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<div class="csl-entry">Prada, E., Igwe, C. L., Müller, D. F., Gisperg, F., Pauk, J. N., Kierein, M., Elshazly, M., Klausser, R., Kopp, J., & Spadiut, O. (2024, April 10). <i>Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence</i> [Conference Presentation]. 8th BioProScale Symposium, Berlin, Germany. http://hdl.handle.net/20.500.12708/208484</div>
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dc.identifier.uri
http://hdl.handle.net/20.500.12708/208484
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dc.description.abstract
Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need of additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labour-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. We implemented intrinsic tryptophan and tyrosine fluorescence as a tool for real-time and in situ monitoring of protein refolding for the first time. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L-1. The change of protein conformation during refolding is reflected as a shift in the position of maximum of tryptophan and tyrosine fluorescence spectrum as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique was demonstrated on the refolding of different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, our methodology poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.
en
dc.description.sponsorship
Christian Doppler Forschungsgesells
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dc.language.iso
en
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dc.subject
Inclusion bodies
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dc.subject
Refolding
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dc.subject
Fluorescence
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dc.title
Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence
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dc.type
Presentation
en
dc.type
Vortrag
de
dc.contributor.affiliation
Process Digitalization - Bioprocessing - Competence Center CHASE GmbH, Austria
