Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-10-06T18:08:45Z No. of bitstreams: 1 DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2017-11-28T12:14:39Z (GMT) No. of bitstreams: 1 DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2017-11-28T12:14:55Z (GMT) No. of bitstreams: 1 DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) / Made available in DSpace on 2017-11-28T12:26:25Z (GMT). No. of bitstreams: 1 DissACP.pdf: 6605106 bytes, checksum: c63dcdec106579e271edaa4dc5a8bdcf (MD5) Previous issue date: 2017-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nowadays, cancer is one of the most feared diseases, affecting each day more and more people worldwide. The importance of new cancer treatment researches is very clear since the ones that has been used are not very effective and may lead to drug resistance, implying in a constant dose increasing which can lead to toxicity issues. Collateral effects and the instability generated in the patient’s organism are also reasons why the necessity of discovering new cancer treatments is imminent. A treatment alternative that has aroused interest is the use of monoclonal antibodies as immunotherapics, since they act by stimulating the patient’s immune system neutralizing the tumor cells in a very efficient and specific way. This kind of antibody can be produced by culturing hybrid animal cells, better known as hybridoma, under strictly controlled conditions so they can be studied and used in human beings. For this reason, the major goal of this project was the production of murine monoclonal antibodies using hybridoma cell culture in order to stablish an efficient culture methodology for hybridomas PC-61 or DTA1 producers of monoclonal antibodies anti-CD25 and anti-GITR, respectively, with high quality and enough amounts using Fetal Bovine Serum (FBS) free medium to, in the future, carry out animal model studies of their potential as therapeutic agents for cancer treatment. Both hybridomas were cultivated on a small scale with RPMI medium and addition of SFB, for comparative purposes and only one was selcted for the second step. The sequential adaptation methodology test, consisted in a gradual percent’s reduction of medium with serum at the same time that increase the percentage of commercial medium without serum, and was selected the medium without SFB in which the hybridoma was better adapted. After was carried out on a laboratory scale in a system type spinner flask (500 ml) with the commercial medium selected in the previous step, in controlled conditions of temperature (37 ° C) and pH (7.2). Based on analyzes of cell culture results, amino acid consumption and monoclonal antibodies quantification , SFM commercial medium SFB-free provided better results for culturing the PC-61 hybridoma, allowing the pilot scale culture reached even higher cell densities than in the standard medium with addition of FBS. / O câncer é uma das doenças mais temidas da atualidade, e atinge cada vez mais pessoas em todo o mundo. A importância de pesquisas sobre novos tratamentos na luta contra o câncer é clara e consensual, uma vez que os que vem sendo utilizados, não são muito eficientes, causam resistência à medicação utilizada o que implica na utilização de doses crescentes que por sua vez podem gerar problemas de toxicidade. Os efeitos colaterais e a instabilidade gerada no organismo do paciente, também são fatores da necessidade de pesquisar novos caminhos para o tratamento do câncer. Uma das alternativas de tratamento que tem despertado interesse é a utilização de anticorpos monoclonais (mAbs) como imunoterápicos, os quais agem estimulando o próprio sistema imune do paciente neutralizando a ação das células tumorais de forma eficiente e específica. A produção de tais anticorpos pode ser feita mediante o cultivo de células animais híbridas, mais conhecidas como hibridomas, sobre condições estritamente controladas para que possam ser estudados e utilizados em humanos. Por essa razão definiu-se como objetivo desse trabalho a produção anticorpos monoclonais murinos por meio de cultivo de hibridomas com a finalidade de estabelecer uma metodologia eficiente de cultivo dos hibridomas PC-61 ou DTA1 secretores dos mAbs anti-CD25 e antiGITR respectivamente, com qualidade e em quantidades suficientes utilizando meios livres de soro fetal bovino (SFB), para a seguir efetuar estudos em modelo animal de seu potencial como agentes terapêuticos no tratamento de câncer. Os dois hibridomas foram cultivados em pequena escala utilizando meio RPMI-1640 e adição de SFB, para fins comparativos e somente um foi selecionado para a próxima etapa. O teste da metodologia de adaptação sequencial, onde houve a redução gradativa da porcentagem de meio RPMI-1640 com 10% de SFB e o aumento da porcentagem de meio comercial sem SFB, e foi selecionado o meio livre de SFB em que o hibridoma melhor se adaptou. Posteriormente foi realizado o cultivo do hibridoma em escala laboratorial em sistema de frasco agitado biorreator do tipo Spinner (500 mL) com o meio livre de SFB selecionado na etapa anterior, sob condições bem controladas de temperatura (37ºC), pH (7,2). Com base nas análises dos resultados dos cultivos celulares, metabolismo de aminoácidos e quantificação de mAbs, o meio comercial SFM livre de SFB proporcionou melhor resultados para o cultivo do hibridoma PC-61, permitindo que o cultivo em escala laboratorial atingisse densidades celulares ainda maiores que no meio padrão com adição de SFB. Como consequência desse vasto crescimento celular em quantidades abundantes de mAbs foram conseguidas para iniciar num futuro próximo ensaios em modelos animais.

