Bøger i Schriftenreihe des Institutes für Bioverfahrenstechnik der Technischen Universität Braunschweig 63 serien

Antibodies and antibody fragments are most important tools for therapeutic and diagnosticapplications. An increasing demand of these high potential drugs makes less costintensive production systems most desirable. Compared to mammalian cell culturemicrobial production platforms are beneficial with regards to reaching high productiontiters, scale up approaches, regulatory aspects and reduced costs of goods. Amongstthese production systems those that are having the ability of an additional productsecretion are most advantageous as the downstream processing costs are decisivelyreduced.In this work Bacillus megaterium, as a Gram positive model organism, was used toextensively study the production and secretion of the antibody fragment D.13 scFv. Forthe bioprocess optimization a holistic approach was followed. First the aim was toestablish a high productive defined cultivation medium. Different media components likecarbon sources, metal ions and ammonium concentrations were screened throughoutvarious cultivation platforms ranging from micro titer plates to shaking flasks. Statisticaldesign of experiments and a genetic algorithm approach were used to establish anappropriate defined high production medium. As a second step the process was transferredto the bioreactor scale of several liter cultivation volume. An optimal bioprocessstrategy based on alternating growth and starvation phases was established to gain highproduct titers of antibody fragment D1.3 scFv. As a final step an up-scale to a 100 Lbioreactor was done accounting for an advanced process control and considering ¿goodmanufacturing practice¿ guidelines. The advanced bioprocess monitoring tool of flowcytometry was used to gain deeper insights on microbial physiology at single cell levelregarding cell viability, cell integrity and production intensity. This knowledge was used fora sophisticated cell physiology based bioprocess development and optimization. Furthermoreculture heterogeneities were measured and characterized for B. megateriumproducing antibody fragment D1.3 scFv under controlled bioreactor conditions. To obtainadditional information about the regulatory processes occurring inside the cell on geneexpression level a transcriptome analysis was performed comparing cells with anincreased production and secretion status to less producing and non-producing cells.These cutting-edge technologies of flow cytometry and transcriptome analysis revealedpossible bottlenecks of the overall bioprocess¿ performance and product secretion ofantibody fragments with B. megaterium as production platform and form a robust basis forrational strain optimization and advanced process designs.