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Transient Transfection of HEK293 Cells in Suspension: Process

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Transient Transfection of HEK293 Cells in Suspension: Process Transient Transfection of HEK293 Cells in Suspension: Process Characterization and Optimization by Applying Invasive Nucleotide and Non-invasive Electronic Nose Technology Doctoral Thesis Maria de los Milagros Bassani Molinas 2005 Transient Transfection of HEK293 Cells in Suspension: Process Characterization and Optimization by Applying Invasive Nucleotide and Non-invasive Electronic Nose Technology Vom Fachbereich für Biowissenchaften und Psychologie der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr.rer.nat.) genehmigte D i s s e r t a t i o n von Maria de los Milagros Bassani Molinas aus Santa Fe, Argentinien 1. Referent: apl. Professor Dr. R. Wagner 2. Referent: Prof. Dr. W.-D. Deckwer eingereicht am: 17.03.2005 mündliche Prüfung (Disputation) am: 17.06.2005 2005 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurde mit Genehmigung der Gemeinsamen Naturwissenschaftlichen Fakultät, vertreten durch die Mentorin oder den Mentor/die Betreuerin oder den Betreuer der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Bassani Molinas MM, Beer C, Hesse F, Wirth M, Durocher Y, Kamen A, Wagner R. 2003. Intracellular nucleotide pools for optimizing product-oriented transient transfection of HEK293 cells in suspension. In: Procedings GVC/DECHEMA Vortrags und Diskussionstagung “Zellkulturen: Vom biologischen System zum Produktionsprozess”. Publisher: VDI-Gesellchaft Verfahrenstechnik und Chemieingenieurwesen GVC, Düsseldorf, Germany, pp: 19-22. Bassani Molinas MM, Nelving A, Beer C, Hesse F, Wirth M, Durocher Y, Kamen A, Wagner R. 2005. Intracellular nucleotide pools for optimizing product-oriented transient transfection of HEK293 cells in suspension. In: Animal Cell Technology meets Genomics. (Gòdia F, Fussenegger M, eds.) Springer, Dordrecht, The Netherlands, pp: 83-85. Patente Wagner R, Bassani Molinas MM, Beer C, Wirth M. 2003. Transfection Process for Mammalian cells. European Patent. 03010206.5-2405. Tagungsbeiträge Bassani Molinas MM, Beer C, Hesse F, Wirth M, Durocher Y, Kamen A, Wagner R. 2003. Intracellular nucleotide pools for optimizing product-oriented transient transfection of HEK 293 cells in suspension. 18th ESACT MEETING. 11-14 May. Granada, Spain. Wagner R, Bassani Molinas MM , Beer C, Wirth M, Durocher Y, Kamen A. 2003. Intracellular nucleotide pools for optimizing product-oriented transient transfection of HEK 293 cells in suspension. GVC/DECHEMA Vortrags - und Diskussionstagung. 26-28 May, Bad Dürkheim, Germany. To my husband and my little son Ignacio A. Favale and Juan Ignacio Favale Bassani To my parents Humberto A. Bassani and Blanca E. Molinas de Bassani Acknowledgments First, I would like to thank my parents, Humberto A. Bassani and Blanca E. Molinas de Bassani, who always have supported me throughout my life. Many thanks for your unconditionally love and for giving me an excellent education, in all aspects, that have made possible my studies here, in Germany. I am very proud of you. I would like to thank my supervisor Prof. Dr. Roland Wagner for give me the opportunity to do my thesis work in the Cell Culture Technology Department of GBF. I also thank him for all his help, not only about work, even more in many personal situations. Many special thanks to my husband, Ignacio (Nacho), who has supported and encouraged me unconditionally to do my thesis work in Germany, helping me with my work, inclusive in the laboratory, as well as in every aspect during all these last years. Enormous thanks to you for your daily love, comprehension, patience and for always being with me. I could not have finished this thesis work without your help. Thanks to my little son (Juan Ignacio) who has taught me (and us), during these last six months, what is the most beautiful and essential in the life. I am very proud of both. Thanks to each member of my family, especially to my sister, Costi, and brother Tito, who have always encouraged and supported me. And many thanks to my grandfather, Alberto José Molinas, who has influenced my life, teaching me what are the most important things in one’s life. Many thanks to all members of the “ex-ZKT” group, Harry, Anja, Nadine, Herbert, Marc, Carsten, Mariela, Samira, Christoph, Corinna, Friedemann, Herr Hammer, Frau Ana, Volker, El-Sayed, Ingrida, Nadia, Anders, Matthias, Sven and Christin, for the nice working atmosphere and for always helping me in many aspects of my life. In particular, thanks to Ing. G.W. (Harry) Piehl for cell cultivation and computer advices, as well as for his unconditionally friendship. Many thanks to Dr. Friedemann Hesse for supervising my work during the first year and for reading the final manuscript, as