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Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions

Authors

  • Ulrich Roessl,

    1. Research Center Pharmaceutical Engineering, Graz, Austria
    2. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria
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  • Johanna Wiesbauer,

    1. Research Center Pharmaceutical Engineering, Graz, Austria
    2. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria
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  • Stefan Leitgeb,

    1. Research Center Pharmaceutical Engineering, Graz, Austria
    2. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria
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  • Ruth Birner-Gruenberger,

    1. Institute of Pathology and Center for Medical Research, Medical University Graz, Graz, Austria
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  • Bernd Nidetzky

    Corresponding author
    1. Research Center Pharmaceutical Engineering, Graz, Austria
    2. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria
    • Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, 8010 Graz, Austria
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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 Cys18, which is the only free cysteine in G-CSF; one of them had an intermolecular Cys18(A)-Cys18(B) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing.

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