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@PHDTHESIS{Steimann:1013898,
      author       = {Steimann, Thomas},
      othercontributors = {Magnus, Jørgen Barsett and Blank, Lars M.},
      title        = {{F}ermentation process for recombinant protein production
                      in {K}omagataella phaffii},
      school       = {Rheinisch-Westfälische Technische Hochschule Aachen},
      type         = {Dissertation},
      address      = {Aachen},
      publisher    = {RWTH Aachen University},
      reportid     = {RWTH-2025-05775},
      pages        = {1 Online-Ressource : Illustrationen},
      year         = {2025},
      note         = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
                      University; Dissertation, Rheinisch-Westfälische Technische
                      Hochschule Aachen, 2025},
      abstract     = {Komagataella phaffii (K. phaffii), formerly known as Pichia
                      pastoris has emerged as a common and robust biotechnological
                      platform organism, to produce recombinant proteins and other
                      bioproducts of commercial interest. This thesis aimed to
                      develop a bioprocess for large-scale recombinant protein
                      production in K. phaffii, focusing on the media, the
                      fermentation process, and product purification. Starting
                      with the media, a key advantage of K. phaffii is growth on
                      simple chemically defined mineral media. These media are
                      preferred in bioprocesses due to their consistent
                      composition, which minimizes batch-to-batch variation.
                      However, at elevated pH values, these media often lead to
                      precipitate formation. The precipitate was identified as
                      struvite, a low-solubility salt that binds key
                      macronutrients. This research demonstrated that struvite can
                      be redissolved under typical fermentation conditions, with
                      particle size significantly influencing the dissolution rate
                      and thus nutrient release. Therefore, struvite formation
                      should be always considered in media development to ensure
                      that dissolution kinetics are faster than the growth
                      kinetics, preventing nutrient limitations. Furthermore, a
                      model-based approach incorporating Monod kinetics was
                      developed to optimize feeding strategies, aiming to enhance
                      space-time yield (STY) and product concentrations to
                      minimize protein production costs. Specific productivity and
                      oxygen demand were derived from experimental data obtained
                      in continuous fermentations. The model also accounted for
                      physical constraints, such as the maximal oxygen transfer
                      capacity, to ensure technical feasibility. Validation
                      against experimental data showed good overall agreement,
                      though oxygen demand was slightly underestimated during the
                      production phase. Implementing a linear feed process
                      extension further improved both STY and product
                      concentration while maintaining oxygen transfer at its
                      maximum capacity. Additionally, this research addressed an
                      overlooked aspect of K. phaffii fermentation processes: the
                      presence of an exopolysaccharide (EPS) impurity in the
                      supernatant. Linked to a mutation in the HOC1 gene, this
                      impurity was found to correlate with biomass formation and
                      was independent of substrate or protein production. Finally,
                      the study explored the impact of glucose feeding strategies
                      on overflow metabolite formation, particularly ethanol, in
                      K. phaffii fermentations. High glucose concentrations caused
                      substantial ethanol accumulation, reducing biomass yield and
                      product formation. By optimizing glucose concentrations and
                      feeding strategies, overflow metabolites were significantly
                      reduced, improving both biomass yield and product
                      concentration.},
      cin          = {416510},
      ddc          = {620},
      cid          = {$I:(DE-82)416510_20140620$},
      typ          = {PUB:(DE-HGF)11},
      doi          = {10.18154/RWTH-2025-05775},
      url          = {https://publications.rwth-aachen.de/record/1013898},
}