title: Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions.
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.
authors.affiliation: Research Center Pharmaceutical Engineering, Graz, Austria; Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Roessl
authors.forename: Johanna
authors.initials: J
authors.lastname: Wiesbauer
authors.forename: Stefan
authors.initials: S
authors.lastname: Leitgeb
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Gruenberger
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D004220:Disulfides / Q000737:chemistry; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008956:Models, Chemical; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor