domain. The Fab consists of the light chain variable and constant domains (VL and CL) and the first two domains of the heavy chain, one variable and one constant (VH and CH1). The Fc portion consists of two constant domains, CH2 and CH3. The hinge region is a short sequence of the heavy chains linking the Fab region to the Fc region (Fig. 4.8). The two heavy chains are linked to each other and to a light chain each by disulfide bonds [109] (Fig. 4.8). The variable domains on the Fab are the ones responsible for the recognition of the antigen. The Fc domain coordinates the pathogen killing through antibody's effector functions such as the antibodydependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). The effector functions can be triggered by the Fc interaction with the FcγR inducing the release of cytotoxic granules resulting in killing of infected cells or triggering the activation of the complement cascade or the activation of phagocytic immune cells.

Fig. 4 . 8

 48 Fig. 4. 8 IgG structure and its glycosylation. The bottom domain is the Fc and the top one is the Fab. VL= variable region of the light chain, CL= constant region of the light chain, VH= variable region of the heavy chain, CH1,2,3= Constant region of the heavy chain, domain 1, 2, 3, Ag= antigen, adapted from [109].

Neu5Gc (Fig 4 . 10 )

 410 including New World primates, Mustelidae, pinnipeds, Procyonidae, hedgehog, bats, spermwhale, and white-tailed deer and humans[START_REF] Dhar | From 'Serum Sickness' to 'Xenosialitis': Past, Present, and Future Significance of the Non-human Sialic Acid Neu5Gc[END_REF]. The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-Nacetylneuraminic acid into CMP-Neu5Gc, which is catalysed by the CMP-Neu5Ac hydroxylase enzyme encoded by CMAH gene. This reaction happens in the cytosol and it is irreversible. Cmah catalyses the transfer of an oxygen atom to CMP-Neu5Ac, generating CMP-Neu5Gc. CMP-Neu5Gc can then be transported into the Golgi apparatus in the same manner as CMP-Neu5Ac (Fig. 4.10 A)

Fig. 4 .

 4 Fig.4. 10 A N-Glycolyl neuraminic acid biosynthesis. B taxa are found in blue, not found in purple and purple and blue where only certain species have lost the capacity to make it, adapted from[START_REF] Altman | Absence of Neu5Gc and Presence of Anti-Neu5Gc Antibodies in Humans-An Evolutionary Perspective[END_REF]

4. 5

 5 Lectins considered in this study 4.5.1 MAAI The first lectin considered for the α2-3/α2-6 LEctPROFILE kit is the sialic-acid binding lectin Maackia amurensis leukagglutinin. It is a L-type lectin extracted from the seeds of the legume tree Maackia amurensis, also referred commercially as MAL or MAA-I. For simplicity we will refer to it as MAAI. It is made of two subunits and in solution it assembles in the two monomers of the "canonical Chapter 4 -Sialic acid 125 legume lectin dimer" containing one carbohydrate binding domain each, as shown in Fig. 4.11. The molecular weight of the dimer is around 74 kDa with one monomer weighing around 35 and the other around 34 kDa. It is inhibited by low concentration of ɑ2-3-sialyllactose (NeuAc2-3Galβ1-

Fig. 4 .

 4 Fig.4. 11 MAA-I in complex with sialyllactose, PDB code 1DBN, adapted from[START_REF] Imberty | An unusual carbohydrate binding site revealed by the structures of two Maackia amurensis lectins complexed with sialic acid-containing oligosaccharides[END_REF]

Fig. 4 .

 4 Fig. 4. 12 SNA in complex with N-Acetylgalactosamine, PDB code 3CA3, adapted from [65].

Fig. 4 .

 4 Fig. 4. 13 structure of VP1. PDB code 4POQ [125].

Fig. 4 .

 4 Fig. 4. 14 3D structure of WGA, adapted from [126] .

Fig 4 .

 4 15B shows the lectin is active and interacts activity α2-3-sialyllactose that is highlighted in the interaction with the neoglycoprotein Neo3'SL. The lectin is highly selective for the α2-3 Sia linkage and does not interact with the neoglycoproteins Neo6'SL, NeoM, NeoF. Moreover, it resulted in no impurities in the end product, as shown in the line 2 of the SDS-PAGE (Fig 4.15A).

Fig. 4 . 15 A

 415 Fig. 4. 15 A SDS PAGE analysis of MAAI. The protein sample was analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: MAAI (35 kDa) B GLYcoPROFILE in direct binding of MAAI interacting with labelled Neo6'SL, Neo3'SL, NeoMa and NeoF at 40 µg/mL.

Fig. 4 . 16 A

 416 Fig. 4. 16 A SDS PAGE analysis of SNA. The protein sample was analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: SNA (33 kDa) B GLYcoPROFILE in direct binding of SNA interacting with labelled Neo6'SL, Neo3'SL and NeoMannose at 40 µg/mL.

Fig.

  Fig.4. 17 GLYcoPROFILE in direct binding representing the interaction between the lectin SNA and the labelled neoglycoprotein Neo6'SL at a starting concentration of 0.6 µM.The curve is obtained from four independent replicates. All measurements were performed in duplicates.

Fig. 4 .

 4 Fig.4.18 GLYcoPROFILE in direct binding representing the interaction between the lectin MAAI and the labelled neoglycoprotein Neo3'SL at a starting concentration of 0.6 µM. The curve is obtained from four independent replicates. All measurements were performed in duplicates.

Fig. 4 .

 4 Fig.4.[START_REF] Bertok | Analysis of serum glycome by lectin microarrays for prostate cancer patients -a search for aberrant glycoforms[END_REF] Inhibition curves of SNA with the inhibitors Neo6'SL, Fetuin, Transferrin at the starting concentration of 10 µM and 6'SL at the starting concentration of 5mM. The labelled tracer Neo6'SL is at the fixed concentration of 20 µg/mL. The curve is obtained from three independent replicates. All measurements were performed in duplicates.

Fig. 4 .

 4 Fig.4.[START_REF] Muthana | Glycan microarrays: Powerful tools for biomarker discovery[END_REF] Inhibition curves of SNA with the inhibitors Neo3'SL and Fetuin at the starting concentration of 10 µM and 3'SL at the starting concentration of 5mM. The labelled tracer Neo3'SL is at the fixed concentration of 20 µg/mL. The curve is obtained from three independent replicates. All measurements were performed in duplicates.

4 . 21 )

 421 and from each y values corresponding to the fluorescence intensity of the native glycoprotein interaction with the lectin, a x value was calculated that corresponds to the equivalent concentration in neoglycoprotein. We have decided to give the fluorescence a quantification in equivalents of neoglycoprotein because the neoglycoproteins are an optimized tool, representing the Sia linkages and they function as a standard of which we know precisely the concentration.The MALDI-TOF/MS with derivatization of sialic acid linkages was performed at the Slovak Academy of Sciences in Bratislava by another PhD student of the synBIOcarb network, Juvissan Aguedo (Description of the method: Chapter 1, Paragraph 1.5.3).

