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@PHDTHESIS{Opdensteinen:956637,
      author       = {Opdensteinen, Patrick},
      othercontributors = {Buyel, Johannes Felix and Schillberg, Stefan Johannes},
      title        = {{A}ssessment of a novel high-throughput process development
                      platform for biopharmaceutical protein production},
      school       = {RWTH Aachen University},
      type         = {Dissertation},
      address      = {Aachen},
      publisher    = {RWTH Aachen University},
      reportid     = {RWTH-2023-04267},
      pages        = {1 Online-Ressource : Illustrationen, Diagramme},
      year         = {2023},
      note         = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
                      University; Dissertation, RWTH Aachen University, 2023},
      abstract     = {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.},
      cin          = {162910 / 160000 / 053400},
      ddc          = {570},
      cid          = {$I:(DE-82)162910_20140620$ / $I:(DE-82)160000_20140620$ /
                      $I:(DE-82)053400_20140620$},
      pnm          = {GRK 2375 - GRK 2375: Tumor-Targeted Drug Delivery
                      (331065168)},
      pid          = {G:(GEPRIS)331065168},
      typ          = {PUB:(DE-HGF)11},
      doi          = {10.18154/RWTH-2023-04267},
      url          = {https://publications.rwth-aachen.de/record/956637},
}