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A proteomics approach was used to identify proteins potentially implicated in the cellular response concurrent with elevated production levels of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line (Darren cells), using zinc and sodium butyrate in the production media to increase expression. To this end, 2-dimensional gel electrophoresis (2-DGE) was utilized. Firstly, several aspects of 2-DGE were developed for this investigation. Gel drying conditions were optimized, and a glycine-free blotting method is described which achieved greater efficiency in rapid transfer of proteins than those previously described. Next, hFSH expression was characterized in Darren cells. An ELISA developed for this investigation examined intracellular (expression) and extracellular (secretion) of hFSH during increased expression. These results show a disproportionate increase in intracellular hFSH (188%) expression above extracellular hFSH (41%).

A proteomics approach was used to identify proteins potentially implicated in the cellular response concurrent with elevated production levels of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line (Darren cells), using zinc and sodium butyrate in the production media to increase expression. To this end, 2-dimensional gel electrophoresis (2-DGE) was utilized. Firstly, several aspects of 2-DGE were developed for this investigation. Gel drying conditions were optimized, and a glycine-free blotting method is described which achieved greater efficiency in rapid transfer of proteins than those previously described. Next, hFSH expression was characterized in Darren cells. An ELISA developed for this investigation examined intracellular (expression) and extracellular (secretion) of hFSH during increased expression. These results show a disproportionate increase in intracellular hFSH (188%) expression above extracellular hFSH (41%).

Natural Killer (NK) cells can recognize and lyse a large variety of tumor cells and have been of interest as a potential cancer treatment option. Our group has developed a particle-based NK cell expansion method that utilizes plasma membrane particles (PM-particles) derived from K562 cells genetically engineered to express membrane bound IL21 and 41BBL(K562-mbIL21-41BBL), two proteins that stimulate growth and activity of NK cells. This method selectively expands highly cytotoxic NK cells > 400-fold in 14 days of culture. Currently NK cells are expanded in vitro using Fetal Bovine Serum (FBS) as a serum-supplement to promote cell growth. While effective, the use of animal products is not preferred in cell cultures grown for clinical purposes. This project tested Human Platelet Lysates (HPL) as a potential replacement for FBS in NK cell culture. NK cells were expanded using PM21-particle based expansion method with either FBS or HPL as supplements. Their growth characteristics, phenotype and functionality were assessed and compared. Results of this study determined that HPL is a viable option to replace FBS in NK cell culture for clinical applications, as there was no significant difference between the two serum supplements.

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

摘 要 本研究主要是探討利用新式生物反應器以固定化細胞培養技術生產日本腦炎病毒(Japanese encephalitis virus；JEV)之研究，首先根據所培養細胞的生長特性與原有生物反應器之缺點，利用已改良設計之新式生物反應器，評估此新式生物反應器適用性、效能，以及所培養細胞之生長代謝情形與病毒力價。整個實驗過程大致分為幾個階段，第一個階段探討細胞固定化培養之最適化培養條件與生長代謝情形，第二個階段找出細胞固定化培養於此新式生物反應器中最佳生長狀態，最後一個階段為病毒的培養。實驗後發現Vero細胞經固定化貼附於FIBRA-CEL®載體上，可擴大培養於新式生物反應器，Vero細胞最佳生長量達到6.6×106cells/mL。希望藉由此改良之新式生物反應器提供細胞與病毒一個良好之生長培養環境，獲得高產量、品質穩定一致之細胞生物製品，以提供ㄧ設備簡單與製程操作容易、低成本、低能源消耗之細胞製品生產基座。 / Abstract In this study, we investigated the production of Japanese encephalitis virus (JEV) by the immobilized cell technology in a novel bioreactor. According to the disadvantages of original bioreactor and growth characteristics of cell culture, we evaluated the suitability and efficiency of a design-improved novel bioreactor as well as the growth and metabolic situation of cultured cells and titers of JEV. All studies including three major stages: (1) investigation of the optimal conditions and metabolic situation for the growth of immobilized cells, (2) finding the optimal conditions for the growth of immobilized cells in this novel bioreactor, and (3) growth of JEV using immobilized cells in this novel bioreactor. Our results showed that after immobilization on the FIBRA-CEL® carries, Vero cells can grow on the novel bioreactor up to the density of 6.6 × 106 cells/mL. Hopefully, the improvement of the novel bioreactor will provide an optimal growth condition for both the cells and viruses. Furthermore, it will also provide the basis for the production of cell products with advantages of simple-equipped, easy-to-operate, low cost, and low energy consumption. / 目 錄 誌謝------------------------------------------------- i 中文摘要 -------------------------------------------- ii 英文摘要 -------------------------------------------- iii 目錄 -------------------------------------------- iv 表目錄 -------------------------------------------- v 圖目錄 -------------------------------------------- vi 第一章 緒論---------------------------------------- 1 第二章 文獻探討------------------------------------ 3 第一節 日本腦炎病毒疫苗---------------------------- 3 第二節 動物細胞的培養------------------------------ 4 第三節 載體上動物細胞的培養------------------------ 5 第四節 動物細胞培養於生物反應器-------------------- 7 第三章 材料與方法---------------------------------- 10 一 細胞株的培養-------------------------------- 10 二 細胞冷凍保存與解凍培養---------------------- 10 三 細胞滾瓶培養-------------------------------- 11 四 病毒株的培養-------------------------------- 12 五 固定化載體材料製備-------------------------- 12 六 載體上細胞數的測定-------------------------- 12 七 細胞貼壁率的計算---------------------------- 13 八 生物反應器結構特性與固定化細胞培養---------- 13 九 日本腦炎病毒力價測定------------------------ 19 十 葡萄糖的測定-------------------------------- 19 第四章 結果與討論---------------------------------- 20 一 固定化載體材料比例對Vero細胞生長的影響------ 20 二 細胞貼附固定化時間對Vero細胞生長的影響------ 23 三 細胞接種量對Vero細胞生長的影響-------------- 24 四 生物反應器培養系統對Vero細胞生長的影響------ 25 五 新鮮培養基更換對Vero細胞生長的影響---------- 27 六 最適化細胞生長條件培養日本腦炎病毒---------- 29 第五章 結論與建議---------------------------------- 30 參考文獻 -------------------------------------------- 31 附錄一 PBS配製方法--------------------------------- 62 附錄二 Medium 199配製方法-------------------------- 62 附錄三 MEM medium配製方法-------------------------- 62