Fig. 4 .

 4 Fig. 4. 21 SNA and MAAI standard curves obtained with labelled Neo6'SL and Neo3'SL.

Fig. 4 .

 4 Fig.4.[START_REF] Suenaga | Monitoring influenza hemagglutinin and glycan interactions using surface plasmon resonance[END_REF] Interaction of MAAI and SNA specific lectins with labelled Fetuin at three different concentrations. The intensity of interaction of each lectin was studied before and after Fetuin digestion by total neuraminidase (NeuT) and α2-3 neuraminidase (Neuα2-3). Two independent assays (each with n = 3 replicates) were carried out.

Fig. 4 .

 4 Fig. 4. 23 MALDI-TOF/MS. A Possible sialylated structures present on Fetuin. B Relative abundance of α2-6 and α2-6+α2-3sialic acid linkages present in sialylated glycans.

Fig. 4 .Table 4 . 5

 445 Fig.4.[START_REF] Reiding | High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification[END_REF] Interaction of MAAI and SNA specific lectins with labelled transferrin at three different concentrations. The intensity of interaction of each lectin was studied before and after transferrin digestion by total neuraminidase (NeuT) and α2-3 neuraminidase (Neuα2-3). Two independent assays (each with n = 3 replicates) were carried out.

Fig. 4 .

 4 Fig. 4. 25 Interaction of MAAI and SNA specific lectins with labelled transferrin native and denatured at three different concentrations. Two independent assays (each with n = 3 replicates) were carried out.

Fig. 4 .

 4 Fig. 4. 26 MALDI-TOF/MS A Possible sialylated structures present on Transferrin. B Relative abundance of α2-6 and α2-6+α2-3sialic acid linkages present in sialylated glycans.

4. 9 . 4

 94 Porcine and Bovine ThyroglobulinThe GLYcoPROFILE for both the Thyroglobulin is shown in Fig.4.27 A and B. In the case of both Porcine and Bovine Thyroglobulin there is more variability in the two experiments considering the error bars. The digestion with NeuT and Neuα2-3 neuraminidase did not seem to completely work, probably because of the big size of these glycoproteins. Overall, we see a ratio α2-3:α2-6 around 55:45 for Porcine Thyroglobulin and 52:48 for Bovine Thyroglobulin.

Fig. 4 . 27 A

 427 Fig. 4.27 A Interaction of MAAI and SNA specific lectins with labelled porcine thyroglobulin at three different concentrations. B Interaction of MAAI and SNA specific lectins with labelled bovine thyroglobulin at three different concentrations. The intensity of interaction of each lectin was studied before and after thyroglobulin digestion by total neuraminidase (NeuT) and α2-3 neuraminidase (Neuα2-3). For each protein two independent assays (each with n = 3 replicates) were carried out.

Fig. 4 .

 4 Fig. 4. 28 MALDI-TOF/MS A Possible sialylated structures present on porcine thyroglobulin. B Relative abundance of α2-6 and α2-6+α2-3sialic acid linkages present in sialylated glycans.

Fig. 4 . 29 A

 429 Fig. 4. 29 A Possible sialylated structures present on bovine thyroglobulin. B Relative abundance of α2-6 and α2-6+α2-3sialic acid linkages present in sialylated glycans.

4. 10

 10 GLYcoPROFILE® of cells 4.10.1 Chinese Hamster Ovary CellsAfter the validation of the kit with commercial glycoproteins, the goal was to have the proof of concept of its exploitability for cell lines, in the perspective of potential research and development applications, such as following the production of a recombinant biotherapeutic glycoprotein in cell lines. The goal of the experiments described here is to highlight the glycosylation and, in particular, the α2-3/α2-6 sialylation of glycoproteins expressed at the surface of CHO cells or released in the culture medium. The brief history of CHO cell line is that in 1957, a parental CHO cell line was initiated from a biopsy of an adult, female Chinese hamster from which the CHO cell line was then derived as a subclone. For this study, we have carried out the GLYcoPROFILE[START_REF] Landemarre | Cell surface lectin array: Parameters affecting cell glycan signature[END_REF] in direct binding of CHO k1 adherent cells, their supernatant and their culture medium. On the same day of the GLYcoPROFILE, cells are harvested and labelled with CFSE (Carboxyfluorescein succinimidyl ester). It is a fluorescent molecule resulting from CFDA-SE (carboxyfluorescein diacetate succinimidyl ester), a cell permeant molecule which diffuse into the cells and it is cleaved by intracellular esterases (Fig 4.30). The CFSE binds indiscriminately with intracellular free amines to generate covalent fluorescent dye-protein conjugates resulting in live cells with an intracellular fluorescent label.

Fig. 4 .

 4 Fig. 4. 30 Intracellular reaction of the non-fluorescent CFDA-SE cells-permeant molecule which diffuses into the cells and it is convertend into the fluorescent CFSE by intracellular esterases.

Fig. 4 . 31 A

 431 Fig. 4. 31 A CHO standard curve with linear regression equation. B GLYcoPROFILE of CHO k1 adherent cells. The data are mean values of three replicates. The wells "without lectins" correspond to the empty wells where only cells are deposed. The percentage of bound cells is calculated based on the standard curve, considering that 2x10 5 cells/well are deposed.

Fig. 4 .

 4 Fig. 4. 32 A. GLYcoPROFILE of CHO supernantant at three different concentrations on a panel of lectins. The supernatant is labelled at a concentration of 1 mg/mL and then serially diluted to 0.25 mg/mL B. GLYcoPROFILE of CHO medium at three different concentrations on a panel of lectins. The medium is labelled at a concentration of 1 mg/mL and then serially diluted to 0.25 mg/mL C Illustrative bar chart of the subtraction of cell supernatant GLYcoPROFILE from which the values of fluorescence intensity of the cell medium GLYcoPROFILE are removed. Below 500 fluorescence units the signal is considered background noise. The subtraction was made only for the highest concentration of 1 mg/mL.

Fig. 4 . 33 A

 433 Fig. 4. 33 A B16 standard curve with linear regression equation. B GLYcoPROFILE of B16 melanoma cells. The data are mean values of three replicates. The wells "without lectins" correspond to the empty wells where only cells are deposed. The percentage of bound cells is calculated based on the standard curve, considering that 2x10 5 cells/well are deposed.

Fig. 4 .