L’activation des cellules NKCD56dim par l’engagement ou non du récepteur FγRIIIA/CD16A entraîne la perte d’expression membranaire de celui-ci par un mécanisme dépendant au moins en partie de la metalloprotéase ADAM17. Celle-ci clive le récepteur entre l’Alanine 195 et la Valine 196 et agit exclusivement en cis. La modulation d’expression du FγRIIIA/CD16A est un marqueur d’activation des cellules NK plus fortement corrélé à la dégranulation qu’à la production d’IFN-γ. L’engagement du récepteur FγRIIIA/CD16A ou le co-engagement des récepteurs activateurs des NKCD56dim induisent de manière non corrélée la dégranulation et la production d’IFN-γ. Cette dichotomie fonctionnelle varie selon les donneurs et dépend de l’expression des récepteurs inhibiteurs spécifiques des molécules du CMH-I. La production d’IFN-γ est ainsi associée à l’expression des KIRs (Killer like-Immunoglobuline Receptor) mais pas à celle du NKG2A. Une meilleure compréhension des réponses effectrices dépendantes du FγRIIIA/CD16A est importante pour améliorer l’efficacité thérapeutique des anticorps monoclonaux à visée anti-tumorale. / FγRIIIA/CD16A-dependent or independent activation of CD56dim NK cells induces down-modulation of this receptor. The mechanism partially involves the ADAM17 metalloprotease, which cleaves the FγRIIIA/CD16A between Alanine 195 and Valine 196 and acts exclusively in cis. FγRIIIA/CD16A downmodulation is a marker of NK cell activation more strongly correlated with degranulation than to IFN-γ- production. FγRIIIA/CD16A engagement or activating receptors co-engagement on CD56dim NK cell induces degranulation and IFN-γ-production, which are not correlated. This functional dichotomy depends on the donor and on the CMH-I-specific inhibitory receptor expression. IFN-γ-production is thus associated with KIRs (Killer like-Immunoglobuline Receptor) but not with NKG2A expression. Understanding the FγRIIIA/CD16Adependent functional responses is essential to improve the efficacy of monoclonal antibodies used in cancer therapy.

La pharmacocinétique (PK) des anticorps monoclonaux (mAbs) est sujette à d’importantes variations interindividuelles. Le récepteur néonatal au Fc des IgG (FcRn) et le statut immun à l’encontre de ces mAbs sont des déterminants de cette PK. La bioconjugaison des mAbs à des cytotoxiques entraîne une altération de leur PK. Nous montrons que le taux de couplage modifie l’affinité de ces espèces pour le FcRn à pH6. La proportion d’agrégats au sein des solutions d’anticorps armés augmente avec le taux de couplage et pourrait entraîner une altération de leur PK. Par ailleurs, cette agrégation est impliquée dans l’immunogénicité des mAbs, et nous avons donc cherché à identifier des acides aminés impliqués dans l’agrégation de mAbs indiqués en clinique. Il apparait que la nature biochimique de résidus des paratopes pourrait augmenter cette agrégation. Les anti-TNF- présentent très peu d'agrégats et figurent pourtant parmi les plus immunogènes chez l’Homme. Nous avons donc exploré le rôle des complexes immuns dans leur immunogénicité chez la souris. Il apparait que la présence du FcRn n’est pas à l’origine de l’immunisation contre ces mAbs, contrairement à celle des complexes immuns. Ces résultats donnent des pistes pour la production de mAbs plus efficients et mieux tolérés. / The pharmacokinetic (PK) profile of monoclonal antibodies (mAbs) shows interindividudal variability. The neonatal Fc receptor (FcRn) and the immounogenicity of these mAbs are determinative factors of mAb PK. Generation of antibody-drug-conjugates alters their PK profile. We show that the the affinity for FcRn at pH6 increases with the drug-to-mAb ratio, as does the amount of aggregates inside the mAb-drug-conjugate. The amount of aggregates could be responsible for an avidity effect towards FcRn. These aggregates are known to cause immunogenicity, so we studied biochemical determinants inside the aminoacid sequence of marketed mAbs. We show that the biochemical nature of some aminoacids inside the paratope has an impact on the amount of aggregation. Anti-TNF- mAbs show very little aggregation but are very immunogenic in humans. We studied the role of the formation of immune complexes in the immunization against anti-TNF- mAbs in mice, and showed that immune complexes, but not FcRn are essential in the immunization process against anti- TNF- mAbs. These results give leads towards the generation of more efficient, better tolerated mAbs.

