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

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.

The production of small therapeutic proteins or peptides in E.coli is advantageous in industry due to the relatively low costs of production. However, a specific limitation of the these therapeutic products is they often have suboptimal biophysiochemical features e.g. short elimination half life, instability and immunogenicity. An approach to improve these characteristics is post-production modification of the protein, which means the attachment of natural or synthetic polymers, and a very established method is PEGylation, the covalent attachment of PEG to a therapeutic protein.

We will develop a E.coli strain which can efficiently produce therapeutic proteins as well as add a site-specific glycan (GalNAc) residue. This glycan will act as the site for PEGylation in vitro using an sialyltransferase enzyme, also optimised in this project.

We have generated a prototype strain which adds the glycan effectively and on an in silico designed interferon alpha acceptance site.

We will use synthetic biology and an iterative metabolic engineering strategy to debottleneck production. At each stage, the exemplar therapeutic protein (IFNa-2b) will be tested in well established fermentation production platforms and tested for biophysical characteristics (i.e. solubility, stability etc.).

This proposal has great potential to generate an economically competitive therapeutic protein production system for the biopharmaceutical sector.

This is an interdisciplinary application at the interface of biological science, biological engineering, and chemistry with several potential impacts as follows.

Economic and societal impacts: Biopharmaceutical compounds in production internationally are valued at over £200 billion, with UK companies owning around ca. £20 billion (TSB, June 2012). Of this, 10% of licensed products sales are represented by biopharmaceuticals, with this figure rising to a third of the products in development (TSB). Industry has a choice of platforms for their manufacture, including animal, plant and microbial cells. Microbial production is industrially vital, and ca. 30% of the genuinely new biopharmaceuticals approved between 2006-10 employed the Escherichia coli platform (Walsh, 2010, Nat Biotechnol, 28, 917-24). We focus on E. coli here.

Academic Impact: Major beneficiaries include academics who study recombinant protein production, microbiology, peptide design and controlled biomolecular assembly, and most importantly industrial biopharmaceutical production. This would also be an exemplar project in the field of synthetic biology and metabolic engineering in the UK. The modular microbial glycosylation process, engineered high expression host strain and sialylation modules that are at the core of this proposal will also be of benefit to the synthetic-biology community more broadly as new parts/components will be generated that could aid in the assembly, production and/or secretion of other industrially important macromolecules. We believe this to be the case because many engineering changes we propose ought to be generic.

Research staff: The proposed development of improved glycoPEGylated proteins requires researchers who will work collaboratively with others from a number of different fields across biological engineering and micro/molecular biology, and the biologics production industries. The project provides opportunities for publishing in new scientific areas, presenting to diverse audiences and strengthening the direction of their careers. The research staff will benefit by meeting with BRIC member companies, publishing in high-impact journals and will be encouraged to be active in the preparation of subsequent funding applications. In addition, we seek, by leveraging the BBSRC-BRIC funding, to build critical mass of researchers in the area of synthetic biology with a focus on applications based research that will impact the area of microbial production of recombinant proteins - especially biopharmaceuticals (where there is a demonstrable need, and we have expertise). Thus any investment will have an impact on the face of interdisciplinary research in the UK.

Industry: Production of cost-effective antibody fragment drugs has great potential for translation into industry. The model we examine here (IFNalpha) appears to be a suitable model (discussed with BRIC member companies). This programme could place the UK at the forefront of using microbial systems to rapidly add site specific modifications to therapeutic antibody fragments and will be a significant advance for the UK to lead, with associated economic benefits.