Nutzung eines 16-fach parallelen Bioreaktorsystems zur Entwicklung von Prozessen für die Herstellung von rekombinanten Proteinen in Mikroorganismen

In this thesis the 16-fold parallel fermentation device fedbatch-pro®, commercially available from the company DASGIP in Jülich, was used to develop processes for the production of recombinant proteins in Hansenula polymorpha. Due to the improvement of the aeration system and the development of a robust fermentation strategy for parallel cultivation, the Hansenula-clone FMD-HSA 56-1 reached dry cell weights of up to 60 g/L and rHSA concentrations of 250 mg/L in the fedbatch-pro® device. After scale-up to a 5-L stirred tank reactor, dry cell weights of 66 g/L and rHSA concentrations of 289 mg/L were measured (values related to total fermentation volume). During co-expression of the chaperones calnexin and proteindisulfideisomerase, a 3.2-fold increase in specific product yield was observed in the parallel fermentation device. In the 5-L stirred tank reactor, the specific product yield was increased 1.8-fold compared to the Hansenula-clone without co-expressed chaperones. By means of several chromatographic methods, rHSA could be purified from Hansenula culture supernatant. While the recovery rate of rHSA was relatively low (values of 27% to 45%), the purity of rHSA was comparable to a commercially available therapeutic HSA product. The formation of a 52 kDa degradation product could neither be prevented by tuning the fermentation conditions nor be separated from the mature 66.5 kDa HSA molecule by chromatographic methods. This degradation product is likely formed by cell wall-associated proteases and might be avoided by targeted knock-out of these proteases. Initially, product concentrations obtained in the parallel fermentation system showed only a low reproducibility. In two 16-fold experiments, statistical spreads in product concentration of 63% and 90% respectively, were noticed. In contrast to this, the cell dry weight data showed only statistical spreads of 7% and 3% respectively. It became apparent that the biomass formation was subject to a more robust mechanism than the product formation. However, with the use of duplicate experiments, adequate reaction conditions could be identified for the production of rHSA. In consideration of data variance, tendencies of different reaction conditions regarding product formation could be recognized and a selection of appropriate fermentation conditions could be carried out. Inaccuracies in the aeration system were identified as one reason for the high deviation in product concentration. Condensate in the off-gas filters led to different aeration rates in the respective vessels. These differences in aeration conditions made an impact on growth and glucose consumption leading to different derepression conditions of the FMD-Promoter and finally resulted in different product concentrations. After removal of the off-gas filters, the statistical spread of product concentration could be reduced to 3% in a four-fold parallel fermentation. For future fermentation experiments the use of off-gas filters with a higher volumetric flow capacity or off-gas condensers should be considered. In addition to the improvement of the albumin process, the process development of a commercially relevant protein was described in this thesis. Using both the fedbatch-pro® device and a 5-L stirred tank reactor, the product concentration could be increased from initially not detectable concentrations to 0.25 g/L in the fedbatch-pro® device and up to 1.87 g/L in the stirred tank reactor within 6 weeks.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Kleebank, Sebastian

Contributors dc:contributor

• Fischer, Rainer

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• Fermentation
• Hansenula polymorpha
• Humanes Serumalbumin
• Fed-batch-Verfahren
• Parallelbetrieb
• parallel fermentation
• HSA
• fed-batch

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123234%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61583