Rational and model-based characterization of heterologous gene expression in biological systems

  • Rationale und modellbasierte Charakterisierung von heterologer Genexpression in biologischen Systemen

Gengenbach, Benjamin Bruno; Buyel, Johannes Felix (Thesis advisor); Blank, Lars M. (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


The production of recombinant proteins for human and animal health is a strong growing business sector with a large demand for antibodies, vaccines, enzymes, and hormones. Next to the already established mammalian and microbial systems, plants pose a cost efficient and easily scalable alternative production platform that is able to produce complex glycoproteins and even peptides that are toxic to mammalian cell lines. Whereas screening platforms for the automated HTP identification of candidates for large scale production already do exist for microbial and mammalian systems, nothing comparable has been developed for plant-based production systems, which is a substantial drawback and relevant market entrance threshold. The goal of this work was to eliminate this drawback, by developing an automated HTP screening system for transient protein expression in plants and to thereby enable plants as productions system to narrow the gap to the established gold standards of biopharmaceutical protein production. To do this, the Plant Cell Pack (PCP) technology, which can be used as model system to predict expression and purification parameters in whole plants, was advanced to be compatible with handling on a conventional laboratory automation system by using Design of Experiment (DoE) approaches and descriptive model building. Suitable methods for generation and incubation of miniaturized N. tabacum BY-2 PCP were identified and the original system was thereby transformed to an automation-compatible 96-well standard plate format. Subsequently, a fully automated protocol was developed that allowed the generation, infiltration and extraction of up 4800 PCPs per day and that is compatible with further optimization cycles by statistical experimental designs. The requirements for manual interventions were limited to an absolute minimum to guarantee a consistent and reliable processing of screening runs, while costs compared to the manual setup were reduced from 0.50 € to 0.23 € per PCP. Additionally, a quality routine regarding BY-2 cell material was implemented to ensure a defined low intra-batch variance of <10% as well as comparability between runs and batches and the capability to immediately sort out potentially comprised runs. Furthermore, an optimized infiltration protocol for the to-be predicted expression in whole plants was developed that increased recombinant protein levels by 20-40% compared to the reference protocol. Finally, the automated PCP system was used for determination of correlation factors with the expression levels in whole plants and a linear correlation of the DsRed model protein with an adjusted r2 of 0.96 over four cellular compartments of N. tabacum was achieved.


  • Department of Biology [160000]
  • Chair of Molecular Biotechnology [162910]