Les anticorps monoclonaux ont révolutionné le traitement de nombreuses pathologies. Cependant, leur pharmacocinétique (PK) et l’influence de leur concentration sur la réponse clinique restent mal connues. Nous avons étudié les sources de variabilité interindividuelle de la PK du cetuximab, un anticorps anti- EGFR, ainsi que l’influence de l’exposition à cet anticorps sur la réponse. Nous avons validé une méthode ELISA de dosage du cetuximab. Dans un modèle murin, nous avons étudié l’absorption pulmonaire du cetuximab. Nous avons étudié la PK du cetuximab chez un patient hémodialysé. Nous avons décrit la PK du cetuximab chez des patients traités pour cancer colorectal métastatique, à l’aide d’un modèle combinant des éliminations d’ordre 0 et 1. Enfin, nous avons identifié la clairance globale du cetuximab, paramètre pouvant être estimé précocement par la concentration résiduelle à J14, comme un facteur influençant la survie sans progression des patients. Nos travaux montrent qu’une description de la PK d’un anticorps par approche compartimentale permet d’identifier les sources de variabilité et d’étudier l’impact de la PK sur la réponse clinique. / Monoclonal antibodies have profoundly modified the treatment of many diseases. However, their pharmacokinetics (PK) and the influence of their concentrations on the clinical response are poorly known. We studied the sources of the interindividual variability of PK of cetuximab, an anti-EGFR, and the influence of the exposure to this antibody on the response. We validated an ELISA technique to measure cetuximab concentrations. We studied the pulmonary absorption of cetuximab in a murine model. We studied cetuximab PK in a hemodialysed patient. In metastatic colorectal cancer patients, we described cetuximab PK with the help of a model combining zero- and first-order eliminations. Finally, we identified the global clearance of cetuximab, a parameter which can be estimated by residual concentration on day 14, as a factor influencing progression-free survival of the patients. Our work shows that the description of the PK of an antibody by compartmental approach allows to identify sources of variability and to study the impact of PK on the clinical response.

Les anticorps monoclonaux (AcM) anti-CD20 sont essentiels pour le traitement du lymphome non hodgkinien et de la leucémie lymphoïde chronique (LLC). Les AcM agissent soit en activant directement la signalisation apoptotique dans les cellules cibles, soit via le système immunitaire. Dans une étude préclinique, nous avons montré que le traitement avec AcM anti-CD20, rituximab et GA101, induit l'expression de la protéine early growth response 1 (EGR-1) (Dalle et al., 2011). EGR-1 est un facteur de transcription régulé par le calcium (Ca2+) et CD20 est impliqué dans la régulation du flux calcique transmembranaire. Nous avons donc étudié le rôle d'EGR-1 et du flux Ca2+ dans l'activité cytotoxique des AcM anti-CD20. Nous avons montré qu'EGR-1 est rapidement induit suite à l'exposition au rituximab et à GA101. La baisse de l'expression d'EGR-1 par shRNA a supprimé l'effet cytotoxique du GA101 à la fois in vitro et in vivo, indiquant qu'EGR-1 est requis pour la mort cellulaire médiée par CD20. De plus, la surexpression d'EGR-1 augmente la sensibilité au GA101 in vitro et in vivo. En outre, nos résultats indiquent que les AcM anti-CD20 induisent un flux Ca2+. Le blocage du flux Ca2+ par inhibiteurs de canaux calciques (ICC) a aboli l'induction d'EGR-1 ainsi que l'efficacité du GA101 in vivo et ex vivo dans des échantillons de LLC. Plus important, nos données indiquent que les patients recevant des ICC ont une moins bonne réponse au traitement par les AcM anti-CD20. En conclusion, nous avons identifié EGR-1 comme potentiel biomarqueur pour prédire la réponse à la thérapie anti-CD20 et démontré que les ICC ont un impact négatif sur l'efficacité des AcM anti-CD20 chez les patients / Anti-CD20 monoclonal antibodies (mAbs) are an essential component of the treatment of patients with non-Hodgkin's lymphoma and chronic lymphocytic leukemia (CLL). They mediate their antitumor effects by activating the immune system or by direct apoptotic signaling in target cells. In a previous preclinical study, we showed that treatment with anti-CD20 mAbs, rituximab and GA101, resulted in upregulated expression of early growth factor 1 (EGR-1) (Dalle et al. 2011). EGR-1 is a calcium (Ca2+) regulated transcription factor and CD20 is hypothesized to regulate transmembrane Ca2+ flux. Therefore, we aimed to assess the role of EGR-1 and Ca2+ flux in the cytotoxic activity of anti-CD20 mAbs. We have shown that EGR-1 expression is rapidly upregulated in CD20+ cells following rituximab and GA101 exposure. Decreasing EGR-1 expression by shRNA abolishes the direct cytotoxic effect of GA101 both in vitro and in vivo, indicating that EGR-1 is required for CD20-mediated cell death. Additionally, the overexpression of EGR-1 enhances the cytotoxic activity of GA101 both in vitro and in vivo. Furthermore, our results indicate that anti-CD20 mAbs induce calcium influx. Blocking the Ca2+ flux with calcium channel blockers (CCB) abolishes EGR-1 induction and impaires the GA101 efficacy in vivo and ex vivo in CLL blood samples. More importantly, our data indicate that patients receiving CCBs and anti-CD20 therapy have worst progression free survival and overall survival. In conclusion we have identified EGR-1 as a potential biomarker to predict response to anti-CD20 therapy. We demonstrated that co-treatement with CCBs negatively impacts the outcome of patients receiving anti-CD20 mAbs

