title: Integrated continuous dissolution, refolding and tag removal of fusion proteins from inclusion bodies in a tubular reactor.
abstract: An integrated continuous tubular reactor system was developed for processing an autoprotease expressed as inclusion bodies. The inclusion bodies were suspended and fed into the tubular reactor system for continuous dissolving, refolding and precipitation. During refolding, the dissolved autoprotease cleaves itself, separating the fusion tag from the target peptide. Subsequently, the cleaved fusion tag and any uncleaved autoprotease were precipitated out in the precipitation step. The processed exiting solution results in the purified soluble target peptide. Refolding and precipitation yields performed in the tubular reactor were similar to batch reactor and process was stable for at least 20 h. The authenticity of purified peptide was also verified by mass spectroscopy. Productivity (in mg/l/h and mg/h) calculated in the tubular process was twice and 1.5 times of the batch process, respectively. Although it is more complex to setup a tubular than a batch reactor, it offers faster mixing, higher productivity and better integration to other bioprocessing steps. With increasing interest of integrated continuous biomanufacturing, the use of tubular reactors in industrial settings offers clear advantages.
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Siqi
authors.initials: S
authors.lastname: Pan
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Monika
authors.initials: M
authors.lastname: Zelger
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria; Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria; Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria. Electronic address: rainer.hahn@boku.ac.at.
authors.forename: Rainer
authors.initials: R
authors.lastname: Hahn
mesh_terms: D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D002479:Inclusion Bodies / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D010447:Peptide Hydrolases / Q000378:metabolism*; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases
keywords: Autoprotease fusion protein; Continuous bioprocessing; Protein precipitation; Protein refolding; Tubular reactor