Roche Kulmbach, GmbH, Kulmbach, Germany.
Anal Biochem. 2011 Jul 1;414(1):47-57. doi: 10.1016/j.ab.2011.02.040. Epub 2011 Mar 2.
Small interfering RNAs (siRNAs) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. The development of siRNA-based therapeutics requires in-depth knowledge of the manufacturing process as well as adequate analytical methods to characterize this class of molecules. Here the impurity formation during the annealing of siRNA was investigated. Two siRNAs containing common chemical RNA modifications (2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-deoxy-ribose, and phosphorothioate linkages) were used to determine major side reactions-such as 2',3'-isomerization, strand scission, and HF elimination-depending on annealing parameters such as RNA concentration, presence of cations, temperature, and time. Individual impurities were characterized using analytical size exclusion chromatography, denaturing and nondenaturing ion-pair reversed-phase high-performance liquid chromatography, differential scanning calorimetry, and ultraviolet spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. The data reported here provide background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics such as aptamers, spiegelmers, and decoy and antisense oligonucleotides.
小干扰 RNA（siRNA）作为一种新的治疗方式，可特异性抑制靶基因表达。siRNA 类药物的研发需要深入了解制造工艺以及足够的分析方法来对这类分子进行特征描述。本文研究了 siRNA 退火过程中杂质的形成。使用两种含有常见化学 RNA 修饰（2'-O-甲基、2'-脱氧-2'-氟、2'-脱氧核糖和硫代磷酸酯键）的 siRNA，以确定主要的副反应，如 2'，3'-异构化、链断裂和 HF 消除，这取决于 RNA 浓度、阳离子存在、温度和时间等退火参数。使用分析性尺寸排阻色谱法、变性和非变性离子对反相高效液相色谱法、差示扫描量热法和紫外光谱法对各个杂质进行了特征描述。本文描述的降解途径可能导致产品质量显著降低和药物活性受损。这里报道的数据为成功解决与 siRNA 及其他核酸治疗药物（如适体、 Spiegelmers、诱饵和反义寡核苷酸）制造相关的挑战提供了背景。