Les bioprocédés industriels mettant en œuvre la culture de cellules animales sont devenus incontournables pour la production d’anticorps monoclonaux (AcM). Cependant, l'état physiologique des cellules et la qualité des AcM produits, en particulier leur glycosylation, sont tributaires des variations intervenant au cours du procédé. Il en découle des risques d'altération de l'efficacité et de la sûreté des AcM. C'est pourquoi, depuis quelques années, l'initiative Process Analytical Technology (PAT) encourage le développement du suivi en ligne de ces procédés, avec l'objectif de mieux les maîtriser et d'assurer la qualité finale des produits. Dans ce contexte, cette thèse propose des approches innovantes pour le suivi en ligne de procédés de culture de cellules CHO (Chinese Hamster Ovary) en bioréacteur, basées sur l'utilisation de trois types de spectroscopies in situ (diélectrique, Raman, proche infrarouge(NIR)). Le premier chapitre présente une nouvelle démarche permettant de prédire en temps réel l'état physiologique des cellules, au travers de la vitesse spécifique de croissance cellulaire (μ). A partir de la mesure en ligne de la permittivité grâce à la spectroscopie diélectrique, la µ a été calculée en temps réel, permettant de détecter le moment critique correspondant au moment où μ diminue. Comparée à une démarche hors ligne, l'utilisation de cette méthode pour le pilotage de cultures en mode recharge-récolte, permet d’assurer à la fois la quantité et la qualité de glycosylation des AcM. Le second chapitre aborde l'utilisation des spectroscopies NIR et Raman in situ combinées à des méthodes chimiométriques. Les performances de ces deux spectroscopies ont été comparées en parallèle. Des modèles en ligne ont été développés pour prédire la concentration de différents paramètres (cellules viables, glucose, lactate, glutamine, ions ammonium, anticorps). L'évaluation de ces modèles par facteurs de mérite (FOM), a révélé certains avantages de la spectroscopie Raman. La combinaison de ces deux spectroscopies par diverses stratégies de fusion de données a été également évaluée. Dans le troisième chapitre, l'intérêt de la spectroscopie Raman a été démontré pour le suivi en ligne, non seulement, de la concentration, mais aussi, de la glycosylation des AcM. Des modèles ont été développés pour la prédiction en ligne, à la fois, de la macro-hétérogénéité (sites de glycosylation), et de la micro-hétérogénéité (structures glycanniques) de la glycosylation des AcM dans le cas de cultures en mode discontinu et recharge-récolte. Le dernier chapitre a utilisé les spectroscopies NIR et diélectrique, en les intégrant à un « capteur logiciel » combinant des équations de bilans de matière. Ce « capteur logiciel », mis en œuvre au cours d'une culture en mode semi-continu pour le contrôle automatique du débit d'alimentation, a conduit à une augmentation de la productivité du procédé ainsi qu'à une meilleure glycosylation des AcM produits / Bioprocesses of mammalian cell culture have become essential for the production of therapeutic recombinant proteins, such as monoclonal antibodies (mAb). However, the physiological state of the cells and the quality of the mAb produced, in particular their glycosylation, may vary during the process, and may lead to the alteration of the safety and efficacy of the final product. Consequently, the Process Analytical Technology (PAT) initiative has encouraged the development of online monitoring techniques, with the aim to better control the process and ensure the quality of the final product. In this context, this thesis proposes innovative approaches for online monitoring of CHO (Chinese Hamster Ovary) cells bioreactor cultures, by using three types of in situ spectroscopic measurements (dielectric, Raman, near infrared (NIR)). The first chapter presents a novel approach to predict in real-time one of the major cell physiological state parameters, the specific growth rate (µ). Based on online permittivity measured by in situ dielectric spectroscopy, the cell concentration was estimated and µ was calculated in real-time, making possible to detect the critical moment when µ begins to decrease significantly. Compared to an offline approach, this online approach allowed to maintain the cells in a stable physiological state, ensuring the glycosylation of the mAb produced in feed-harvest cultures. The second chapter shows the use of in situ NIR and Raman spectroscopies combined with chemometric methods. For the first time, the performances of these two spectroscopies were compared in parallel in the same cultures. Online models were developed to predict in real-time the concentration of different parameters (viable cells, glucose, lactate, glutamine, ammonium ions and antibodies). The evaluation of these models by the multivariate Figures of Merit (FOM) revealed some of the advantages of Raman spectroscopy. The combination of the two spectroscopies by various data fusion strategies has also been evaluated. In the third chapter, the interest of Raman spectroscopy for the online monitoring of both the quantity and the glycosylation of the mAb was demonstrated. Models were developed for online prediction of both macroheterogeneity (glycosylation site occupancy) and microheterogeneity (glycan structures) of mAb glycosylation in batch and feed-harvest cultures. The last chapter used models previously developed for NIR and dielectric spectroscopies, to integrate into a “soft sensor” by combining with cell metabolic and mass balance equations. This “soft sensor”, implemented in a fed-batch cell culture for the automatic control of the feed rate, leads to an increased mAb productivity and better mAb glycosylation

