Development of novel purification technologies for therapeutic peptides

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.