well as for his suggestions and comments. Thank you, Nadine Konisch, for helping me with the first bioreactor cultivations and thanks to Ing. Joachim Hammer for the amino acids analysis and for your invitations to different cultural activities. I am grateful to Mrs. Kristin Anastassiadis for the nucleotide analysis and for teaching me this technique. Thank you, Herbert Kraft, for helping me with some cultivations and to freeze my cells. Many thanks to Carsten Griesel for teaching me about flow cytometry analysis and to Nadya Zoghul for reading and correcting the Introduction section. Special thanks to Corinna Mauth for helping me to understand all the German documents to submit this Thesis to the University and to be the first to teach me this difficult language. Thanks to Dr. Manfred Wirth and Dr. Christiane Beer for teaching and helping me in molecular biology. Special thanks to all my friends from GBF, Mariela, Marcelo, Pablo, Guillermina, Sylvia, Howard, Rosa Lila, Felipe, Magally, Faiza, Melita, Stefan, Maria Margarita, David, Lily, Ariana, Beatriz, Roberto, Alejandra, Carlos, Alicia, Alexandre, Sylvana, Gonçalo and David, to share very nice and important moments and to encourage me with my work. Many thanks to all the people who have helped me to finish this thesis work, especially: Mariela, for manuscript correction, for your time to discuss my thesis, for your suggestions and comments, and mainly, for your unconditionally friendship. Thanks to you and Marcelo for supporting me, especially during the first year in Germany. Maricel, thank you for reading the first thesis version, for the grammatical English correction and for your unconditionally friendship. Howard, I am grateful to you for manuscript correction and for teaching me some computer programs, as well as for helping me with some of them. Thanks to David, for correcting and controlling my English in the last thesis version and to Felipe, for helping me with the edition of the final manuscript. Finally, thanks to all these persons and friends from Argentine who have supported and encouraged me to come to Germany to do my thesis work. Thanks to be always with me despite of the distance. Table of contents i Table of contents Abreviations ....................................................................................................................v Units ........................................................................................................................... vii Summary........................................................................................................................ ix 1. Introduction ...........................................................................................................1 1.1 Animal cell culture: historical outline............................................................1 1.2 Animal cell culture: importance in protein production..................................2 1.3 Gene expression in mammalian cells.............................................................4 1.3.1 Transient transfection versus stable transfection......................................5 1.3.2 Large-scale transient transfection .............................................................7 1.4 Monitoring of cultivation process................................................................10 1.4.1 Nucleotides and bioprocess control ........................................................12 1.4.2 Electronic nose and bioprocess control ..................................................13 1.4.3 Multivariate analysis...............................................................................18 1.4.3.1 Principal component analysis (PCA)................................................18 1.5 Problems in production of large amounts of r-proteins - Aims of the work ..............................................................................................................22 2. Materials and Methods .......................................................................................24 2.1 Equipments used ..........................................................................................24 2.2 Materials used ..............................................................................................27 2.3 Cell line and bacterial strains.......................................................................28 2.3.1 Cell line...................................................................................................28 2.3.2 Bacterial strains.......................................................................................29 2.4 Culture media..................................................................................................29 2.4.1 For mammalian cells:..............................................................................29
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