Les anticorps monoclonaux (AcM) sont des protéines ayant une reconnaissance antigénique spécifique utilisée pour le développement de réactifs thérapeutiques et diagnostiques. La production commerciale est réalisée en cultivant des cellules hôtes dans des bioréacteurs spécifiques. La densité cellulaire et le métabolisme cellulaire sont des paramètres clés pour le rendement élevé des AcM. Bioréacteur à fibres creuses (HFB), une cartouche contenant des fibres poreuses emballées, est l'un des systèmes de production disponibles dans le commerce. Si la densité de cellules obtenue peut conduire à un rendement élevé, le coût de l'ensemble du dispositif, y compris les pompes et les cartouches très coûteuses, empêche son utilisation de petites unités. Comme une alternative économique, nous avons proposé ici d'étudier le potentiel des modules de dialyse de polysulfone du commerce, classiquement employés dans le traitement de l'insuffisance rénale en phase terminale. Cependant, la membrane de polysulfone native a démontré une adsorption de protéines non spécifique significative préjudiciable à la production d'AcMs. De plus des enjeux normatifs viennent se greffer à ces problématiques scientifiques et technico-économiques, avec le cas des normes (ISO 13485/ AC S99-104/ GMP FDA /BPF…) qui imposent des méthodes de travail normalisées. Ce travail de thèse consiste en la conception d’un bioréacteur jetable, sur la base d’une cartouche de dialyse médicale à fibres creuses. Ce système doit offrir toutes les garanties en termes de production, de facilité d’utilisation, de stérilité, et permettre de concentrer les produits de cytoculture. La méthodologie scientifique a été couplée à une démarche qualité. La gestion de ce projet a été couplé à l’analyse de risques. En effet ce projet a été divisé en ses 5 composantes élémentaires décrite par Ishikawa par la méthode des 5 M. L’analyse de risque a consisté au calcul d’indice de criticité par la méthode AMDEC de chacune de ses familles de risques. Cette approche nous a permis de formaliser deux axes de recherche : i) la mise en œuvre d’une oxygénation efficace du milieu de culture (chapitre 4) et ii) les moyens de limiter le colmatage dans le module à fibres creuses pour obtenir une culture cellulaire conforme aux objectifs (chapitre 5). Le taux d’oxygénation est un facteur à prendre en compte dans un processus de culture cellulaire. L’oxygène peut être supplémenté selon deux modes, le mode passif ou le mode actif [Ozturk et Palsson 1990; Zhang S. et al 1992]. Il existe 2 types de système d’oxygénation : les système dit passif ou les échanges se font a travers une paroi de silicone et un système actif par aération directe dans le milieu de culture. Ce système est de loin l'opération la plus simple pour fournir de l'oxygène. Cependant, lorsque celui-ci est utilisé pour apporter de l’oxygène à des cultures de cellules mammifères, cela peut engendrer des altérations cellulaires. Des agents protecteurs chimiques peuvent être utilisés pour réduire les dommages cellulaires et la formation de mousse [Kamase et Moo-yung 1990; van Der pol L.A et al 1993]. Nos études ont démontré que l'addition d'agents anti-mousse peut entraîner une diminution du coefficient de transfert de masse d’O2 en phase liquide (Kl) [Kamase et Moo-yung 1990]. Nous avons établi l’efficacité de l’utilisation d’un polymère silice/silicone pour éliminer la mousse sur des cultures bactériennes et de cellules mammifères. Afin de limiter ces phénomènes de colmatage, les fibres de polysulfone ont été traitées avec plusieurs tensioactifs (acide pluronique F127, D-limonen et différentes huiles de silicone) qui ont conduit à une diminution significative de l'adsorption protéique. L'effet de ces surfactants sur les performances de filtration et sur la cytotoxicité a été étudié. Certains d'entre eux n'ont pas influencé ces paramètres alors que d'autres ont présenté des effets négatifs. / Monoclonal Antibodies (mAbs) are proteins with specific antigen recognition used for development of both therapeutic and diagnostic reagents. Commercial production is achieved by growing host cells in specific bioreactors. Cell density and cell metabolism are key parameters for high yield of mAbs. Hollow fiber bioreactor (I-IFB), a cartridge containing packed porous fibres, is one of the system for production commercially available. If the cell density achieved can lead to high yield, the cost of the whole device, including pumps and very expensive cartridges prevents its use of small units. As an economical alternative, we proposed here to investigate the potential of commercial polysulfone dialysis modules, classically employed in the treatment of end stage renal failure. However, the native polysulfone membrane demonstrated a significant non-specific protein adsorption detrimental to mAbs production. Moreover normative issues are added to these scientific and techno-economic issues, with the case of standards (ISO 13485 / AC S99-104 / GMP FDA / BPF ...) which impose standard working methods. This thesis consists of the design of a disposable bioreactor, based on a hollow-fiber medical dialysis cartridge. This system must offer all the guarantees in terms of production, ease of use, sterility, and allow to concentrate the cytoculture