Roche Kulmbach GmbH, Kulmbach, Germany.
Nucleic Acid Ther. 2011 Dec;21(6):383-93. doi: 10.1089/nat.2011.0317. Epub 2011 Oct 21.
A manufacturing and purification process for duplex oligonucleotides was established, which shortens and simplifies currently used procedures, yielding a product of higher purity. The reported procedure is based on nondenaturing anion-exchange (AEX) chromatography, which is performed on the annealed duplex rather than the individual single strands. The duplex is formed early in the process by annealing of the crude single strands directly after solid-phase synthesis. Two 30 μmol manufacturing runs using duplex purification were performed on 2 different AEX resins and compared with a manufacturing run of the same scale using conventional single-strand chromatography. The same pooling strategy was employed for all purifications. Content of optimal duplex (duplex exclusively comprising full-length single strands) was 90.5% and 90.2% for the batches obtained by duplex purification and 86.1% for the batch obtained by single-strand purification. Maximum chromatographic recoveries were 67% for the duplex purification and 68% for the single-strand purification. Hence, the manufacture of small interfering RNA (siRNA) using duplex purification was simpler and faster than conventional single-strand purification and provided better purity and similar yield of final siRNA.
建立了一种双链寡核苷酸的制造和纯化工艺，该工艺缩短并简化了目前使用的程序，得到了更高纯度的产物。所报道的方法基于非变性阴离子交换（AEX）层析，该方法在退火的双链上进行，而不是在单个单链上进行。该双链在固相合成后直接退火粗单链的早期过程中形成。使用两种不同的 AEX 树脂进行了两次 30 μmol 的制造运行，以双链纯化进行，与使用常规单链层析进行的相同规模的制造运行进行了比较。所有的纯化都采用相同的汇集策略。通过双链纯化获得的批次的最佳双链（仅由全长单链组成的双链）含量为 90.5%和 90.2%，通过单链纯化获得的批次的最佳双链含量为 86.1%。双链纯化的最大色谱回收率为 67%，单链纯化的最大色谱回收率为 68%。因此，使用双链纯化制造小干扰 RNA（siRNA）比传统的单链纯化更简单、更快，并且提供了更好的纯度和类似的最终 siRNA 产率。