 4 37A, the SDS-PAGE shows the pure lectin at the expected molecular weight of 17 kDa. No impurities were detected. The bar chart in Fig. 4.37 B shows the selectivity of the protein for the NeoNeuGc neoglycoprotein but not for the NeoNeuAc and NeoF underlying the selectivity of the lectin for glycolyl neuraminic acid.

Fig. 4 . 36 A

 436 Fig. 4. 36 A SDS PAGE analysis of HPyL. The protein sample was analysed under denaturing conditions on 14% polyacrylamide gel. Line 1: Protein marker, Line 2: HPyL (35 kDa). B GLYcoPROFILE in direct binding of HPyL interacting with labelled NeNeuGc, NeoNeuAc, and NeoFucose at2.3 µM.

Fig. 4 .

 4 Fig. 4. 37 GLYcoPROFILE in direct binding of WGA interacting with labelled NeoNeuAc, NeoNeuGc and NeoFucose at 0.6 µM.

Fig. 4 . 38 A

 438 Fig.4. 38 A GLYcoPROFILE in direct binding representing the interaction between the lectin WGA and the labelled neoglycoprotein NeoNeu5Ac at a starting concentration of 2 µM.The curve is obtained from three independent replicates. All measurements were performed in duplicates. B GLYcoPROFILE in direct binding representing the interaction between the lectin HPyL and the labelled neoglycoprotein NeoNeu5Gc at a starting concentration of 2 µM.The curve is obtained from three independent replicates. All measurements were performed in duplicates

Fig. 4 .

 4 Fig.4.[START_REF] Elliott | Control of rHuEPO biological activity: The role of carbohydrate[END_REF] Inhibition curves of WGA with the inhibitors NeNeuAc and Transferrin at the starting concentration of 10 µM. The labelled tracer NeoNeuGc is at the fixed concentration of 50 µg/mL. All measurements were performed in duplicates.

  For the fluorescent plates, after washing to remove the excess of streptavidin-DTAF solution, PBS was added in the plates and the fluorescence was read at λexc = 485nm and λem = 520nm with the help of a fluorescent reader. The fluorescence intensity is directly correlated to the concentration of biotinylated neoglycoprotein that interacts with the lectin. The fluorescence background of the lectin was corrected by removing the fluorescence intensity obtained of the lectin with only PBS and without any glycoconjugate.Moreover, the data were corrected by the gain of the plate measured by the plate reader. In the absorbance test, the plates were washed to remove the excess of the extrAvidin-peroxidase. Then, OPD peroxidase substrate was added and incubated in the dark for 15 minutes more. The reaction was stopped with 1M HCl . The plate was read at 450nm with the absorbance plate reader. The absorbance is directed correlated with the concentration of biotinylated neoglycoprotein that interacts with the lectin. The absorbance signal was corrected for the background by removing the absorbance value obtained with only PBS and without any glycoconjugate. Both for fluorescence and for absorbance test, a positive internal control to verify the correct running of the experiment was added.

  °C under gentle agitation sheltered from the light. CFSE is a fluorescent molecule resulting from CFDA-SE (carboxyfluorescein diacetate succinimidyl ester), a cell permeant molecule which diffuse into the cells and it is cleaved by intracellular esterases (Fig 5.2). The CFSE binds indiscriminately with intracellular free amines to generate covalent fluorescent dye-protein conjugates resulting in live cells with an intracellular fluorescent label.

Fig. 5 .

 5 Fig. 5. 1Intracellular reaction of the non-fluorescent CFDA-SE cells-permeant molecule which diffuses into the cells and it is convertend into the fluorescent CFSE by intracellular esterases. After washing with PBS without cations, cells were incubated on the previously prepared LEctPROFILE plate at a concentration of 2x10 5 cells/well for 1 hour at room temperature. After washing, the fluorescence intensity was measured using the microplate reader. The fluorescence values was normalized by the gain after removal for each well of the autofluorescence . The results were expressed in fluorescence intensity, cells number and cells bound percentage after having recorded a standard curve of the labelled cells.

  level of expertise in different fields, brought in the network by all beneficiaries and partners. My role in the network, at the company GLYcoDiag, has been in the part of the development of a new lectin array technology, based on the GLYcoPROFILE platform, for the in-process monitoring of biotherapeutic glycoproteins throughout all stages of the manufacturing process. This goal has taken a new shape in due course, and it has become possible to speculate upon other interesting applications requiring the use of a fast, high-throughput LEctPROFILE kit. This lectin array technology permits a wide variety of compounds to be screened at one time, in contrast to the nature of existing well-established analytical technologies, such as HPLC or CE.The focus of the research was placed mainly on two glycan motifs: the Galili antigen and the sialic acid. The kits development was approached in several steps. First of all, we purified highly-selective lectins: MOAβT, MAAI, HPyL, all of them carefully controlled for their concentration, purity and functionality. The purification of MOAβT, performed with Dr. Simona Notova at CERMAV (CNRS, Grenoble) contribute d to the production and characterisation of the Janus lectin RSL-MOA, described in the paper "Extending Janus lectins architecture: characterization and application to protocells", at the end of Chapter 3.Then, we undertook the synthesis of neoglycoproteins by click-chemistry reaction between the BSA functionalized with an alkyne or azido group and the sugars of interest functionalized with the same groups. We synthetized five neoglycoproteins, NeoGalili, Neo6'SL, Neo3'SL, NeoNeuAc and NeoNeuGc with a good yield and high purity. These neoglycoproteins allowed us to have reference molecules in order for us to define the kits characteristics. In conclusion, the work we published in collaboration with Professor's O. Renaudet's group was an extension of the initial synthesis done for neoglycoprotein. The paper, "Homoand Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins" (Chapter 2), in which we synthetized neoglycoproteins with clusters of glycans distributed at different position, opens the way for heterofunctionalization of neoglycoproteins.