products. Scientific methodology has been coupled with a quality approach. The management of this project was coupled with the risk analysis. Indeed this project was divided into its 5 elementary components describe by Ishikawa by the 5M method. The risk analysis consisted in the calculation of the criticality index by the AMDEC method of each of its families of risks. This approach allowed us to formalize two research axes: i) the implementation of an effective oxygenation of the culture medium (chapter 4) and ii) the means of limiting the clogging in the hollow fiber module to obtain a culture consistent with the objectives (Chapter 5). The rate of oxygenation is a factor to be taken into account in a cell culture process. Oxygen can be supplemented in two modes, passive mode or active mode [Ozturk and Palsson 1990; Zhang S. et al 19921. There are two types of oxygenation system: the so-called passive system or the exchanges are made through a silicone wall and an active system by direct aeration in the culture medium. This system is by far the simplest operation for providing oxygen. However, when it is used to supply oxygen to mammalian cell cultures, this can cause cellular damage. Chemical protective agents can be used to reduce cell damage and DKamase and Moo-yung 1990 foam formation; van Der pol L.A. et al 1993 Cl. Our studies have shown that the addition of antifoaming agents can lead to a decrease in the liquid phase (K2) mass transfer coefficient of D Kamase and Moo-yung 1990C]. We have established the effectiveness of using a silica / silicone polymer to remove foam on bacterial and mammalian cell cultures. In order to limit these adsorption phenomena, polysulfone fibers were treated with several surfactants (pluronic acid F 127, D-limonen, and different silicone oils) which led to a significant decrease in protein adsorption. The effect of such surfactants on the filtration performances and on cytotoxicity were investigated. Some of the them did not influence these parameters while some presented negative effects. Finally, different cell culture parameters (cells densities, production yield, flow properties, fouling) were studied, as well as the performance of the bioreactor in perfusion continuous mode. The bioreactor was maintained in continuous mode for fifteen days and the production yield per batch was 250 mg of AcMs. The results obtained in this work allowed us to define the next steps to be taken, and are the subject of the Perspectives section.

Le virus de l’hépatite C (VHC) est l’une des causes majeures de cirrhose du foie et de carcinome hépatocellulaire. Au courant de la première partie de ma thèse, nous nous sommes intéressés à caractériser plus en détail le rôle de SR-BI dans l’infection par le VHC. Bien que les mécanismes impliquant SR-BI dans la liaison du virus à l’hépatocyte aient été partiellement caractérisés, le rôle de SR-BI dans les étapes suivant la liaison du VHC reste encore largement méconnu. Afin de mieux caractériser le rôle de l’interaction VHC/SR-BI dans l’infection par le VHC, notre laboratoire à généré une nouvelle classe d’anticorps monoclonaux anti-SR-BI inhibant l’infection virale. Nous avons pu démontrer que SR-BI humain jouait un rôle dans le processus d’entrée du virus à la fois lorsde l’étape de liaison du virus à la cellule hôte mais aussi au cours d’étapes suivant cette liaison. Ainsi il serait intéressant de cibler cette fonction de SR-BI dans le cadre d’une stratégie antivirale pour lutter contre l’infection parle VHC. Dans la seconde partie de ma thèse, nous avions pour but de caractériser les mécanismes moléculaires intervenant dans la réinfection du greffon lors de la transplantation hépatique (TH). Nous avons ainsi identifiés 3 mutations adaptatives dans la glycoprotéine d’enveloppe E2 responsables de l’entrée virale augmentée du variant hautement infectieux. Ces mutations influent sur la dépendance au récepteur CD81 du VHC résultant en une entrée virale accrue. L’identification de ces mécanismes va nous permettre une meilleure compréhension de la pathogénèse de l’infection par le VHC, et est un premier pas pour le développement d’une stratégie préventive antivirale ou vaccinale. / Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. In the first part of my PhD, we aimed to further characterize the role of scavenger receptor class B type I (SR-BI) in HCV infection. While the SR-BI determinants involved in HCV binding have been partially characterized, the post-binding function of SR-BI remains remained largely unknown. To further explore the role of HCV-SR-BI interaction during HCV infection, we generated a novel class of anti-SR-BI monoclonal antibodies inhibiting HCV infection. We demonstrated that human SR-BI plays a dual role in the HCV entry process during both binding and post-binding steps. Targeting the post-binding function of SR-BI thus represents an interesting antiviral strategy against HCV infection. In the second part of my PhD, we aimed to characterize the molecular mechanisms underlying HCV re-infection of the graft after liver transplantation (LT). We identified threeadaptive mutations in envelope glycoprotein E2 mediating enhanced entry and evasion of a highly infectious escape variant. These mutations markedly modulated CD81 receptor dependency resulting in enhanced viral entry. The identification of these mechanisms advances our understanding of the pathogenesis of HCV infection and paves the way for the development of novel antiviral strategies and vaccines.

