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Quantitative analysis of recombinant protein release using the Bacterial Ghost platform technology / Daniela Ehgartner
AuthorEhgartner, Daniela
CensorHerwig, Christoph ; Sagmeister, Patrick
Description145 S. : Ill., graph. Darst.
Institutional NoteWien, Techn. Univ., Dipl.-Arb., 2013
Document typeThesis (Diplom)
Keywords (DE)Bacterial Ghost Technologie, Downstream process, bone morphogenetic protein 2, multivariate Datenanalyse, Soft-Sensor, Design of experiment
Keywords (EN)Bacterial Ghost platform technology, downstream processing, bone morphogenetic protein 2, multivariate data analysis, soft-sensor, design of experiment
URNurn:nbn:at:at-ubtuw:1-96416 Persistent Identifier (URN)
 The work is publicly available
Quantitative analysis of recombinant protein release using the Bacterial Ghost platform technology [9.25 mb]
Abstract (English)

Bone morphogenetic protein (hBMP-2) is a bone growth factor applied on bone defects and for osteoporosis treatment. In recombinant production of hBMP-2 the downstream process is very time-consuming and costly. One common step in downstream processing is cell rupture, for instance homogenization. Bacterial ghosts are envelopes of Gram negative bacteria, which emerge by the formation of a lysis tunnel through the phage protein E. pBAD is an expression system providing triggering of protein production by the amount of L-arabinose in the medium and therefore avoiding high metabolic loads. The aim of the study was to investigate the applicability of the bacterial ghost platform for the release of recombinant protein in a fermentation process. Furthermore, the applicability of a soft-sensor in process development was examined. A DoE using specific D-glucose and L-arabinose uptake rates as critical process parameters was conducted. During induction phase a mixed feed system with D-glucose as the primary carbon source and L-arabinose as the secondary carbon source and inducer was applied. The specific substrate uptake rates were controlled by a first principle soft-sensor. Lysis efficiency was optimal (99%) at high total specific substrate uptake rates and low concentrations of L-arabinose in the feed. The maximal bone morphogenetic protein titer (5.61.5g/l) and the highest purity (714%) were reached at a low total specific substrate uptake rate (0.1g/g/h). There was no difference between protein titers comparing homogenization and E-lysis as cell rupture methods. Purity was higher in homogenized samples, but E-lysed samples were purer than samples without cell rupture. An optimal point for the fusion of bone morphogenetic protein production using the pBAD system and the bacterial ghost platform was found at 0.2g/g/h specific D-glucose and 0.05g/g/h specific L-arabinose uptake rate (92.520% lysis efficiency, 4.550.84g/l protein titer). Although the physiological state of the cells at the end of production phase needs to be considered, the bacterial ghost platform provides a micro-biological alternative to classical mechanical cell rupture methods. The soft-sensor was successfully applied for the control of critical process parameters in the process development.

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