  interesting to use other glycoproteins to go on with the comparative experiments of quantification of the α2-3/ α2-6 linkages with the LEctPROFILE kits and the MALDI-TOF/MS with linkages derivatization. The development of the Neu5Ac/Neu5Gc LEctPROFILE kit; which has been started, needs to be continued.The ITN synBiocarb offered me the opportunity to build constructive collaborations: I spent one month at the Cermav in Grenoble in January 2020 in Dr. Anne Imberty's group. There, as mentioned before, together with Dr. Simona Notova, we purified the recombinant lectin MOAβT that was of great use for my experiments on the LEctPROFILE kit and that is included in the paper at the end of Chapter 3. In addition, I had the chance to compare the kit performances to the MALDI/TOF/MS with derivatization of sialic acid linkages performed by Juvissan Aguedo at the Slovak Academy of Sciences. The synBIOcarb ITN provided me with high-level scientific training in the field of synthetic glycobiology, that helped me to build the knowledge to go ahead during the PhD program. Moreover, the network gave us many training in scientific communication: we had the opportunity to improve our presenting and writing skills and we got the chance to learn the basics of videomaking and to make a video speaking about our project to a lay audience. The covid epidemic outburst was something completely unexpected and new that had a great impact on people's lives all over the world and naturally, on us as young scientists as well. Unfortunately, our scheduled synBIOcarb biannual meetings were only three and many congresses I was supposed to participate in, were cancelled. No matter this, we were given the training remotely and I experienced to present online and, once travelling was permitted again, I had the chance to go to conferences to present my work (listed below). Last but not least, being in an industrial PhD program gave me the opportunity to look at my research from a more pragmatic and applicable way and gave me the chance to engage in multiple collaborations resulted in publications (listed below).Conclusion finale et perspectivesL'objectif du projet Innovative Training Networks (ITN) « synBIOcarb » est d'étudier les interactions glycobiologiques afin de concevoir des systèmes de délivrance de médicaments et des outils de diagnostic ciblés. Cet objectif ambitieux nécessite un haut niveau d'expertise dans différents domaines, qui est constitué par le réseau devtous les bénéficiaires et partenaires de ce projet. Mon rôle dans le réseau, au sein de la société GLYcoDiag, a été de participer au développement d'une nouvelle technologie de lectine array, basée sur la plateforme GLYcoPROFILE, pour le suivi en cours de production des glycoprotéines biothérapeutiques et à toutes les étapes du processus de fabrication. Cet objectif aévolué au cours du temps pour s'adapter également à d'autres applications intéressantes nécessitant l'utilisation d'un kit LEctPROFILE rapide et à haut débit. Cette technologie de réseau de lectines permet de cribler une grande variété de composés en une seule fois, sans nécessiter d'une forte expertise de la technologie, contrairement à la nature des technologies analytiques existantes et bien établies, telles que la spectrométrie de masse, la chromatographie (HPLC) ou l'électrophorèse capillaire (CE). La recherche s'est principalement concentrée sur deux motifs glycaniques : l'antigène Galili et l'acide sialique. Le développement des kits a été abordé en plusieurs étapes. Tout d'abord, nous avons purifié des lectines hautement sélectives: MOAβT, MAAI, HPyL, toutes soigneusement contrôlées pour leur pureté et leur fonctionnalité. La purification de MOAβT, réalisée avec le Dr Simona Notova au CERMAV (CNRS, Grenoble), a contribué à la production et à la caractérisation de la « Janus » lectine RSL-MOA, décrite dans l'article "Extending Janus lectins architecture : characterization and application to protocells", à la fin du chapitre 3. Par la suite, nous avons entrepris la synthèse de néoglycoprotéines par réaction de chimie du click entre la sérum albumine bovine (BSA) fonctionnalisée par des groupements alcyne ou azido et des glycannesd'intérêt fonctionnalisés par les mêmes groupements chimiques. Nous avons ainsi synthétisé cinq néoglycoprotéines, NeoGalili, Neo6'SL, Neo3'SL, NeoNeuAc et NeoNeuGc avec un bon rendement, une grande pureté et selon des protocoles particulièrement reproductibles. Ces néoglycoprotéines nous ont permis de disposer de molécules de référence afin de définir les caractéristiques des kits que nous allions développer ensuite. En conclusion de cette partie, nous présentons le travail que nous avons publié en collaboration avec le groupe du Professeur O. Renaudet, qui représentait une extension du travail intial mené autour de la synthèse des néoglycoprotéines. L'article "Homo-and Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins" (Chapitre 2), dans lequel nous avons synthétisé des néoglycoprotéines avec des clusters de glycannes distribués sur différentes positions, ouvre la voie à l'hétérofonctionnalisation des néoglycoprotéines. Le développement du kit LEctPROFILE ciblantl'antigène Galili a été le suivant : tout d'abord, nous avons étudié la quantité optimale de lectine MOAβT/puits à immobiliser sur la plaque de microtitration et le meilleur type de plaque. Nous avons choisi initialement c une liaison d'amination médiée par l'EDC et une détection par absorbance car c'était celle qui donnait le signal le plus robuste. Ensuite, nous avons également adapté l'utilisation de la lectine MOAβT une détection par fluorescence : la sensibilité du kit est plus faible en fluorescence mais elle est beaucoup plus exploitable. En effet, la détection par fluorescence supprime l'étape de l'ajout du substrat de l'extravidine-peroxydase ce qui rend le kit moins plus simple et plus rapide d'utilisation. De plus, si la lectine MOAβT est inclue dans un kit plus large contenant plus de lectines, la fluorescence est avantageuse car certaines lectines réagissent de manière croisée avec la peroxydase. La lectine GSLIB4 a également été étudiée en fluorescence pour le développement de ce kit. Ayant une haute spécificité pour la néoglycoprotéine NeoGalili et une très bonne sensibilité, les deux lectines MOAβT et GSLIB4 peuvent être considérées comme des outils complémentaires sur le même kit : MOAβT a une forte sélectivité pour l'antigène Galili mais elle a une faible affinité pour les monosaccharides et disaccharides alors que GSLIB4 a une affinité plus élevée pour les monosaccharides et disaccharides également liés en 1,4 et 1,6. Le développement du LEctPROFILE α2-3/α2-6 a été réalisé en fluorescence à l'aide de la lectine MAAI spécifique de la liaison acide sialique α2-3 et de la lectine SNA spécifique de la liaison acide sialique α2-6. Le kit est performant en mode de liaison directe, où la quantité de liaisons glycosidiques α2-3 et α2-6 peut être exprimée en équivalents de néoglycoprotéine ou en ratio de liaisons α2-3/α2-6. En mode d'inhibition/compétition, où les résultats peuvent être exprimés en potentiel d'inhibition relative (RIP), permettant ainsi de comparer aisément l'avidité de nombreux composés glycosylés simultanément. Cette partie de reconnaissance desacide sialiques a également fait l'objet d'une autre étude menée en collaboration avec l'équipe du Dr S. Gouin et basée sur l'évaluation des IC50 d'inhibiteurs multivalents potentiels de sialidases. Ces résultats ont été publiés dans l'article " Polymers of a transition-state sialyl cation strongly Inhibit Bacterial Sialidases ", ajouté à la fin du chapitre 4. Dans l'ensemble, au vu des travaux et des résultats présentés dans cette thèse, l'objectif de développement de kits a été atteint. Cependant, plusieurs perspectives à court et à long terme doivent être envisagées pour les travaux futurs. La purification d'un plus grand nombre de lectines qui pourrait étendre le criblage à une plus grande variété de motifs glycanniques, tels que le « corefucose », qui est une autre caractéristique importante à contrôler pour la fonction effectrice des anticorps monoclonaux thérapeutiques (mAbs). En plus des néoglycoprotéines déjà disponibles chez GLYcoDiag, la synthèse de néoglycoprotéines contenant des motifs glycanniques plus complexesest aussi àenvisager. De plus, il serait très intéressant de tester le kit avec un plus grand nombre de macromolécules. Par exemple, plus de mAbs, IgG provenant d'individus sains et d'individus pathologiques à différents stades inflammatoires afin de suivre les changements de glycosylation de la maladie. Il serait également intéressant d'utiliser d'autres glycoprotéines pour poursuivre les expériences comparatives de quantification des liaisons α2-3/ α2-6 avec les kits LEctPROFILE et le MALDI-TOF/MS avec dérivation des liaisons. Le développement du kit LEctPROFILE Neu5Ac/Neu5Gc, qui a été commencé, doit être également poursuivi. L'ITN synBiocarb m'a offert l'opportunité de construire des collaborations constructives : J'ai passé un mois au CERMAV de Grenoble en janvier 2020 dans le groupe du Dr Anne Imberty. Là, comme mentionné précédemment, avec le Dr. Simona Notova, nous avons purifié la lectine recombinante MOAβT qui a été d'une grande utilité pour mes expériences sur le kit LEctPROFILE « Galili » . En outre, ce travaila fait l'objet d'une publicationau travers de la « Janus » lectine MOAβT -RSLdans l'article inclus à la fin du chapitre 3. J'ai également eu l'occasion, dans le cadre d'une glycoprotéine modèle, de comparer les performances du kit LEctPROFILE α2-3/α2-6 aux résultats obtenus parMALDI/TOF/MS avec dérivatisation des liaisons d'acide sialique, réalisé par Juvissan Aguedo à l'Académie des Sciences de Slovaquie. L'ITN synBIOcarb m'a fourni une formation scientifique de haut niveau dans le domaine de la glycobiologie synthétique, ce qui m'a aidé à acquérir les connaissances nécessaires pour poursuivre mon programme de doctorat. De plus, le volet formationde cet ITN nous a permis de suivrede nombreuses formations en communication scientifique : nous avons eu l'occasion d'améliorer nos compétences en matière de présentation et d'écriture et nous avons eu la chance d'apprendre les bases de la réalisation de vidéos et de faire une vidéo parlant de notre projet à un public profane. Le déclenchement de la pandémie de Covidétait quelque chose de complètement inattendu et nouveau qui a eu un grand impact sur la vie des gens dans le monde entier et, naturellement, sur nous aussi en tant que jeunes scientifiques. Malheureusement, les réunions semestrielles de SynBIOcarb que nous avions prévues ont été réduites àtrois et de nombreux congrès auxquels je devais participer ont été annulés. Malgré tout, nous avons reçu une formation à distance et j'ai appris à faire des présentations en ligne pour plusieurs réunions et conférences. Une fois que les voyages ont été à nouveau autorisés, j'ai eu la chance d'assister à des conférences pour présenter mes travaux (voir la liste ci-dessous). Enfin, le fait d'être dans un programme de doctorat industriel m'a donné l'occasion de considérer mes recherches d'une manière plus pragmatique et applicable et m'a donné la chance de m'engager dans de multiples collaborations qui ont abouti à des publications (voir ci-dessous).

