<div class="csl-bib-body">
<div class="csl-entry">Slouka, C. (2018). <i>Custom made inclusion bodies: effects on quality attributes by variation of critical process parameters and control strategies</i> [Dissertation, Technische Universität Wien]. reposiTUm. https://doi.org/10.34726/hss.2018.60188</div>
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dc.identifier.uri
https://doi.org/10.34726/hss.2018.60188
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dc.identifier.uri
http://hdl.handle.net/20.500.12708/1888
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dc.description.abstract
E. coli is the host of choice for about 30% of the worldwide production of recombinant pharmaceuticals. In a multitude of processes, recombinant proteins are expressed as inclusion bodies (IB) in E. coli - an (in)active aggregate of misfolded produced protein. Respective IB Quality attributes (QA) should ideally be modified by the cultivation conditions in the upstream process as they have severe impact on the performance of the subsequent process chain, like the centrifugation or refolding unit operations. However, only a very few studies deal with the general quality attributes of IBs. The goal of this work was to create a process development platform for an industrial relevant recombinant produced protein, produced as IB in E. coli. The work followed a three-step approach according to the generally known and accepted process development cycle: In the first step we aimed for understanding the impact of process parameters on different IB quality attributes. We performed a quantitative process development based on the interaction of different IB QAs like size, titer and purity and their interactions on critical process parameters temperature, pH and specific growth rate (qs). These were altered using design of experiment (DoE) approaches and analyzed with different analytical techniques, like SEM, Bioanalyzer based chip technology and HPLC. QAs were monitored as a function of induction time and analyzed in a time resolved manner. It was shown, that different QAs already showed a strong time dependence within a single cultivation run and were highly correlated to each other. In the second step the knowledge on the interactions onto IB quality attributes was used to develop physiological control strategies to obtain optimized titer and size during the process. With trigger indicators like the cumulative sugar uptake, we were able to receive custom made IBs with optimized performance for further process steps. In the third step we established a process analytical tool for stable processing. As product degradation in the investigated process was also accompanied or caused by decrease in viable cell concentration, we aimed for direct determination of the viable cell concentration during the cultivations. The developed sensor is based on electrochemical impedance spectroscopy in the low frequency regime and tested on different model microorganism like E. coli and S. cerevisiae. The different parts - starting with analytical techniques for IB quality attribute determination, control of the IB production process and advances in process analytical tools - sum up to an overall closing of the process development cycle for the given product and could be seen as overall achievement of this work. The given platform knowledge of IB based cultivations may help to facilitate further process developments for new IB based products in E. coli and result in a robust downstream processing.
en
dc.language
English
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dc.language.iso
en
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dc.rights.uri
http://rightsstatements.org/vocab/InC/1.0/
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dc.subject
E. coli
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dc.subject
Upstream Development
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dc.subject
Inclusion Bodies
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dc.subject
Quality Attributes
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dc.title
Custom made inclusion bodies: effects on quality attributes by variation of critical process parameters and control strategies
en
dc.type
Thesis
en
dc.type
Hochschulschrift
de
dc.rights.license
In Copyright
en
dc.rights.license
Urheberrechtsschutz
de
dc.identifier.doi
10.34726/hss.2018.60188
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dc.contributor.affiliation
TU Wien, Österreich
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dc.rights.holder
Christoph Slouka
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dc.publisher.place
Wien
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tuw.version
vor
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tuw.thesisinformation
Technische Universität Wien
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tuw.publication.orgunit
E166 - Institut für Verfahrenstechnik, Umwelttechnik und technische Biowissenschaften