Nanostar Sieving for Oligonucleotides Manufacture (NanoSieveOligo)

寡核苷酸药物通过调节蛋白质的表达和基因的功能发挥作用。目前市场上有9种批准的寡聚药物，还有更多正在开发中，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。这个项目将探索一种新的精密聚合物制造概念--NanSTAR筛分--是否可以用于生产寡聚分子。大自然通过将不同的单体(核苷酸)按规定的顺序连接起来来生产寡聚分子。核苷酸的准确顺序对寡核苷酸的功能是绝对关键的。在工业上，通过将单体顺序添加到生长中的低聚物中来制造低聚物，在下一个周期之前小心地去除残留的未反应的单体，以便在序列中没有错误。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将生长中的寡聚连接到固体载体上，在添加下一个核苷酸之前，用干净的溶剂洗涤固体载体以去除残留物。当寡聚生长完成时，它从固体支撑物中分离出来。然后移除所有其他侧链保护基团，然后我们继续测试最终寡聚的纯度--是否添加了所有必需的核苷酸？由于固体载体上的反应没有完成，通常会发现60-80%的所需n-聚寡聚，以及缺失1、2、3或更多核苷酸的n-1、n-2、n-3聚体较短的寡聚“阶梯”，因此常常会出现“缺失”单体。必须移除梯子，这需要广泛而昂贵的色谱分析。固相齐聚物合成是在实验室快速制造大量低聚物的好工具，但对于每年生产数百公斤甚至数吨的量来说是个缺点。三个主要问题是：(I)人们不容易知道每个反应的程度，因为不能在固相上进行在线分析；(Ii)随着寡聚分子的生长，新鲜核苷酸扩散和反应的空间变得紧张-导致不完全偶合和因此n-1，n-2错误；以及，(Iii)很难扩大固体床的规模。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，在英国制造，并在全球范围内应用于从石化到制药的各种行业。使用OSN膜，我们最近开发了一种新的工艺，NanSTAR筛分。关键的创新是使用OSN膜将不断增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。通过将三种正在生长的聚合物连接到一个中心枢纽分子，我们创建了一个大型的NanSTAR复合体，增强了膜保留率并促进了高效分离。我们已经使用纳米STAR筛分来生产聚乙二醇酯，这是一种广泛用于药物的合成聚合物，对纯度的控制前所未有。我们还没有成功地使用纳米STAR筛分来制造寡聚糖，要做到这一点，必须克服一些挑战。在这里，我们寻求解决这些挑战--(I)通过表面修饰配体来改善我们的膜；(Ii)使用UV-Vis和31P核磁共振的在线分析来优化反应，以确保反应最终完成；以及(Iii)通过开发具有新的、提高溶解性的侧链保护基团的亚磷酰胺单体，在寡聚物长度增长时保持NanSTAR络合物的溶解性，而不需要混合溶剂。我们的“延伸”目标将是利用这项技术附着靶向部分，以增强药物输送。如果我们成功，该项目将带来一种新的寡聚制造技术，并将导致更纯、更具成本效益的寡聚可规模化应用于医疗保健和其他领域。

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.