title: Disulfide-bridging PEGylation during refolding for the more efficient production of modified proteins.
abstract: Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide-bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG-mono-sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide-bridging conjugation to give the PEGylated proteins. Interferon-β1b (IFN-β1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging-conjugation during refolding. The PEG-IFN-β1b was isolated by ion-exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN-β1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN-β1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.
authors.affiliation: UCL School of Pharmacy, London, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Ginn
authors.affiliation: PolyTherics Ltd, Babraham Research Campus, Babraham, Cambridge, UK.
authors.forename: Ji-Won
authors.initials: JW
authors.lastname: Choi
authors.affiliation: UCL School of Pharmacy, London, UK. steve.brocchini@ucl.ac.uk.
authors.forename: Steve
authors.initials: S
authors.lastname: Brocchini
mesh_terms: D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D000068576:Interferon beta-1b / Q000737:chemistry / Q000302:isolation & purification; D008958:Models, Molecular; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000737:chemistry*; D058849:Protein Refolding; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D006163:Ribonuclease T1 / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D004220:Disulfides; D011506:Proteins; D000068576:Interferon beta-1b; D011092:Polyethylene Glycols; D006163:Ribonuclease T1; D003545:Cysteine
keywords: Disulfide-bridging PEGylation; Process integration; Protein modification; Refolding PEGylation