title: Antibody production in Bacillus megaterium: strategies and physiological implications of scaling from microtiter plates to industrial bioreactors.
abstract: Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5-100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft-sensor for biomass estimation to establish a μ-oscillating time-dependent fed-batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH(4))(2)SO(4), and inductor concentrations. For bioprocess design, an adapted oscillating fed-batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed-batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale-free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.
authors.affiliation: Institute for Biochemical Engineering, Technische Universität Braunschweig, Braunschweig, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: David
authors.forename: Miriam
authors.initials: M
authors.lastname: Steinwand
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.forename: Kathrin
authors.initials: K
authors.lastname: Bohle
authors.forename: Anton
authors.initials: A
authors.lastname: Ross
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
authors.forename: Ezequiel
authors.initials: E
authors.lastname: Franco-Lara
mesh_terms: D000645:Ammonium Sulfate; D001410:Bacillus megaterium / Q000235:genetics / Q000276:immunology*; D019149:Bioreactors; D002244:Carbon; D060846:Cell Engineering / Q000379:methods*; D003470:Culture Media; D005434:Flow Cytometry; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007218:Industrial Microbiology / Q000379:methods*; D010865:Pilot Projects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D003470:Culture Media; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D002244:Carbon; D000645:Ammonium Sulfate