Kelly Ronan, Parga Catalina, Ferguson Steven
Department of Chemical and Bioprocess Engineering, University College Dublin, D04 V1W8 Dublin 4, Ireland.
SSPC, The Research Ireland Centre for Pharmaceuticals, School of Chemical and Bioprocess Engineering, University College Dublin, D04 V1W8 Dublin 4, Ireland.
Org Process Res Dev. 2025 May 13;29(6):1577-1592. doi: 10.1021/acs.oprd.5c00117. eCollection 2025 Jun 20.
In this article, a new one-pot liquid-phase oligonucleotide synthesis (OP-LPOS) route enabled by organic solvent resistant (OSR) ceramic membranes is described. This approach was demonstrated through the synthesis of 6mer and 18mer 2'-OMe phosphorothioate oligonucleotides with high stepwise filtration yields (97-100%), and high crude purity (∼72% for 18mer) using just 1.5 equiv of phosphoramidites. Ceramic organic solvent nanofiltration (OSN) and ultrafiltration (OSU) membranes were used to selectively retain the growing oligonucleotide, which is reversibly tethered to a 4-arm branched PEG support, facilitating lower molecular weight reaction byproducts to permeate to waste. This is the first application of ceramic ultrafiltration membranes in such an application, which enables purification of intermediate products in just 5 diavolumes with high permeance (13 Lm h bar). We employ a one-pot approach that integrates sequential coupling, sulfurization, and detritylation steps, followed by a single membrane purification step per chain extension cycle. Analysis of the methodology indicates that the homogeneous reactions and separation performance, which use commercially available reagents and highly scalable membrane systems, represent a promising alternative to solid-phase oligonucleotide synthesis (SPOS) for large-scale manufacturing of therapeutic oligonucleotides. Furthermore, the combination of OP-LPOS with membrane separation increases intermediate product purity and yield. It reduces the number of unit operations, cycle times, and process mass intensity (PMI) compared to the previous state-of-the-art membrane-based LPOS.
本文描述了一种由耐有机溶剂（OSR）陶瓷膜实现的新型一锅法液相寡核苷酸合成（OP-LPOS）路线。通过合成6聚体和18聚体2'-O-甲基硫代磷酸酯寡核苷酸证明了该方法，其逐步过滤产率高（97-100%），且仅使用1.5当量的亚磷酰胺时粗品纯度高（18聚体约为72%）。陶瓷有机溶剂纳滤（OSN）膜和超滤（OSU）膜用于选择性保留不断增长的寡核苷酸，该寡核苷酸可逆地连接到四臂支化聚乙二醇载体上，促进低分子量反应副产物渗透到废料中。这是陶瓷超滤膜在该应用中的首次应用，其能够在仅5倍体积置换下以高渗透通量（13 Lm h bar）纯化中间产物。我们采用一锅法，该方法整合了顺序偶联、硫化和脱三苯甲基步骤，随后在每个链延伸循环中进行单个膜纯化步骤。对该方法的分析表明，使用市售试剂和高度可扩展的膜系统的均相反应和分离性能，对于大规模生产治疗性寡核苷酸而言，是固相寡核苷酸合成（SPOS）的一种有前景的替代方法。此外，OP-LPOS与膜分离的结合提高了中间产物的纯度和产率。与先前基于膜的最先进的LPOS相比，它减少了单元操作数量、循环时间和过程质量强度（PMI）。
