Assessment of a novel high-throughput process development platform for biopharmaceutical protein production

  • Einschätzung einer neuen Hochdurchsatz-Prozessentwicklungsplattform für die biopharmazeutische Proteinproduktion

Opdensteinen, Patrick; Buyel, Johannes Felix (Thesis advisor); Schillberg, Stefan Johannes (Thesis advisor)

Aachen : RWTH Aachen University (2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2023


Plants can complement dominant expression systems such as Escherichia coli and Chinese hamster ovary (CHO) cells with additional production capacity in response to emerging infectious diseases, but compared to these hosts few high-throughput screening tools that facilitate biopharmaceutical development have been available in plants. To advance this situation, high-throughput techniques were implemented during cloning, expression, purification and quantification, thus increasing the screening throughput across the entire development process. This concept was applied to a transient expression in Nicotiana spp., which allows to establish production processes in as little as 3 weeks and thus quickly react to changing demands. In combination with statistical design of experiments, the established high-throughput screening tools allowed to rapidly clone and test libraries of expression cassette elements such as promotors, 5′ UTRs and signal sequences and identify combinations thereof that maximize target protein accumulation. This strategy was successfully employed to produce interleukins, polyphosphate kinases, IgG3 antibodies, biofilm degrading enzymes and endolysins selected for a multilayered strategy directed against methicillin-resistant Staphylococcus aureus (MRSA). Notably, IgG3 accumulation levels achieved by systematically screening expression cassette elements were threefold higher than the literature. Using the same strategy, dispersin B accumulation levels equivalent to E. coli were reached. Further improvement can be expected in the future by expanding the set of expression cassette elements used for screening, particularly with synthetic promotors, 3′ UTRs and terminators. Plant-derived target proteins were functional, except for a reduced enzymatic activity of polyphosphate kinases, indicating that Nicotiana spp. can supply recombinant proteins to counter emerging MRSA. Using the high-throughput screening tools established herein, additional plant-made proteins can be rapidly assessed for their usefulness against MRSA in the future.In accordance with a recent approach in biopharmaceutical development, data generated with the different target proteins were used to identify parameters that can guide the selection of candidate proteins and even optimization strategies. For instance, the target protein origin had a major impact on the ideal expression compartment, thus allowing to pre-select suitable expression compartments and reduce the screening workload. Assessing intrinsic protein stability parameters allowed to sort out unsuitable candidate proteins, albeit currently limited to comparisons within the same protein class. A parameter that can guide the optimization of plant cell cultivation media is the medium osmolality, essentially controlling the uptake of water into plant cells. Characterization of host cell proteins that persist after chromatography allowed to derive purification strategies that facilitate their removal.


  • Fraunhofer Institute for Molecular Biology and Applied Ecology - IME [053400]
  • Department of Biology [160000]
  • Chair of Molecular Biotechnology [162910]