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.
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