Orientador: Elenice Deffune / Banca: Mirthes Ueda / Banca: Rejane Tommasini Grotto / Resumo: O Vírus Sincicial Respiratório humano (VSR) é o principal agente causal das Infecções Respiratórias Agudas (IRAs) em lactantes e pré-escolares. Apresenta dois subgrupos - A e B - sendo o primeiro responsável por quadros clínicos mais graves. Os RNA mensageiros virais codificam 11 proteínas conhecidas, das quais a proteína F é responsável pela fusão viral à célula e se apresenta na forma de F0 com peso molecular de 67kDa, podendo ser clivada em duas unidades: F2 de 20kDa e a F1 de 47kDa. A proteína F recombinante (Fr), expressa em E.coli (BL21A no vetor pET28a), foi cedida por pesquisadores da UNESP de São José do Rio Preto. Com intuito de produzir anticorpos monoclonais contra a proteína Fr para fins de diagnóstico laboratorial, inoculamo-la em camundongos isogênicos Balb/c. Durante o desenvolvimento do monoclonal, foi necessário a eliminação da contaminação de cultura por Mycoplasma ssp. Após ajuste de dose de inoculação e descontaminação do antígeno (hipótese: contaminação com endotoxinas provenientes de E.coli), seis clones secretores de anticorpos monoclonais foram produzidos (5 do tipo IgM e 1 do tipo IgG2a) e testados, simultâneamente, contra toxina bacteriana e proteína Fr (40μg/mL) por técnica de ELISA indireto. Paralelamente, testes por citometria de fluxo foram realizados, incubando-se os clones obtidos com E.coli (bactéria com membrana permeabilizada e in natura). Os clones (VIRSV2-87A74 e VIRSV2-87A80) apresentam um reconhecimento inespecífico de proteínas da E. coli e não foi possível caracterizar os clones obtidos em função da dificuldade de se obter novas amostras de proteína Fr. Para o futuro, os anticorpos obtidos deverão ser marcados com isotiocinato de fluoroceína e comporem um amplo teste epidemiológico em paralelo com o kit disponível em mercado respeitando a sazonalidade da doença. Novos ensaios para caracterização... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Human Respiratory Syncytial Virus (HRSV) is the main cause of Acute Respiratory Insufficiency in suckling child and children below 5 years old. It has 2 subgroups - A and B - being the first one responsible for more dangerous clinical situations. The viral messenger RNA codifies 11 known proteins, which the F protein is responsible for the viral fusion and it is presented as F0 with molecular weight of 67kDa, but once is broke it generates two units: F2 with 20kDa and F1 with 47kDa. The recombinant F (Fr) protein were expressed on E.coli (BL21A, on vector pET28a), and it was given to us by researches from UNESP of São José do Rio Preto. Wishing to produce monoclonal antibodies (Mab) against Fr protein to laboratorial diagnosis, mice (Balb/c) were inoculated with de antigen (Fr protein). During the development of the Mab, it was necessary to eliminate, from the culture, Mycoplasma spp. After we adjusted the necessary amount to inoculate on the mice and eradicated the contamination of the antigen (hypotheses: there were endotoxins from the E.coli), six clones that secrete Mab were produced (5 of the kind IgM, and 1 of the kind IgG2a) and they were tested, simultaneously, against bacterium toxin and Fr protein (40μg/mL) using ELISA technique. Together, flow cytometry test were performed when we incubated the clones with E. coli (bacterium with permeable membrane and in natura). The clones (VIRSV2-87A74 and VIRSV2-87A80) presented an unspecific recognition for the proteins from E.coli and it was not possible to characterize the cells because we couldn't get more samples of Fr protein. For the future, the Mab should be conjugated with FITC and then, they must be part of a wide epidemiologic test side by side from the commercial kit, always respecting the seasonal disease. New assays to characterize antibodies (like Western blotting and imunepreciptation) are already being done... (Complete abstract click electronic access below) / Mestre

