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Updated: Jul 15, 2025

Chemical Triphosphorylation of Oligonucleotides
Published on: June 2, 2022
Establishing stereochemical comparability in phosphorothioate oligonucleotides with nuclease P1 digestion coupled
Zifan Li1, Fei Tong1, Li Xiao1
1Pharmaceutical Operations & Technology, Biogen, 225 Binney Street, Cambridge, MA 02142, USA. zifan.li@biogen.com.
Abstract:
Stereochemical comparability is critical for ensuring manufacturing consistency in therapeutic phosphorothioate oligonucleotides. Currently, analytical methods for this assessment are limited. We hereby report on a novel protocol capable of detecting a stereochemistry change in a single phosphorothioate linkage by employing nuclease P1 digestion of the oligonucleotide with subsequent LCMS analysis of the resulting fragments. The method proves valuable for establishing stereochemical comparability and for ensuring manufacturing consistency of oligonucleotide therapeutics.
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