Nanostar Sieving for Oligonucleotides Manufacture (NanoSieveOligo)

寡核苷酸（oligo）药物通过调节蛋白质的表达和基因的功能发挥作用。现在市场上有9种已批准的寡聚药物，还有更多的药物正在开发中，人们越来越需要一种高效的制造技术来制造这些高价值的分子。该项目将探索精密聚合物的新制造概念Nanostar Sieving是否适用于生产寡聚分子。大自然通过将不同的单体（nucelotide）按规定的顺序连接在一起来制造寡聚物。核苷酸的确切顺序对寡核苷酸的功能至关重要。寡聚物在工业上是通过将单体依次加入到生长的寡聚物中来制备的，在下一个循环之前注意去除残留的未反应的单体，使得序列中没有错误。这需要在每个耦合周期结束时进行出色的分离。一种非常有效的方法是将生长中的寡核苷酸附着在固体载体上，在加入下一个核苷酸之前，用干净的溶剂洗涤固体载体以去除残留物。当寡聚物生长完成时，将其从固体支持物上裂解。然后去除所有其他侧链保护基团，我们继续测试最终寡核苷酸的纯度-是否添加了所有所需的核苷酸？通常存在“缺失的”单体，因为在固体支持物上的反应没有完成，并且通常发现60-80%的所需n聚体寡核苷酸，以及缺失1、2、3或更多个核苷酸的n-1、n-2、n-3聚体较短寡核苷酸的“梯”。必须去除阶梯，这需要大量且昂贵的色谱法。固相寡聚物合成是在实验室中快速制备大量寡聚物的重要工具，但每年生产数百公斤甚至数吨的量存在缺点。三大问题是：（ii）随着寡核苷酸的生长，用于新鲜核苷酸扩散和反应的空间变紧-导致不完全偶联，因此产生n-1、n-2个错误;以及，（iii）难以按比例放大固体床。帝国理工学院的研究开创了有机溶剂纳滤（OSN），使用在有机溶剂中稳定的膜将小分子与大分子分离。这些膜已经商业化，在英国制造，并在全球范围内用于从石油化工到制药制造的行业。使用OSN膜，我们最近开发了一种新的工艺，Nanostar筛分。关键的创新是使用OSN膜将生长的聚合物与未反应的单体分离。这是在液相中进行的，分析相对简单。通过将三种生长的聚合物连接到一个中心分子上，我们创造了一个大型的纳米星复合物，增强了膜的保留能力，促进了有效的分离。我们已经使用Nanostar Sieving生产PEG，这是一种广泛用于药物的合成聚合物，对纯度进行了前所未有的控制。我们尚未成功地使用Nanostar Sieving生产寡核苷酸，并且必须克服许多挑战。在这里，我们寻求解决这些挑战-（i）用表面改性配体改善我们的膜;（ii）使用UV-Vis和31 P NMR的在线分析来优化反应并确保它们达到完成;和（iii）通过开发具有新的，溶解度增强侧链保护基团。我们的“延伸”目标将是使用该技术连接靶向部分以增强药物递送。如果我们成功，该项目将为寡核苷酸制造带来一种新技术，并将导致更纯净，更具成本效益的寡核苷酸在医疗保健等领域的应用中大规模可用。

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.