  melanomas, show an increase of Neu5Gc and Neu5Gc antibodies. The LEctPROFILE kits focused on sialic acid can give a discrimination between the two linkages or between Neu5Ac and Neu5Gc in order to provide a semi-quantification and have a rapid tool for potential biomarker discovery. Key words: lectins, lectin array, biotherapeutics, glycobiology, Galili antigen, sialic acid, neoglycoproteins GLYcoDiag, 2 Rue du Cristal, 40100 Orléans

Table 3 .

 3 1 MOA βT characteristics. __________________________________________________________ 66 Table 3. 2 BC50 and Bmax calculated for MOAβT based on the experimental curves shown in Fig 3.13. ____________________________________________________________________________________ 71 Table 3. 3 Characteristics of mAbs. _________________________________________________________ 79 Table 3. 4 Calculation of Cetuximab equivalents of neoglycoprotein based on the standard curves in Annex I. _________________________________________________________________________________ 80

Table 4 .

 4 1 IC50 and RIP values calculated for all inhibitors tested with the lectins SNA and MAAI. The calculation is based on the inhibition curves in Fig.4.16 and 4.17. ______________________________ 131

Table 4 .

 4

2 Characteristics of glycoproteins considered in the study.

____________________________ 134

Table 4 .

 4 3 Equivalents of neoglycoprotein and ratio α2,3/ α2,6 on Fetuin, calculated at the maximum concentration based on the standard curve Fig. 4.18. ________________________________________ Table 4. 4.Summary of glycosidic linkages relative abundances and ratio α2,6/ α2,3 on Fetuin. ___ Table 4. 5 Equivalents of neoglycoprotein and ratio α2,3/ α2,6 on Transferrin, calculated at the maximum concentration based on the standard curve Fig. 4.18. _______________________________ Table 4. 6 Comparison of equivalents of neoglycoprotein and ratio α2,3/ α2,6 on Transferrin native and denatured, calculated at the maximum concentration based on the standard curve Fig. 4.18. _ Table 4. 7 Summary of glycosidic linkages relative abundances and ratio α2,6/ α2,3 on Transferrin.141 Table 4. 8 Equivalents of neoglycoprotein and ratio α2,3/ α2,6 on Transferrin, calculated at the maximum concentration based on the standard curve Fig. 4.18. _______________________________ Table 4. 9 Summary of glycosidic linkages relative abundances and ratio α2,6/ α2,3 on both Porcine and Bovine Thyroglobulin. ________________________________________________________________ Table 4. 10 Sample 1, 2, 3, 4 obtained by EFS and purified on Protein A. The concentration of purified IgG was estimated by Bradford Protein Assay. ______________________________________________ Table 4. 11 Quantity of α2,3 sialic acid and α2,6 sialic acid expressed in equivalents of negolycoproteins. The calculation is made only for the highest IgG experimental concentration. ___ Table

