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Non‐native aggregation of recombinant human granulocyte‐colony stimulating factor under simulated process stress conditions
Author(s) -
Roessl Ulrich,
Wiesbauer Johanna,
Leitgeb Stefan,
BirnerGruenberger Ruth,
Nidetzky Bernd
Publication year - 2012
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201100436
Subject(s) - chemistry , protein aggregation , cysteine , recombinant dna , biophysics , intramolecular force , aeration , chromatography , denaturation (fissile materials) , biochemistry , stereochemistry , enzyme , organic chemistry , nuclear chemistry , biology , gene
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.