Abstract
The number of therapeutic oligonucleotides on the market and in clinical trials has increased gradually over the last two decades but little change has been seen in their commercial synthesis. Until recently, the demand for oligonucleotides has been fulfilled by traditional solid-phase oligonucleotide synthesis (SPOS) which suffers from scalability limitations. Liquid phase oligonucleotide synthesis (LPOS) uses a soluble support and is a possible scalable alternative. Organic solvent nanofiltration (OSN) membrane assisted synthesis of oligonucleotides using a PEG soluble support has been demonstrated as a potential solution to the scalability issues with SPOS.
Current LPOS methods require a minimum of 2 reaction steps and 2 separation steps by either precipitation, liquid-liquid extraction or membrane filtration. In this work a one-pot liquid phase oligonucleotide synthesis (OP-LPOS) method was developed which allows sequential one-pot coupling, sulfurization and deprotection followed by a single membrane separation step per nucleotide extension cycle. In-line reaction and purification monitoring were performed by HPLC to ensure reactions and filtration go to completion. The growing oligonucleotide is reversibly tethered to a 5 kDa 4-arm branched PEG soluble support to improve separation. A ceramic nanofiltration membrane (750 Da MWCO) selectively retains the growing PEG-oligo and permeates the smaller reaction by-products and impurities. Combining OP-LPOS with membrane separation is beneficial as it reduces the number of unit operations and solvent consumption. Phosphorothioate oligonucleotides were produced using this method with high stepwise filtration yields (84-94%) and lose to quantitative product rejection by the membrane (99.9%). This process is readily scalable due to the use of LPOS, standard phosphoramidite reagents and commercially available soluble support and nanofiltration membrane.