Chapter 1 -Introduction and thesis objective

  Description du projet et structure de la thèse La glycobiologie synthétique est un domaine en plein essor visant l'ingénierie et la reconception des glycanes, des enzymes actives sur les glycanes et des protéines de liaison aux glycanes pour la recherche fondamentale, la biomédecine et la biotechnologie. Ma thèse

	de doctorat a été réalisée dans le cadre du réseau de formation innovant (ITN) synBIOcarb
	(https://synbiocarb.science/) faisant partie du programme de recherche et d'innovation Les chapitres 2, 3 et 4 contiennent les principaux résultats et sont conçus pour être lus
	Horizon 2020 de l'Union européenne, sous la convention de subvention Marie Skłodowska indépendamment les uns des autres: chacun a son propre résumé, son introduction, ses
	Curie n° 814029. SynBIOcarb a commencé en 2019 et c'est un consortium de bénéficiaires résultats, sa discussion et sa conclusion.
	académiques et industriels européens où 15 doctorants, appelés Early Stage Researchers Le chapitre 2 est le prélude aux chapitres 3 et 4 car il contient les résultats concernant la
	(ESR), ont été formés. Dans le réseau, il y a d'autres partenaires, dans le but de contribuer synthèse des néoglycoprotéines qui, avec les lectines, sont les principaux outils pour le
	au consortium, en donnant aux étudiants le soutien adéquat requis et la possibilité d'effectuer développement des kits. À la fin de ce chapitre est joint le document rédigé en collaboration

6 1.1.1 des détachements. Sur la carte de la Fig. 1.1, sont mis en évidence les pays européens qui participent au RIT synBIOcarb et une liste des bénéficiaires et des partenaires du réseau ; en rouge sont mis en évidence nos principaux collaborateurs. SynBIOcarb est un réseau multidisciplinaire et chacun des participants possède une grande expertise en chimie, biochimie, biologie moléculaire et ingénierie des protéines, biologie structurale, biologie cellulaire, outils bioanalytiques. L'objectif final commun est le développement et l'exploitation de la glycobiologie synthétique pour le diagnostic et l'administration ciblée de médicaments grâce à la mise en place de collaborations. SynBIOcarb est organisé en 4 workpackages, présentés schématiquement dans la Fig. 1.2 . Ma thèse fait partie du workpackage 3 et s'est déroulée au sein de la société GLYcoDiag avec l'Université d'Orléans comme institution partenaire. GLYcoDiag a été fondée par le Dr. Ludovic Landemarre en 2005 et est une société spécialisée dans la glycoanalyse ; la première à lancer la recherche, l'application et les services de lectin array en France. Dans mon projet de thèse, la méthode déjà établie basée sur les réseaux de lectines appelée technologie GLYcoPROFILE® [1] (décrite dans le paragraphe 1.4.1) a été adaptée pour servir l'objectif de la thèse qui est de construire un kit de lectines pour la détection des principales structures glycaniques indésirables trouvées principalement sur les glycoprotéines biothérapeutiques. L'objectif est de permettre le suivi et le contrôle de qualité en temps réel de ces produits biothérapeutiques, mais aussi d'autres applications potentielles (contrôle de qualité des vaccins, diagnostics). La recherche a été principalement axée sur deux glycanes : l'antigène Galili et l'acide sialique. Étant donné le large éventail de possibilités offertes par ce domaine, mon projet s'est élargi au cours des trois dernières années. Il est devenu intéressant pour nous d'imaginer de nouveaux domaines autres que le contrôle de qualité, dans lesquels un kit de lectine rapide pourrait s'avérer d'une grande aide pour la détection des glycanes cibles. Pour aider le lecteur à s'orienter dans cette thèse, nous décrivons ici brièvement sa structure: Le chapitre 1 est une introduction générale sur le sujet, particulièrement axée sur l'étude des glycanes et des interactions lectine-glycane et sur l'importance du contrôle rapide de la qualité des produits biothérapeutiques. avec le Dr Goyard et le professeur Renaudet de l'Université de Grenoble-Alpes : "Homo-et Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins". Le chapitre 3 est entièrement consacré à l'antigène Galili. À la fin de ce chapitre est joint l'article rédigé en collaboration avec le Dr Simona Notova du Cermav à Grenoble : "Extending Janus lectins architecture : characterization and application to protocells". Le chapitre 4 est entièrement consacré à l'acide sialique. À la fin de ce chapitre est joint l'article rédigé en collaboration avec le Dr Gouin de l'Université de Nantes: " Polymers of a transition-state sialyl cation strongly Inhibit Bacterial Sialidases ". Le chapitre 5 est consacré aux matériaux et méthodes et il est commun à toutes les expériences car les techniques utilisées sont les mêmes pour les trois chapitres de résultats. Le chapitre 6 est un bref résumé des principales réalisations de la thèse et des perspectives d'avenir. Le travail expérimental peut être divisé en quatre grandes parties qui, mises ensemble, ont permis d'atteindre l'objectif final de construire les kits de lectines. Le flux de travail est représenté dans la Fig. 1.3. Les quatre étapes sont : 1. Purification de lectines hautement spécifiques. Cette partie du travail avait pour but de cibler avant tout les lectines qui sont spécifiques aux glycanes d'intérêt. Les lectines sont soit recombinantes, soit de source naturelle. Concernant les lectines recombinantes, la première qui a été purifiée est MOAβT et cela a été fait en collaboration avec le Dr Simona Notova du réseau synBIOcarb, lors de mon détachement au Cermav-CNRS de Grenoble en janvier Chapter 1 -Introduction and thesis objective 8 2020. L'autre recombinant est le HPyL spécifique du Neu5Gc, purifié chez GLYcoDiag. Concernant les lectines naturelles, la lectine MAAI a été purifiée par mes soins, et la lectine SNA a été purifiée au GLYcoDiag avant mon arrivée.

Table 3 .

 3 1 MOA βT characteristics.

Clinical use Production Type of glycans based on literature

				TNF-α with	Psoriatic
				high affinity	arthritis
				Binds to	Treatment of	Chinese	G0, G1F,
				protein CD20,	patients with	Hamster	G2F, low
				on the	stage III-IV	Ovary cells	levels of
				surface of	follicular		high
				immune	lymphoma		mannose
	Rituximab	Rituxan		system B cells	who are chemoresistan		and NANA sialic acid
			145 kDa	triggering cell	t or are in their		are
				death	second or		detected
					subsequent		as well [71]
					relapse after
					chemotherapy
							G0F, G1F,
							G2, high
							mannose,
							hybrid
							structures,
					Treatment of		Low levels
			152 kDa	EGFR	metastatic	Murine	of G2Fgal1
	Cetuximab	Erbitux	including	inhibitor	colorectal	myeloma	(Asn299
			carbohydra	(Paragraph	cancer and	cells	Fc
			tes	3.2.1)	head and neck		domain),
					cancer		three
							glycan
							species
							with NGNA
							(Asn88 Fd
							domain)
							[71].
				Inhibition of	Chron's		M3G0F,
				the pro-	desease,		M5, G1,
	Infliximab	Remicade		inflammatory cascade	Rheumatoid Arthritis,	Mouse myeloma	G1F, M6, G2, G2F
			149 kDa	signalling via	Ankylosing	cells (SP2/0	[88]
				binding to	Spondylitis,	cells)

Table 3 . 3

 33 Characteristics of mAbs.

Table 3 .

 3

4

Calculation of Cetuximab equivalents of neoglycoprotein based on the standard curves in Annex I.

