Nanostar Sieving for Oligonucleotides Manufacture (NanoSieveOligo)

Oligonucleotide (oligo) medicines work by modulating the expression of proteins and the functioning of genes. There are now 9 approved oligo drugs on the market and many more in development, and there is a growing need for an efficient manufacturing technology to make these high value molecules. This project will explore whether a new manufacturing concept for precise polymers, Nanostar Sieving, can be adapted to produce oligo molecules.

Nature makes oligos by joining different monomers (nucelotides) in a prescribed sequence. The exact order of the nucleotides is absolutely crucial to the oligo function. Oligos are made industrially by sequential addition of monomers to growing oligos, taking care to remove residual, unreacted monomer before the next cycle, so that there are no errors in the sequence. This requires excellent separation at the end of each coupling cycle. A very effective way of doing this is to attach the growing oligo to a solid support, which is washed with clean solvents to remove residuals, before the next nucleotide is added. When oligo growth is complete, it is cleaved from the solid support. All other side chain protecting groups are then removed, and we proceed to test the purity of the final oligo - have all the required nucleotides been added? Often there are "missing" monomers because the reactions on the solid support did not go to completion, and it is typical to find 60-80% of the desired n-mer oligo, together with a "ladder" of n-1, n-2, n-3 mer shorter oligos which are missing 1, 2, 3 or more nucleotides. The ladder must be removed, and this requires extensive, and expensive, chromatography.

Solid Phase Oligo Synthesis is a great tool for rapidly making lots of oligos in the lab, but has drawbacks for manufacturing hundreds of kg or even multi-ton quantities per year. The three major problems are: (i) one cannot know the extent of each reaction easily, because in-line analysis cannot be done on the solid phase; (ii) as the oligo grows, the space for the fresh nucleotides to diffuse in and react gets tight - leading to incomplete couplings and so n-1, n-2 errors; and, (iii) it is hard to scale up the solid beds.

Research at Imperial College has pioneered Organic Solvent Nanofiltration (OSN), using membranes that are stable in organic solvents to separate small molecules from large molecules. These membranes have been commercialised, and are manufactured in the UK and employed globally in industries ranging from petrochemicals to pharmaceutical manufacture. Using OSN membranes, we have recently developed a new process, Nanostar Sieving. The key innovation is to use OSN membranes to separate a growing polymer from unreacted monomers. This is carried out in the liquid phase and analysis is relatively straightforward. By connecting three growing polymers to a central hub molecule, we create a large nanostar complex, enhancing membrane retention and promoting efficient separation. We have used Nanostar Sieving to produce PEG, a synthetic polymer used widely for medicines, with unprecedented control over purity.

We have not yet been successful at making oligos using Nanostar Sieving, and to do so have to overcome a number of challenges. Here we seek to address these challenges - (i) to improve our membranes with surface modifying ligands; (ii) to use in-line analysis with UV-Vis and 31P NMR to optimise reactions end ensure they reach completion; and (iii) to maintain the solubility of the nanostar complex as the oligos grow in length, without the need for mixed solvents, by developing phosphoramidite monomers with new, solubility-enhancing side chain protecting groups. Our "stretch" goal will be to use the technology to attach targeting moieties to enhance drug delivery. If we are successful, the project will result in a new technology for oligo manufacture, and will lead to purer, and more cost-effective oligos becoming available at scale for applications in healthcare and beyond.

The economic benefits of the research proposed are the business around manufacturing and selling the high value oligonucleotides that Nanostar Sieving produces, and the economic and societal benefits of the applications of these materials.

The Livingston Group at Imperial College has a demonstrated record in the commercialisation of membranes derived from its research. Evonik MET, based in West London, is the leaading dedicated manufacturing facility for organic solvent nanofiltration membranes in the world. The fundamental processes they use were developed at Imperial College with EPSRC research funding. Therefore the group has experience in the development of manufacturing processes and commercialisation of research, and specifically research based around membranes. In 2018 a group comprising AGL (the PI), PRJG (the Researcher Co-I), Imperial Innovations and 11 other current and former post-docs and PhDs from Livingston Group, founded Exactmer Ltd, a new start-up whose initial aim is to develop the production of exact PEG polymers via Nanostar Sieving. The first products offered by Exactmer are unimolecular PEG of 5kDa molecular weight, heterobifunctionalised with end groups suitable for PEGylation and for use in antibody drug conjugates.

If the proposal is successful, it will show that Nanostar Sieving has high potential in oligonucleotide manufacturing. The most immediate likely interest in this technology and the materials it produces will come from pharmaceutical manufacturers working on development programmes with oligo drugs, and companies actively manufacturing and supplying oligo molecules to the pharmaceutical industry. One route to commercial impact is through Exactmer, who might develop and scale up the technology. Since it is based on liquid phase synthesis, it should be easy to retrofit a membrane separation unit to an existing reaction vessel to create a viable process. Alternatively, there may be the opportunity for direct licensing, where this makes more economic sense. In either case, economic benefits, including investment in capital equipment and creation of skilled jobs, will derive from the manufacture of oligos and from installation of suitable assets for the manufacturing process.

The second economic impact will come through the application of oligos to healthcare and other sectors. We envisage that they will be used as biologically active agents for medicines, with obvious patient benefits. The rate of new approvals of oligo drugs is growing rapidly, with 3 FDA approvals in 2018 and 9 oligo drugs in total on the market now. It is estimated that there are 130 oligo based drugs currently undergoing clinical trial programmes, which is an indication of the potential scale of the impact on the healthcare industry. Beyond healthcare, oligos produced by nanostar sieving might also find application in DNA nanotechnology, or for information storage where there is a growing interest in using strands of DNA to store information and reduce reliance on data centres, which are becoming major energy consumers. Oligo manufacture could be developed as a specialist high value sector of the UK chemical economy. This would lead to a major impact on jobs and capital investment across a broad part of the UK pharma / chemical sector.

These economic benefits have parallel social benefits. The installation of manufacturing processes, and the manufacture of high value polymers, creates high level and knowledge intensive employment, and improves the national accounts through exports achieved.

Finally, there would be substantial benefits to other UK academic research groups ho who would be invited to use the Nanostar Sieving Synthesiser to study the formation of oligos of interest and produce samples for research.