Carloni Laure-Elie, Deschrijver Tiny, Ryvers Kirsten, Noten Bart, Stratmann Lukas M, De Vijlder Thomas
Therapeutics Development & Supply, Janssen Research & Development, NV, a Johnson & Johnson company, Beerse, Belgium.
Therapeutics Development & Supply, Janssen Research & Development, NV, a Johnson & Johnson company, Beerse, Belgium.
J Pharm Sci. 2025 Feb;114(2):1359-1367. doi: 10.1016/j.xphs.2024.12.009. Epub 2024 Dec 16.
Small interfering RNAs (siRNAs) have emerged as a highly promising class of therapeutics, capable of effectively treating a wide range of indications, including previously challenging targets. To correctly characterize the duplex content of siRNA therapeutics, a careful design of the analytical conditions is required. This is due to the weak interactions governing the duplex formation and thermal stability of these double-stranded oligonucleotides. In this study, we demonstrate that the reliability of duplex content analyses can be compromised by denaturation or hybridization artifacts caused by environmental factors related with sample preparation or with the 'non-denaturing' chromatographic analysis method. To address this issue, we propose to characterize the siRNA duplex in various analytical media with unbiased techniques such as circular dichroism spectrophotometry and use the results to evaluate potential artifacts in the 'non-denaturing' method, developed to determine the duplex content. Through this approach, one can optimize the sample preparation and develop 'non-denaturing' method conditions to minimize the influence of environmental factors on the duplex content, and thereby determine the assay of siRNA duplex with no bias.
小干扰RNA（siRNA）已成为一类极具前景的治疗药物，能够有效治疗多种适应症，包括以前颇具挑战性的靶点。为了正确表征siRNA治疗药物的双链体含量，需要精心设计分析条件。这是因为这些双链寡核苷酸的双链体形成和热稳定性受弱相互作用支配。在本研究中，我们证明，与样品制备或“非变性”色谱分析方法相关的环境因素导致的变性或杂交假象，可能会影响双链体含量分析的可靠性。为解决这一问题，我们建议采用圆二色分光光度法等无偏技术，在各种分析介质中表征siRNA双链体，并利用结果评估为测定双链体含量而开发的“非变性”方法中的潜在假象。通过这种方法，可以优化样品制备并制定“非变性”方法条件，以尽量减少环境因素对双链体含量的影响，从而无偏地测定siRNA双链体的含量。