Table 4 .

 4 1 IC50 and RIP values calculated for all inhibitors tested with the lectins SNA and MAAI. The calculation is based on the inhibition curves in Fig.4.16 and 4.17.

		IC50	RIP	Compound	IC50 (µM)	RIP
	6'SL	200	1	3'SL	500	1
	Neo6'SL	0.6	333	Neo3'SL	0.6	833
	Fetuin	0.15	1333	Fetuin	1.12

Table 4 . 2

 42 Characteristics of glycoproteins considered in the study.

Table 4 . 3

 43 Equivalents of neoglycoprotein and ratio α2-6:α2-3 on fetuin, calculated at the maximum concentration based on the standard curve Fig.4.21.

Table 4 .

 4 4.Summary of glycosidic linkages relative abundances and ratio α2-6:α2-3 on calculated according to MALDI-TOF/MS with linkages derivatization.

3:α2-6

	Glycoprotein	n (µg/mL)		(µg/mL)
			SNA	MAAI
	Transferrin native	40	0.42	0.016	99:1
	Transferrin denatured	40	1.4	1.1	56:44

Table 4 . 6

 46 Comparison of equivalents of neoglycoprotein and ratio α2-6:α2-3 on transferrin native and denatured, calculated at the maximum concentration based on the standard curve Fig.4.21.

Table 4 . 7

 47 Summary of glycosidic linkages relative abundances and ratio α2-6:α2-3 on transferrin calculated according to MALDI-TOF/MS with linkages derivatization.

	Linkage	Relative abundance %	Ratio α2-6:α2-3
	α2-6 sialyllactose	56.6
			65:35
	α2-3 sialyllactose	29.83
	α2-6 + α2-3 linkage	80.7

Table 4 . 8

 48 Equivalents of neoglycoprotein and ratio α2-6:α2-3 on both thyroglobulins, calculated at the maximum concentration based on the standard curve Fig.4.21.

Polymers of a transition-state sialyl cation strongly Inhibit Bacterial Sialidases

  D'autre part, le développement du kit α2-3/α2-6 LEctPROFILE a été efficace et nous avons obtenu une vue complète. Selon le mode de réalisation du test, les résultats peuvent être exprimés différemment. Dans le mode de liaison directe, la quantité des liaisons glycosidiques α2-3 et α2-6 peut être exprimée en équivalents de néoglycoprotéine ou en rapport α2-3/α2-6 alors que dans le mode d'inhibition, les résultats peuvent être exprimés en RIP. Ces caractéristiques déterminent ensemble la valeur de l'échantillon et permettent d'évaluer des composés inconnus. L'avantage de cette méthodologie est le fait que les glycanes n'ont pas besoin d'être détachés de l'échantillon et, en inhibition, l'échantillon n'a même pas besoin d'être étiqueté.Un tel outil peut élucider non seulement les liaisons α2-3/α2-6, mais en incluant plus de lectines dans le test, d'autres structures de glycanes peuvent être élucidées avec des exigences minimales en matière d'échantillons et sans étapes supplémentaires de préparation des échantillons. Un exemple est le GLYcoPROFILE de cellules, qui peut être appliqué soit pour la détection de parties de glycanes dans le contrôle de qualité en cours de fabrication de produits biothérapeutiques, soit dans la recherche sur le cancer pour la découverte de biomarqueurs.

	GNA WFA Paper 3 -functionality of the neoglycoproteins was checked by GLYcoPROFILE in direct binding after labelling Galanthus nivalis Terminal mannoses. Mana1-3Man ; α2-macroglobulin ; mannopentaose (Forssman antigen) > GalNAc with biotin.
				Galβ1-4GlcNAcβ1-2Man, the bisecting
	PHA E GSLIB4	isoB4 Phaseolus vulgaris Griffonia simplicifolia	GlcNAcb1-4Man is essential α-Gal
			Phaseolus vulgaris	Galβ1-4GlcNAcb1-6Man of branched
	PHA L MOAβT	part) agglutinin (β-Trefoil Marasmius oreades	structures of N-glycans, Galb1-4GlcNAcb1-2Man Gal-α-1,3-Gal
			Arachis hypogaea
		PNA	Datura stramonium	Lactose, T-antigen GlcNAcβ4GlcNAc oligomers, Galb4GlcNAc
		DSA
			Pisum sativum	Man > Glc ; enhanced by Fuca1-6 on the core
		PSA		GlcNAc-Asn N-glycopeptides, IgM1A mouse
			Dolichos biflorus	Terminal GalNAc, GalNAca3GalNAc
		DBA		(Forssman), blood group A trisaccharide
			Sambucus nigra	Neu5Acα2-6Gal
	Annex II	SNA
		GSLII	Griffonia simplicifolia	Terminal GlcNAc
	Short Name MAAI BC2LA	Maackia amurensis Common name	Neu5Acα2-3Ga Glycans structures specificity Manα1-2, Manα1-3, Manα1-6, dimanoside
			Burkholderia
			cenocepacia lectin A
	AIA HPyL WGA	Artocarpus intergrifolia Human Polyomavirus 9 VP1 Triticum vulgare	Galα1-6 or Galβ1-3GalNAc (T-antigen)>> lactose Neu5Gc GlcNAc; GlcNAcβ4 oligomers, core of Asn linked oligasacchide; Neu5Ac
	Con A UEAI	Canavalia ensiformis Ulex europeus	Man > Glc ; branched mannoses Fucose
			Wisteria floribunda	GalNAc ; GalNAca6Gal > GalNAcα3GalNAc

En outre, nous avons testé des IgG provenant de sérum humain. Il serait très intéressant de poursuivre ces études et de tester les IgG de personnes atteintes de pathologies telles que la polyarthrite rhumatoïde à différents stades ou afin de surveiller les changements de glycosylation de la maladie. En outre, un plus grand nombre de patients serait nécessaire afin d'identifier un modèle répétitif. Parallèlement, il serait également intéressant de tester la transferrine provenant de patients alcooliques pour voir les différences de sialylation avec les individus sains. Toutes ces informations pourraient permettre le dépistage d'un grand nombre d'échantillons en peu de temps.

  protein concentration is determined by Bradford Protein Assay. This test is based on the on a colour change of Coomassie Blue reagent (from BioRad) when it reacts with proteins. It gives a blue colour, which has a maximum of absorbance between 465 and 595 nm. The colour intensity is directly proportional to the protein concentration. Briefly, the test was performed in a flat bottom microtiter plate. The samples were diluted in PBS directly on the microplates in serial dilutions. The standard curve was prepared in triplicate, using a BSA solution prepared at 100 µg/ml in PBS. 50 μL of Coomassie Blue reagent is added to the plate, immediately followed with measurement of absorbance at 595 nm with a microplate reader. The concentration of sample was calculated by reporting the absorbance value from the linear equation of the standard curve.

