Light-mediated control and analysis of recombinant protein production in microscale cultivations

  • Lichtbasierte Steuerung und Analyse der Produktion von rekombinanten Proteinen in Kultivierungen im Mikromaßstab

Wandrey, Georg Benjamin; Büchs, Jochen (Thesis advisor); Pietruszka, Jörg (Thesis advisor)

Aachen (2017)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2017

Abstract

The efficient production of technical or pharmaceutical proteins requires a large number of cultivation experiments during process development. These experiments are often miniaturized and parallelized and can be monitored with established measurement techniques. However, control options are still lacking in this scale. It was therefore the aim of this work to apply light, which is so far used for non-invasive optical measurements, to also reactivate photocaged inducer molecules and thereby enable optical process control.An optical online monitoring system was constructed based on the well-known BioLector setup. The capabilities of this device were demonstrated by optimizing the sequence specific incorporation of non-natural amino acids into recombinant proteins for click-chemistry applications. A sevenfold increased target protein concentration was achieved by a considerable increase of the unnatural amino acid concentration. In order to enable optical process control, the setup was expanded with a newly constructed UV-A LED module establishing an intensive yet individual illumination option for each well of a 48-well microtiter plate. The application of a 6-nitropiperonal photocaged derivate of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) demonstrates that a minimally invasive optical induction can be achieved and that convetional induction can be replaced this way.Light-mediated control over protein formation with caged compounds can not only replace conventional methods but also offers new control options for microscale cultivations. By releasing a second caged compound - photocaged methionine (NVOC-Met) - with complex illumination schemes, it is demonstrated that heterologous protein formation in a methionine repressible S. cerevisiae strain can be started, paused or stopped by optical means. A combination with the online measurements of the BioLector system establishes a closed-loop control setup for the gradual regulation of protein formation rates in each well of a microtiter plate which has not been achieved so far. Thereby, parallelized light-mediated control over recombinant protein formation is combined with light-mediated analysis, opening up a vast number of promising applications in bioprocess development.