Ce travail apporte une contribution au développement de la stratégie PAT pour les procédés de culture de cellules animales. Il propose l'amélioration de la compréhension et de la maîtrise de la culture de cellules Vero, dédiées à la production de vaccins viraux, et cultivées sur microporteurs dans un milieu sans sérum. Une première partie a permis de cribler les effets de certains groupes de composés du milieu de culture par le suivi de la croissance en microplaques. Puis, des études cinétiques et métaboliques plus approfondies, réalisées en spinners, ont montré que le métabolisme carboné des cellules Vero est saturé par l'accumulation intracellulaire de pyruvate et qu’il est peu orienté vers la croissance. Alors que le renouvellement du milieu ou l'ajout ponctuel de glutamine amélioraient la croissance cellulaire sans ré-équilibrer le métabolisme, la substitution du glucose et de la glutamine a permis de réduire l’apoptose et d'améliorer les performances métaboliques et de croissance. Par ailleurs, les spectroscopies diélectrique et proche-infrarouge ont été évaluées pour le contrôle en-ligne du procédé, en prenant en compte les particularités des cellules adhérentes. Nous avons montré leur capacité à évaluer les concentrations de cellules, de composés du milieu, et à détecter l'apoptose. Enfin, les principales améliorations par substitution de la glutamine ont été appliquées en bioréacteurs, à la production d'un vaccin prototype contre la dengue, dans des conditions proches de celles d'un procédé industriel. Ceci a permis de limiter les renouvellements de milieu pendant l’expansion cellulaire, sans compromettre la production de particules virales infectieuses / This work contributes to the development of the PAT strategy for animal cell culture processes. The aim of this study was to improve the understanding and the control of Vero cell culture, dedicated to the production of viral vaccines, and grown on microcarriers in serum-free medium. An initial study was performed to screen the effects of certain groups of compounds of the culture medium, by the cell growth monitoring in microplates. Then, kinetic and metabolic studies conducted in spinners flasks allowed to go further and to show that the Vero cell metabolism is saturated through the pyruvate intracellular accumulation and that it is not oriented toward growth. While media renewal or punctual addition of glutamine improve the cell growth without improving the metabolism balance, the substitution of glucose and glutamine allowed to reduce apoptosis and to improve growth and metabolic performances.Furthermore, dielectric and near-infrared spectroscopies have been evaluated for the in-line process monitoring, taking into account the particularities of adherent cells. We have demonstrated their ability to quantify cell concentrations, medium component concentrations, and to detect apoptosis. Finally, major improvements by substitution of glutamine have been applied to bioreactor culture to produce a dengue vaccine prototype, with culture conditions close to industrial process. In these cases, medium renewal during the cell expansion was removed without compromising the production of infectious viral particles