Scalable Membrane Enabled One-Pot Liquid-Phase Oligonucleotide Synthesis

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^-2 h^-1 bar^-1). 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.