Orientador: Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-06T20:08:56Z (GMT). No. of bitstreams: 1 Bresolin_IgorTadeuLazzarotto_M.pdf: 4686617 bytes, checksum: ed7a688421b5d854024279523878c12a (MD5) Previous issue date: 2006 / Resumo: Anticorpos monoclonais são imunoglobulinas secretadas por clones de linfócitos B, normais, tumorais ou obtidos pela tecnologia de hibridomas. Os anticorpos monoclonais têm sido utilizados nas áreas analítica e terapêutica, o que implica na necessidade de sua obtenção com pureza superior a 95%. Muitos estudos têm sido realizados visando à purificação de anticorpos monoclonais, destacando-se as técnicas de adsorção seletiva, como as cromatografias de troca iônica, hidrofóbica e de afinidade. Neste trabalho aplicou-se a cromatografia de afinidade em membranas de álcool polietileno-vinílico (PEVA), comparando o desempenho dos agentes quelantes (AQ) ácido iminodiacético (IDA), ácido aspártico carboximetilado (CM-Asp) e tris-2(aminoetil)amina (TREN) com íons metálicos imobilizados na purificação de anticorpos monoclonais anti-TNP, isotipo IgG1 a partir de sobrenadante de cultura celular precipitado e dialisado. Para determinar as melhores condições de adsorção e eluição na presença de diferentes sistemas tamponantes, foram testados os quelatos AQ-Ni2+, AQ-Zn2+ e AQ-Co2+, bem como os agentes quelantes sem metal imobilizado como grupos ionogênicos. De acordo com eletroforeses SDS-PAGE e análises ELlSA das frações dos picos de proteína obtidos, as melhores condições utilizadas para a purificação foi o uso de membranas PEVA-IDA-sem metal e PEVA-CM-Asp-Zn2+, em presença de tampão Tris-HCI 50 mM a pH 7,0 e eluição por aumento de concentração de Tris, atingindo fatores de purificação de 138,9 e 103,8 e pureza de 92,3% e 68,1%, respectivamente. A partir das isotermas de adsorção, determinou-se a capacidade máxima de adsorção e a constante de dissociação dos complexos IDA-lgG1 e CM-Asp-Zn2+ -lgG1 que, de acordo com o ajuste dos parâmetros pelo modelo de Langmuir, mostraram alta capacidade de adsorção e constantes de dissociação características de sistemas de afinidade média. Com o módulo de fibras ocas, construído em nosso laboratório, determinaram-se as curvas de ruptura por meio de experimentos de filtração para os processos propostos, e os resultados obtidos demonstraram que os sistemas de fibras ocas PEVA-IDA e PEVA-GM-Asp-Zn2+ são factíveis para a purificação de anticorpos monoclonais do isotipo IgG1 / Abstract: Monoclonal antibodies are immunoglobulins produced by normal, tumoral or hybrids Iymphocytes 8 obtained by hibridoma technology. Hybridomas are resulted from the fusion of Iymphocytes B with malignant myeloma cells which express both the Iymphocyte's property of specific-antibody production and the immortal characteristic of the myeloma cells. Monoclonal antibodies have been used in analytic and therapeutic areas. This application needs highly pure antibodies. Many techniques have been studied focusing monoclonal antibodies purification. These techniques include ion exchange, hydrophobic and affinity chromatography. In this study, we applied polyethylenevinyl alcohol (PEVA) membranes in the purification of monoclonal antibody from cel! culture supematant comparing the chelating agents Iminodiacetic Acid (IDA), Carboxy-methyl Aspartic Acid (CM-Asp) and Tris-2(aminoethyl)amine) (TREN) with different immobilized metal ios, Ni2+, Zn2+ and C02+, and with different buffers. We also evaluated the adsorption and purification of monoclonal antibodies using the chelating agents as ionogenic groups. According to SDS-PAGE electrophoresis and ELlSA analysis, the higher selectivity was obtained in the presence of 50 mM Tris-HCI buffer, pH 7,0 and with elution by increasing Tris concentration, with CM-Asp-Zn2+ and IDA as ionogenic group, which provided the purification of IgG with traces of albumin, reaching purification factors of 138.9 and 103.8 and purities of 92.3% and 68.1, respectively. The adsorbent capacity and the dissociation constant of the complexes IDA-lgG1 e CM-Asp-Zn2+ -lgG1 were determinate from adsorption isotherms. According to Langmuir model, the results indicated that the matrix presents high adsorption capacity and a dissociation constant characteristic for intermediate affinity systems. We also evaluated the breaktrough curves for the proposed processes. These breaktrough curves are important to scale up procedure / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química