The Insect Cell - Baculovirus Expression Vector System (IC-BEVS) is widely used for the production of complex recombinant (glyco)proteins. The simplicity of insect cell cultivation in suspension serum-free media and the easy construction of recombinant baculovirus vectors have made the BEVS quite an effective expression system. On the other hand, the BEVS is a transient lytic system that may present some drawbacks in purification and potential degradation of the products. Among the various insect cell lines, the High-Five cell line has a great potential for the production of recombinant proteins using the BEVS in stirred bioreactors, reaching high cell densities and high protein production levels. Moreover, these cells can tolerate environmental stresses and can be cultivated on a large scale (Chapter 1). Unfortunately, up to now, there have been limited data available regarding suitable culture conditions and the metabolism of High-Five cells, a key requirement for the rational development of new processes.
The overall goal of the present work was the study of these High-Five cells, in order to develop sophisticated new processes as alternatives to batch cultivation. The original contributions have been developed along two axes. The first axis concerns the study of the physiology and metabolism of High-Five cells. At first, we undertook a study aiming to prevent cell ring formation on suspension culture recipient walls (Chapter 2). Next, we analyzed environmental factors affecting insect cell growth and death, by comparing and developing methods able to distinguish between apoptosis and necrosis of cells (Chapter 3). The comprehensive study of the extended metabolism of High-Five cells was done using a metabolic flux network that takes account of the catabolism but also the anabolism of uninfected and baculovirus-infected cells (Chapter 4).
The second axis was the application of the previously gained knowledge on High-Five cells to develop high-density systems specifically adapted to them: a fed-batch feeding strategy consisting of different pulses developed to increase the productivity of cells during infection (Chapter 5) and a fixed-bed reactor system (Chapter 6), as an alternative to classic perfusion, adapted to High-Five cells for recombinant protein production.
In sum, new physiological and metabolic knowledge has been translated into new process options for High-Five cells.
| Identifer | oai:union.ndltd.org:BICfB/oai:ucl.ac.be:ETDUCL:BelnUcetd-01032007-161307 |
| Date | 07 February 2007 |
| Creators | Drugmand, Jean-Christophe |
| Publisher | Universite catholique de Louvain |
| Source Sets | Bibliothèque interuniversitaire de la Communauté française de Belgique |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://edoc.bib.ucl.ac.be:81/ETD-db/collection/available/BelnUcetd-01032007-161307/ |
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