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.

OPTIMA aims at demonstrating photonic payloads for telecommunication satellites by joining the efforts of industrial and academic European actors from both the worlds of space and terrestrial communications. In the near future, a major increase in telecoms satellites capacity is required to address the challenges of the Digital Agenda for Europe, and to remain in line with the skyrocketing evolution of terrestrial communications, in a globally connected world. A major technological breakthrough is needed to meet the capacity increase objectives within the mass, size and power envelope allowed by the foreseen evolution of launchers and satellite platforms. Photonics has largely contributed to the revolution of Information Technology for ground applications and is the most promising technology to overcome the issues faced by Satcoms, thanks to the compact, lightweight and low-power nature of optical-fibre based equipment. However, great efforts are required to bring these benefits to the world of telecoms payloads as all the photonics equipment used on ground need to be adapted for space. In OPTIMA, Airbus Defence and Space (UK, FR), a world-leading satellite prime manufacturer, will define, assemble and a test photonic payload demonstrator based on building blocks developed, adapted for space and provided by other members of the consortium: DAS Photonics (ES), Linkra (IT), SODERN (FR), IMEC (BE) and Polatis (UK). By gathering all these actors around a concrete project, in a real-world industrial environment, OPTIMA will provide a strong initial impulse to make photonics technology available to the Satcom industry and pave the way towards an in-orbit demonstration as early as 2020. This will not only allow the European space industry to address the challenges of the DAE 2020, but also strengthen its position in a very competitive, worldwide market and create new opportunities for each of the members of the consortium (new applications, products and markets).

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.