	5.4	Sodium	dodecyl	sulphate-polyacrylamide	gel	electrophoresis	(SDS-
	PAGEelectrophoresis)
	Proteins were controlled for their purity by SDS-PAGE electrophoresis using stacking gel (4%, w/v)
	and running gel (14%, w/v) in Tris/glycine buffer. The samples were diluted (4:1) in Laemmli sample
	buffer, boiled at 96 °C for 5 min and loaded into the wells. Electrophoresis was conducted using Bio-
	Rad Mini-PROTEAN® Electrophoresis System at constant voltage (200 V). All samples sizes were
	compared to Thermo Scientific PageRuler Plus Pre-stained Protein Ladder. Protein were fixed and
	visualized on the gel by incubating for approximately 30 min in a staining solution of Coomassie

  GraphPad Prism software (San Diego, CA, USA) was used for data analysis and graphing. The data presented are mean values of experiments performed in triplicate or duplicate. If the standard deviation did not exceed the 10% of the mean value, error bars are not shown in the graphs.5.6 Mammalian cells culture and GLYcoPROFILE® of cellsAll the steps before the labelling of cells and GLYcoPROFILE were performed in sterile Before reaching the confluence, the cells were harvested using Tripsin/EDTA 1X and after harvesting, cells were washed in PBS pH 7.4 without cations, collected by centrifugation and resuspended in PBS pH 7.4 without cations. The cells were counted using the Trypan blue 0.4% , dye exclusion method. To perform the cells count, the Malassez counting chamber was used. After counting, all cells were diluted to 2x10 6 cells/mL and labelled with CFSE at 40 μM for 15 min at 37

	conditions under a laminar flow hood. All reagents for culture cells (media and reagents) were
	purchased from Lonza. CFSE (Carboxyfluoresceinsuccinimidyl ester) for labelling of the cells was
	purchased from Sigma Aldrich. CHO K1 were obtained from Promogene and B16 were obtained
	from LGC Standard.
	5.6.1 CHO K1
	Adherent CHO K1 cells were grown in medium RPMI 1640 + L-glutamine 2mM + heat
	inactivated foetal bovine serum 5% + antibiotics. They were kept in the incubator at 37 °C with 5%
	of CO2.

Acknowledgments

Acknowledgments

According to the SDS-PAGE in Fig. 4. [START_REF] Dumont | Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives[END_REF], the IgGs coming from there four samples after elution are relatively pure due to the presence of two main bands corresponding to the heavy and light chains, respectively at around 55 kDa and 25 kDa. In Table 4.11 the quantity of α2-3 sialyllactose α2-6 sialyllactose sialic acid is expressed in equivalents of neoglycoprotein, and the ratio α2-6:α2-3 is calculated. Calculations are based on the standard curve (Fig. 4.21).

Sample

Equivalent of neoglycoprotein (µg/mL)

Ratio α2-6:α2-3 From this data it is difficult to understand whether the significant glycosylation change on the IgG of sample 4 is due to the disease, especially because we do not have data for this patient in healthy

Conclusion

The main objective of this chapter was to build a sialic acid-targeted LEctPROFILE kits and more specifically, one for the recognition of Neu5Ac and Neu5Gc and the other for the discrimination of α2-3/α2-6 linkages. The first one mentioned only underwent preliminary studies, thus future investigations are required to establish all the required parameters and to verify the feasibility of the assay.

On the other side, the development of the α2-3/α2-6 LEctPROFILE kit was efficient and we have obtained a complete view. Depending of the mode in which the assay is performed, results can be expressed differently. In the direct binding mode, the quantity of the glycosidic α2-3 and α2-6 linkages can be expressed as equivalents of neoglycoprotein or as ratio α2-3/α2-6 whereas in the inhibition mode the results can be expressed as RIP. These characteristics together determine the value of the sample and allow to evaluate unknown compounds. The advantage of this methodology is the fact that the glycans do not need to be detached from the sample and, in inhibition, the sample doesn't even need to be labelled.

Such a tool can elucidate not only α2-3/α2-6 linkages, but including more lectins in the assay, other glycan structures can be elucidated with minimal sample requirements and without additional sample preparation steps. One example is the GLYcoPROFILE of cells, that can be applied either for the detection of glycan moieties in the in-process quality control of biotherapeutics, either in cancer research for biomarker discovery.

Moreover, we have tested IgG from human serum. It would be of great interest to further these studies and test IgG from individuals with pathologies like Rheumatoid arthritis at different stages or in order to monitor the glycosylation changes of the disease. In addition, a bigger pool of patients would be needed in order to identify a repetitive pattern. Along with this it would be also interesting to test transferrin coming from alcohol abusive patients to see the sialylation differences with healthy individuals. All this information could allow the screening of a large number of samples in a short time.

L'objectif principal de ce chapitre était de construire des kits LEctPROFILE ciblés sur les acides

MPA

Maclura pomifera

Galb3GalNAc (T antigen), Gala6Glc

ACA

Amaranthus caudatus Galb3GalNAca-O-R (T-antigen)

Table 4. 12 List of all lectins used through this thesis and corresponding specificities.

Chapter 5 -Materials and methods

Materials and methods

The ultrapure water, purified through the Aqua Service Distribution (ASD) water purification system, was used for all solutions and buffer preparation. The buffer were filtered at 0.2 µm (Millipore filters) using a filtration system and those used for mammalian cells culture were sterilised by autoclaving (autoclave Vapour line, VWR) prior to use. When not otherwise specified, PBS (Phosphate buffer saline) pH 7.4 was supplemented with cations CaCl2 1M, and MgCl2 0.5 M. was purchased from H2B/Seqens. Sephadex used for the purification of neoglycoproteins was obtained from Cytiva.

5.1.1Mass spectrometry

(MALDI-TOF/MS) analysis was recorded on an UltrafleXtreme from Bruker by The Facility for Mass Spectrometry and Proteomics at the Centre for Molecular Biophysics (CBM) in Orléans.

Functionalization of BSA with an alkyne or azido group

A solution of Propargyl-NHS ester or Azido-NHS ester (60 equivalents) was added to a solution of BSA in PBS (50 mM, pH 8). The reaction mixture was stirred for 5 h at room temperature.

BSA-alkyne or BSA-azide was purified on a Sephadex G25 and its purity was controlled by SDS-PAGE. The protein concentration was determined by Bradford. The number of alkyne or azido group per BSA was determined by MALDI-TOF/MS. Mots clés : lectines, lectin array, biothérapies, glycobiologie, antigène Galili, acide sialique, néoglycoprotéines

Synthesis of neoglycoprotein by click chemistry