This proposal addresses a current bottleneck on research and industrial scale protein production, by removing the need for column chromatography . The majority of recombinant protein purification on the research scale now uses affinity purification (for which column chromatography is pre-requisite) and the approach is also used widely on a process scale (e.g. therapeutic antibody preparation). New downstream processing techniques, such as those proposed are of considerable value to biomanufacturers to provide higher throughput, ease of use and economy. Techniques are sought particularly to avoid end-user column packing and qualification. This proposal seeks to eliminate column chromatography from affinity purifications and replace it with novel, generic Affinity-based Aqueous Two-Phase Systems (Af-ATPS), which will be easily scalable. Two commonly-used affinity tags, glutathione-S transferase (GST) and maltose binding protein (MBP) will be used to develop Ab-ATPS. Rather than using standard model proteins, we will develop these systems using therapeutically-relevant peptides. As an exemplar, we have selected GLP-1, a peptide relevant to new diabetic therapies. The timely relevance of this peptide is demonstrated by current multiple Phase III of clinical trials of commercial analogues of GLP-1. The primary impact of the work is to deliver a novel, generic protein-purification strategy that will deliver cheaper ways to manufacture therapeutically relevant products. This work will require a combination of molecular biologists, bio-chemical engineers and synthetic chemists. ATPS (affinity or standard) comprise two non-miscible aqueous solutions. Owing to their different densities, one solution (phase) floats on top of the other. ATPS can be used to separate complex mixtures of molecules, such as crude cell lysates. This involves mixing the protein preparation vigorously with the ATPS, which is then allowed to separate back into two layers. When the layers have re-separated, some of the components of the preparation will have segregated (partitioned) into each of the phases, depending upon the chemical natures of the phase and the component itself. Affinity ATPS is already known, but generally refers to bespoke systems designed for individual proteins. We aim to generate Af-ATPS that can exploit the existing affinity domains in common usage. Surprisingly, such generic application of ATPS is rare and, to the best of our knowledge, is limited to a few examples in which IDA-Cu2+ ligands have been linked either covalently to PEG or EOPO or else non-covalently to PVP. Alternatively, conventional IMAP resins have been added to ATPS, to 'pull out' His-tagged proteins. Finally, although it does not involve a small ligand, there is one example of a mannose binding domain being used to direct a protein to the upper phase of a galactomannan / hydroxypropyl starch ATPS, where generic application is suggested, but not demonstrated. Within this project, we aim to generate a generic, 'two-shake' system to purify any recombinant peptide that has been expressed as a fusion to GST or MBP. In essence, a bacterial lysate (there is no requirement for centrifugation) will be shaken in a first ATPS. The upper phase, containing the fusion protein, will be removed, added to a fresh lower phase and a modified protease added. Digestion of the fusion protein will proceed in the upper phase. Thereafter, a second shake/separation will leave the fusion domain and protease in the upper phase, while the required peptide segregates to the lower phase, from which it can be isolated.

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.

