Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Nihon Waters KK, Kitashinagawa, Shinagawa, Tokyo 140-0001, Japan.
J Am Soc Mass Spectrom. 2024 Sep 4;35(9):2156-2164. doi: 10.1021/jasms.4c00197. Epub 2024 Jul 31.
Therapeutic oligonucleotides such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA) are among the most remarkable modalities in modern medicine. ASOs and siRNA are composed of single- or double-stranded 15-25 mer synthesized oligonucleotides, which can be used to modulate gene expression. Liquid chromatography-mass spectrometry (LC/MS) is a necessary technique for the quality control of therapeutic oligonucleotides; it is used to evaluate the quantities of target oligonucleotides and their impurities. The widely applied oligonucleotide therapeutic quantitation method uses both ultraviolet (UV) absorbance and the MS signal intensity. Peaks separated from the main peak, which contains full-length product, are generally quantitated by UV. However, coeluting impurities, such as - 1 shortmers, abasic oligonucleotides, and PS → PO (phosphorothiate to phosphodiester) oligonucleotides, are quantitated by MS. These coeluting impurities can also be comprised of various isomers with the same modification, thus increasing the difficulty in their separation and relative quantitation by LC/MS. It is possible that a specific isomer with a certain structural form induces toxicities. Therefore, characterization of each isomer separation is in high demand. In this study, we separated and characterized oligonucleotide isomers by employing a cyclic ion mobility mass spectrometry (cyclic IMS) system, which allows the separation of ions with the same / ratio based on their structural differences. Patisiran antisense and sense strands and their - 1 and abasic isomers were used as sample sequences, and their ratio characterization was achieved by cyclic IMS. In addition, we evaluated the PS → PO conversion isomers of the antisense strand of givosiran, which originally contained four PS modification sites. The PS → PO isomers exhibited specific and distinguishable mobiligram patterns. We believe that cyclic IMS is a promising method for evaluating therapeutic oligonucleotide isomers.
治疗性寡核苷酸，如反义寡核苷酸（ASO）和小干扰 RNA（siRNA），是现代医学中最显著的治疗方法之一。ASO 和 siRNA 由单链或双链 15-25 个碱基组成的合成寡核苷酸组成，可用于调节基因表达。液相色谱-质谱联用（LC/MS）是治疗性寡核苷酸质量控制的必要技术；它用于评估目标寡核苷酸及其杂质的数量。广泛应用的寡核苷酸治疗定量方法同时使用紫外（UV）吸光度和 MS 信号强度。从包含全长产物的主峰中分离出的峰通常通过 UV 进行定量。然而，共洗脱的杂质，如 -1 短链、无碱基寡核苷酸和 PS→PO（磷硫代酯到磷酸二酯）寡核苷酸，通过 MS 进行定量。这些共洗脱的杂质也可以由相同修饰的各种异构体组成，从而增加了通过 LC/MS 进行分离和相对定量的难度。特定的异构体可能具有特定的结构形式会引起毒性。因此，需要对每个异构体的分离进行特征描述。在这项研究中，我们通过使用循环离子淌度质谱（cyclic IMS）系统分离和表征寡核苷酸异构体，该系统允许根据结构差异分离具有相同 / 比的离子。使用 patisiran 反义链和有义链及其 -1 和无碱基异构体作为样品序列，并通过循环 IMS 实现它们的比率特征。此外，我们评估了 givosiran 反义链的 PS→PO 转换异构体，该异构体最初含有四个 PS 修饰位点。PS→PO 异构体表现出特定且可区分的淌度图谱。我们相信循环 IMS 是评估治疗性寡核苷酸异构体的一种很有前途的方法。
