Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
Roessl, U; Wiesbauer, J; Leitgeb, S; Birner-Gruenberger, R; Nidetzky, B.
Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions.
Biotechnol J. 2012; 7(8):1014-1024
Doi: 10.1002/biot.201100436
Web of Science
PubMed
FullText
FullText_MUG
- Co-Autor*innen der Med Uni Graz
- Birner-Grünberger Ruth
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- Abstract:
- Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate. G-CSF was precipitated rapidly from solutions that were aerated or stirred; only small amounts of soluble aggregates were found. Exposed hydrophobic surfaces were a characteristic of both soluble and insoluble G-CSF aggregates. Using confocal laser scanning microscopy, the aggregates presented mainly a circular shape. Their size varied according to incubation time and stress applied. The native intramolecular disulfide bonds in the insoluble G-CSF aggregates were largely disrupted as shown by mass spectrometry. New disulfide bonds formed during aggregation. All involved Cys(18) , which is the only free cysteine in G-CSF; one of them had an intermolecular Cys(18(A)) -Cys(18(B)) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing.
- Find related publications in this database (using NLM MeSH Indexing)
- Bioreactors -
- Biotechnology - methods
- Disulfides - chemistry
- Granulocyte Colony-Stimulating Factor - chemistry
- Humans -
- Microscopy, Confocal -
- Models, Chemical -
- Models, Molecular -
- Recombinant Proteins - chemistry
- Find related publications in this database (Keywords)
- Granulocyte-colony stimulating factor
- Process conditions
- Protein aggregation
- Protein pharmaceuticals