Antiphospholipid syndrome (APS) is an autoimmune disease. This means that it is a disease in which the immune system of the body, which is designed to protect us against infections, instead starts to attack parts of the body itself causing the disease process. Different autoimmune diseases attack different parts of the body and have different symptoms. In APS, the problem is that the immune system makes antibodies called antiphospholipid antibodies (aPL) which interact with various different types of cells. The main cells affected are in blood vessels or in the womb, so the main effects of APS are to cause clots in blood vessels, strokes in the brain and/or recurrent miscarriages. APS is one of the main causes of these problems; for example it is one of the most important causes of stroke in people under 50. The only treatments currently available to prevent clots, strokes or miscarriages in patients with APS are drugs that thin the blood and stop it from clotting. These drugs are called anticoagulants, and include warfarin and heparin. However, they have side-effects, notably a risk of bleeding, because they oppose all clotting - even the helpful clotting that occurs after an injury to stop bleeding from a wound. We seek to develop an entirely new form of treatment for APS, which does not thin the blood but which directly targets the aPL themselves. The main way in which aPL cause their harmful effects in APS is to attach themselves to a protein in the blood called beta-2-glycoprotein I (beta2GPI). Beta2GPI is present in everyone and is harmless in the absence of aPL. When aPL combine with beta2GPI, however, this combination can bind to the surfaces of cells in the blood vessels or womb, change the behaviour of these cells and thus promote clotting or miscarriage. We are developing a drug that will be designed to stop aPL binding to beta2GPI to prevent this harmful process from occurring. Beta2GPI is composed of five parts, called domains, arranged end to end like beads on a string. We know that aPL primarily attach to the end domain (Domain I or DI). Over the last 10 years our research group has developed the only system in the world for making DI in bacteria. We are now able to grow these bacteria in large quantities and purify DI from the bacterial cultures. This can be done in high-yield with the DI at over 95% purity. We have shown that this purified DI can be used to block binding of aPL from patients with APS to human beta2GPI on plastic plates and also to stop human aPL from causing clots in mice. However, DI is a small molecule, which makes it unsuitable for use as a drug because it would only be retained in the body for a few hours. To circumvent this problem we need to modify our DI to make it larger. We are doing this by a process called PEGylation, in which large polyethylene glycol (PEG) molecules are joined to smaller molecules. We have been working with a biotechnology company called PolyTherics to achieve this. PolyTherics have developed technology to PEGylate small molecules at precisely determined points on their surface. We have achieved production of three different variants of PEGylated DI, which have PEG of different sizes. Larger PEGs could be good to make the DI last longer in the body after injection but could also block the effects of DI on aPL. Therefore we need to do tests comparing all three variants to see which is best. We have already proved that our PEG-DI blocks effects of aPL from patients with APS on binding to beta2GPI, on clotting in a test tube and on formation of clots in mice. In this project we will carry out further tests to find out which form of PEG-DI is best at blocking effects of aPL then take that form forward to tests in animals. These tests will determine how long it is retained in the body and whether it has any toxic side-effects. Assuming no toxicity is found we will develop production of this PEG-DI at large scale in a form pure enough for human trials.

The bioprocess industry manufactures novel macromolecular drugs, proteins, to address a broad range of chronic and debilitating human diseases. The complexity of these protein-based drugs brings them significant potential in terms of potency against disease, but they are also much more labile and challenging to manufacture than traditional chemical drugs. This challenge is continuing to increase rapidly as novel technologies emerge and make their way into new therapies, such as proteins conjugated to chemical drug entities, DNA, RNA or lipids, or fusions of multiple proteins, which increase their potency and targeted delivery in patients. The UK holds a leading position in developing and manufacturing new therapies by virtue of its science base and has unique university capabilities underpinning the sector. Whilst revenues are large, ~£110bn in 2009 on a worldwide basis, there are huge pressures on the industry for change if demands for healthcare cost reduction and waste minimisation are to be met, and populations are to benefit from the most potent drugs becoming available. A sea change in manufacturing will be needed over the next decade if the potential of modern drugs are to make their way through to widespread distribution. Moreover there is a widely accepted skills shortage of individuals with fundamental "blue-skies" thinking capability, yet also with the manufacturing research training needed for the sector. The proposed EPSRC CDT will deliver a national capability for training the next generation of highly skilled future leaders and bioprocess manufacturing researchers for the UK biopharmaceutical sector. They will be capable of translating new scientific advances both in manufacturing technologies and new classes of macromolecular products into safely produced, more selective, therapies for currently intractable conditions at affordable costs. This is seen as essential where the rapid evolution of biopharmaceuticals and their manufacturing will have major implications for future medicine. The CDT will be a national resource linked to the EPSRC Centre for Innovative Manufacturing (CIM) in Emergent Macromolecular Therapies (EP/I033270/1), which aims to tackle new process engineering, product stability, and product analysis challenges that arise when manufacturing complex therapies based on radically new chemistry and molecular biology. The CDT will embed PhD students into the vibrant research community of the top UK Institutions, with collaborations overseen by the EPSRC CIM, to enable exploration of new process engineering, modelling, analysis, formulation and drug delivery techniques, and novel therapies (e.g. fusion proteins, and chemical drugs conjugated to antibodies), as they emerge from the international science and engineering community. Alignment to the EPSRC CIM will ensure projects strategically address key bioprocess manufacturing challenges identified by the industrial user group, while providing a cohort-based training environment that draws on the research excellence of the ESPRC CIM to maximise impact and knowledge transfer from collaborative partners to research led companies.