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Protein PEGylation of unfolded proteins

ReferenceBB/F018185/1
Principal Investigator / Supervisor Professor Stephen Brocchini
Co-Investigators /
Co-Supervisors
Dr Antony Godwin
Institution University College London
DepartmentPharmaceutics
Funding typeSkills
Value (£) 80,540
StatusCompleted
TypeTraining Grants
Start date 08/10/2008
End date 07/10/2012
Duration48 months

Abstract

unavailable

Summary

Protein based medicines are now the fastest growing sector of the medicinal biotechnology sector with over 250 million patients receiving such treatments. Increasingly the majority of all new approved medicines are protein based with over 300 candidates currently in clinical trial. Unfortunately protein based medicines suffer from limitations due to rapid clearance and toxicity (especially immunogenicity). Rapid clearance necessitates increased dosing frequency which impinges on patient complience and increases the propensity for immunogenic side effects. One of the few clinically proven strategies to address these limitations of protein based medicines is based on the concept of protein PEGylation. Poly(ethylene glycol) (PEG) which is widely used in healthcare, pharmaceutical formulation, and consumer products is covalently bound to the thereapeutic protein (hence the term protein PEGylation). Understandably the first generation PEGylated proteins that are clinically use also suffer limitations in terms of cost, product performance and homogeneity, and ease of manufacture. The PEGylation technology known as disulfide bridging PEGylation which is being developed by PolyTherics addresses these issues for a large number of therapeutic proteins. This new technology which has been described peer reviewed articles in 2006-2007 (Nature Chem Bio, Nature Protocols, Bioconjugate Chemistry) has recent been found to be useful for proteins very early in their manufacture prior to purification. The PolyTherics conjugation of PEG to a protein occurs via a thermodynamic pathway that results in the annealing of native disulfide bonds from their constituent free cysteine residues. A 3-carbon bridge connects the two cysteines with PEG attached to the bridge. This methodology allows for the exploitation of the chemical efficiency and site selectivity of sulphur based addition reactions. Crucially in the case of many therapeutic proteins (e.g. cytokines, antibody fragments, cyclic peptides), there is generally an accessible disulfide near the protein's surface. Often such disulfides aid in maintaining the stability of the protein and can be modified with the insertion of a PEG linked bridge. We have published these findings in peer reviewed articles in 2007 (e.g. Theoretica Chimica Acta,additional article in Nature Protocols, Advanced Drug Delivery Reviews). To extend this PEGylation technology we have found that it is possible to exploit the thermodynamics of the conjugation reaction by achieving the conjugation during protein folding. This key finding can be exploited to site-specifically PEGylate proteins much earlier in their manufacturing processing. The technology also potentially allows for conjugation of proteins. Thus the main hypothesis of the project is to PEGylate during protein folding. We have unfolded proteins and have found the efficiency and site-specificity of he PEGylation reaction is maintained. If successful this PhD project offers the student a multidisciplined training opportunity in protein chemistry, protein expression, synthesis of PEG reagents and conjugation chemistry. Characterisation techniques both physicochemically and biologically will also be learned. If successful PEGylation during folding will be useful to aid in the PEGylation of non-glycosylated proteins. In the case where glycosylated proteins are also functional in their non-glycosylated state (e.g. erythropoietin, which will be the test protein of the project), then this approach may find considerable utility. Such proteins tend to aggregate easily in their folded state. Since PEGylation minimizes aggregation, it is felt that PEGylating during the folding process will allow efficient and site-specific PEGylation to occur for proteins that are proned to aggregation.
Committee Not funded via Committee
Research TopicsX – not assigned to a current Research Topic
Research PriorityX – Research Priority information not available
Research Initiative X - not in an Initiative
Funding SchemeTraining Grant - Industrial Case
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