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起作用。目前市场上有9种经批准的低聚药物，还有更多的药物正在开发中，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。该项目将探索一种新的精密聚合物制造概念，纳米星筛分，是否可以适用于生产低聚分子。大自然通过按规定的顺序连接不同的单体（核苷酸）来制造寡核苷酸。核苷酸的确切顺序对寡核苷酸的功能至关重要。工业上，低聚物是通过向生长的低聚物中依次添加单体来制造的，在下一个循环之前，要注意去除残留的、未反应的单体，这样就不会出现序列错误。这需要在每个耦合周期结束时进行良好的分离。一种非常有效的方法是在添加下一个核苷酸之前，将生长的寡核苷酸附着在固体载体上，用干净的溶剂清洗以去除残留物。当寡糖生长完成时，它就从固体支撑中分离出来。然后去除所有其他侧链保护基团，然后我们继续测试最终寡核苷酸的纯度-是否添加了所有需要的核苷酸？通常会有“缺失”单体，因为固体载体上的反应没有完成，通常会找到60-80%所需的n-mer低聚物，以及n-1、n-2、n-3短聚物的“阶梯”，这些低聚物缺失1、2、3或更多核苷酸。梯子必须被移除，这需要大量的，昂贵的色谱。固相低聚物合成是在实验室中快速制造大量低聚物的好工具，但对于每年制造数百公斤甚至数吨的数量有缺点。三个主要问题是：(1)由于不能对固相进行在线分析，人们不能轻易地知道每个反应的程度；（ii）随着寡核苷酸的增长，新核苷酸扩散和反应的空间变得更紧，导致不完全偶联，从而导致n-1， n-2错误；(3)固体层难以按比例放大。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来分离小分子和大分子。这些膜已经商业化，并在英国制造，并在全球范围内应用于从石油化工到制药制造等行业。利用OSN膜，我们最近开发了一种新的工艺，纳米星筛分。关键的创新是使用OSN膜将生长中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枢纽分子，我们创建了一个大的纳米星复合物，增强了膜保留并促进了有效的分离。我们已经使用纳米星筛分生产聚乙二醇，一种广泛用于药物的合成聚合物，具有前所未有的纯度控制。我们还没有成功地使用纳米星筛分制造寡聚物，要做到这一点，必须克服许多挑战。在这里，我们寻求解决这些挑战- (i)用表面修饰配体改善我们的膜；（ii）使用UV-Vis和31P NMR在线分析来优化反应末端，确保它们完成；(3)随着低聚物长度的增长，不需要混合溶剂，通过开发具有新的增强溶解度的侧链保护基团的酰胺磷酸单体来保持纳米星复合物的溶解度。我们的“扩展”目标将是使用该技术附着靶向部分来增强药物输送。如果我们取得成功，该项目将带来低聚物制造的新技术，并将导致更纯净、更具成本效益的低聚物大规模应用于医疗保健等领域。

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.