Improving biopharmaceutical production in microbial systems: Engineering GlycoPEGylation in E.coli

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We aim to produce an example therapeutic protein (medicine) in the bacterium Escherichia coli (E. coli) that can be purified and then efficiently modified to improve its biological and physical characteristics and thus overall effectiveness. Although ca. 30% of the genuinely new biopharmaceuticals (protein-based medicines) approved between 2006-10 employed E. coli, there is an opportunity to improve this host system. For example, smaller proteins or protein fragments such as antibody fragments can be made more efficiently in E. coli compared to mammalian or plant cell systems due to relatively inexpensive growth requirements, high cell densities and high protein yields. Although effective as medicines, the smaller size of these proteins means they have a higher clearance rate in humans (ie the drug is removed by the kidneys), reducing overall efficacy of the dose. This project builds on the concept of post-production modification to increase the circulatory half-life of these type of proteins (the drug lasts longer in the body). An inert, synthetic polymer, polyethylene glycol (PEG) is commonly used in industry and will be employed here - its attachment to the drug is known as PEGylation. The target protein IFN-a2b (a member of the interferon family of medicine known as cytokines) will serve as the exemplar 'drug' for this project as it is a well understood and widely manufactured therapeutic agent. In addition, it has been PEGylated previously and it's selection has been supported by BRIC industrial partners (Lonza and Fuji Diosynth). Optimising the process of PEGylation has received a lot of attention as the efficiency directly translates into manufacturing costs (high efficiency means reduced manufacturing costs). Traditional methods have led to random PEGylation that means many different protein forms are made, reducing productivity (and increasing costs). Several site-directed methods were subsequently proposed including one where the protein is purified from E. coli and then two enzymes (biological catalysts) are used in a separate process outside E .coli after the protein has been made (in vitro) to add a sugar (enzyme 1) and then sugar-PEG (enzyme 2). The process is referred to as glycoPEGylation. This project builds on this concept and exploits the newly discovered ability of E. coli to glycosylate proteins (add sugar groups to the protein in the cell) using enzymatic machinery from another microorganism (BRIC1 - funded). By designing a sugar (glycosylation) attachment site into the protein target, we have shown that the sugar-adding (glycosylation) machinery in E. coli can recognise and add a specific sugar to the site (with IFN-a2b and other proteins such as GFP). We propose that this modified protein can be purified and then used in a one step reaction outside the cell where PEG is added. The requires a specific enzyme that will be designed and optimised.We will use a combination of cutting edge biological engineering techniques, now considered part of an emerging field known as synthetic biology, to manipulate E. coli to produce the modified protein target IFN-a2b. We will employ in-house metabolic engineering strategies (forward and backward/inverse) to improve yields. To improve PEGylation efficiency, the sugar acceptance site in IFN-a2b will be varied to optimise enzyme recognition of the added sugar. For rapid translation to industry the optimised cell system and protein will be tested in bioreactors which we have already shown increases antibody fragment production yields in E. coli. We wish to gain insight into how easy this product would be to manufacture (manufacturability) and we will design fermentation with E. coli and discuss this with BRIC members.For quality control, the modified IFN-a2b will be tested for biophysical stability throughout using a combination of tools. Also, cost comparisons to the existing site-directed glycoPEGylation methodology, will be performed throughout.