Establishing stereochemical comparability in phosphorothioate oligonucleotides with nuclease P1 digestion coupled with LCMS analysis

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 phosphorothi...

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Bibliographic Details
Published in:Analyst (London) 2023-10, Vol.148 (21), p.5361-5365
Main Authors: Li, Zifan, Tong, Fei, Xiao, Li, Larson, Nicholas R, Zhou, Xuan, Zhang, Yueheng, Immel-Brown, Jonas P, Bou-Assaf, George M
Format: Article
Language:English
Subjects:
Consistency
Digestion
Manufacturing
Nuclease
Oligonucleotides
Stereochemistry
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Summary: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. NP1 digestion coupled with UHPLC-HRMS can provide valuable information on stereochemical comparability between phosphorothioate oligonucleotides by examining digestion fragments.
ISSN:0003-2654
1364-5528
DOI:10.1039/d3an01392h