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La survenue d'anticorps neutralisant le facteur VIII de la coagulation (FVIII) chez les patients hémophiles A constitue un échec thérapeutique majeur. Si les étapes effectrices de la réponse immunitaire, la nature des cellules immunitaires impliquées et les propriétés des anticorps ont été largement documentées, les étapes précoces de la réponse immunitaire restent mal connues et constituent l'objet de ma thèse. La première partie de ma thèse aborde le rôle du microenvironnement rencontré par le FVIII une fois administré in vivo, notamment le rôle des molécules du complément. Acteur majeur de l'immunité innée, le système du complément participe également aux réponses immunitaires adaptatives et peut interagir avec la cascade de la coagulation. Alors que le complément est impliqué dans les réponses immunitaires contre des agents pathogènes, son implication dans les réponses immunitaires dirigées contre les protéines thérapeutiques n'a jamais été décrite jusqu'à présent. Je me suis donc intéressé au potentiel rôle des molécules du complément dans l'immunogénicité du FVIII thérapeutique.La seconde partie de ma thèse aborde l'implication de la structure du FVIII pour sa reconnaissance par le système immunitaire. Ainsi, a-t-il été démontré l'importance du domaine C1 du FVIII pour sa prise en charge par les cellules du système immunitaire in vitro et pour son immunogénicité in vivo. En raison des fortes homologies de structure entre les domaines C1 et C2, je me suis intéressé au rôle potentiel du domaine C2 dans la capture du FVIII par les cellules présentatrices d'antigènes et l'élaboration de la réponse immunitaire anti-FVIII. / Occurrence of pro-coagulant factor VIII (FVIII) neutralizing antibodies in hemophilia A patients constitutes a major therapeutic failure. While the effector steps of the immune response, the nature of the involved immune cells and the antibodies properties have been well-reported, the early stages of the immune response are poorly described and constitute the topic of my PhD thesis. The first part of my thesis deals with the role of the microenvironment encountered by FVIII once administered in vivo, especially the role of complement system molecules. While complement system is a major actor of the innate immunity, it also participates to adaptive immune responses and can interact with the coagulation cascade. Though complement is involved in immune responses against pathogens, its contribution to immune responses against therapeutic proteins has never been reported so far. Therefore I have investigated the potential role of the complement system in the immunogenicity of therapeutic FVIII. The second part of my thesis focuses on the involvement of FVIII structure for its recognition by the immune system. It has been demonstrated that FVIII C1 domain plays a major role of its uptake by immune cells in vitro and its immunogenicity in vivo. Because of strong structural homologies between C1 and C2 domains, I investigated the potential role of FVIII C2 domain for its endocytosis by antigen presenting cells and the elicitation of the anti-FVIII immune response.

Proteins, participating in a myriad of biological function, are at the core of all cellular activities occurring within living organisms. Therapeutic proteins, hence constitute a major part of the pharmaceutical industry. The glycoprotein hormones follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH) and human chorionic gonadotropin (CG) regulate various reproductive and metabolic functions in humans and hence have high therapeutic potentials. The increasing demand of recombinant proteins for therapeutic uses drives the development of better expression systems. The methylotrophic yeast Pichia pastoris, has been termed as an industrial workhorse for heterologous protein expression. However, the N-glycosylation in yeast is of the high mannose type, resulting in a reduced serum half-life of the recombinant proteins. In the current work, we have re-engineered the Pichia N-glycosylation pathway to mimic the human type of N-glycosylation. Towards this end, we abolished the yeast native N-glycosylation and introduced enzymes from various eukaryotic sources into the system. These modifications resulted in the conversion of the yeast Man9-20GlcNAc2 glycan structure to a more human like GlcNAc2Man3GlcNAc2 form on over 70 % of the heterologous expressed proteins. In order to demonstrate the application of these strains as efficient protein expression hosts, the glycoengineerd Pichia was used for large scale expression of the glycoprotein hormones, hCG and FSH. The purified recombinant hormones were found to have binding affinities and structure similar to that of the natural hormones. These recombinant hormones were also able to elicit over two fold responses in animal models compared to buffer controls and the activity was comparable to the natural counterparts. Thus, we report the generation of a glycoengineered Pichia pastoris, which can be considered as a serious contender for the expression of glycosylated proteins of therapeutic importance.

<p>The main aim of the thesis was to explore the potential of supercritical fluid (SF) techniques in the field of drug delivery. In particular, the relatively recently developed solution-enhanced dispersion by supercritical fluids (SEDS) technology has been employed in the preparation of particles/powders. </p><p>The manufacturing, stability and bioavailability of a dosage form strongly depend on the physicochemical properties of the formulation particles. For example, dry powder inhalation (DPI) for administering drugs to the respiratory tract require particles in a narrow size range (1-5 μm) to be effective. The identification of polymorphs and control of purity are also important issues since the physicochemical properties and therapeutic effects of the alternative forms of a drug may differ substantially. Solvent-based traditional crystallisation processes provide the product that may require further down-stream processing to obtain particles for advanced drug delivery applications. This can result in unwanted changes in the physicochemical properties of the particles and thus affect the performance of the dosage form. SF processing has addressed many of the challenges in particle formation research. Among several SF technologies developed for particle processing over the last decade, the SEDS process with its specially designed co-axial nozzle with mixing chamber has resulted in improved control over the particle formation process. Carbon dioxide (CO₂) was used as the SF, because it has low critical points and is non-toxic, non-flammable and relatively inexpensive. </p><p>The initial part of the thesis concerns the formation of particles of model drugs such as hydrocortisone, budesonide and flunisolide using SEDS technology and the determination of the influence of processing conditions and solvents on particle characteristics such as size, shape and crystal structure. Particles of model drugs of differing shapes in a size range suitable for inhalation delivery were prepared. In the process, two new polymorphic forms of flunisolide were identified. This was the first report of SEDS technology being shown as a polymorph-screening tool. The remainder of the thesis deals with the development of SEDS technology for precipitating therapeutic proteins such as recombinant human growth hormone (hGH) from aqueous solutions. Powders of hGH were precipitated using SEDS without significant changes in the chemical or physical stability of the protein. The addition of sucrose to hGH in the feed solution promoted precipitation and minimised the detrimental effects of the solvent and/or the process on the physical aggregation of the protein. </p><p>In conclusion, this thesis highlights the applicability of the SEDS process in drug delivery research and advances general understanding of the particle formation phenomenon. The SEDS process may also prove to be a potential alternative technology for the precipitation of stable powders of therapeutic proteins.</p>

The main aim of the thesis was to explore the potential of supercritical fluid (SF) techniques in the field of drug delivery. In particular, the relatively recently developed solution-enhanced dispersion by supercritical fluids (SEDS) technology has been employed in the preparation of particles/powders. The manufacturing, stability and bioavailability of a dosage form strongly depend on the physicochemical properties of the formulation particles. For example, dry powder inhalation (DPI) for administering drugs to the respiratory tract require particles in a narrow size range (1-5 μm) to be effective. The identification of polymorphs and control of purity are also important issues since the physicochemical properties and therapeutic effects of the alternative forms of a drug may differ substantially. Solvent-based traditional crystallisation processes provide the product that may require further down-stream processing to obtain particles for advanced drug delivery applications. This can result in unwanted changes in the physicochemical properties of the particles and thus affect the performance of the dosage form. SF processing has addressed many of the challenges in particle formation research. Among several SF technologies developed for particle processing over the last decade, the SEDS process with its specially designed co-axial nozzle with mixing chamber has resulted in improved control over the particle formation process. Carbon dioxide (CO2) was used as the SF, because it has low critical points and is non-toxic, non-flammable and relatively inexpensive. The initial part of the thesis concerns the formation of particles of model drugs such as hydrocortisone, budesonide and flunisolide using SEDS technology and the determination of the influence of processing conditions and solvents on particle characteristics such as size, shape and crystal structure. Particles of model drugs of differing shapes in a size range suitable for inhalation delivery were prepared. In the process, two new polymorphic forms of flunisolide were identified. This was the first report of SEDS technology being shown as a polymorph-screening tool. The remainder of the thesis deals with the development of SEDS technology for precipitating therapeutic proteins such as recombinant human growth hormone (hGH) from aqueous solutions. Powders of hGH were precipitated using SEDS without significant changes in the chemical or physical stability of the protein. The addition of sucrose to hGH in the feed solution promoted precipitation and minimised the detrimental effects of the solvent and/or the process on the physical aggregation of the protein. In conclusion, this thesis highlights the applicability of the SEDS process in drug delivery research and advances general understanding of the particle formation phenomenon. The SEDS process may also prove to be a potential alternative technology for the precipitation of stable powders of therapeutic proteins.