Assessment of Stereochemical Comparability in Phosphorothioated Oligonucleotides by CD, ³¹P NMR, and NP1 Digestion Coupled to LC-MS

Substitution of a nonbridging oxygen for sulfur on the phosphodiester backbone of an oligonucleotide enhances its pharmacokinetic properties. This substitution creates a new chiral center at the phosphorus atom of the linkage. Oligonucleotides with >10 phosphorothioate linkages are therefore mixtures of thousands to hundreds of thousands of diastereomers. From a chemistry, manufacturing, and control perspective, it is essential to ensure manufacturing consistency and product quality by controlling the factors that influence stereochemistry during synthesis and by implementing the proper analytical tools to analyze the stereochemical composition of phosphorothioated oligonucleotides. Here, we evaluated three orthogonal methods─circular dichroism (CD), ³¹P-nuclear magnetic resonance (³¹P NMR), and nuclease P1 (NP1) digestion followed by liquid chromatography coupled to mass spectrometry (LC-MS)─for their sensitivity to detect a change in stereochemical composition and therefore their ability to monitor batch-to-batch stereochemical comparability. Whereas all three methods are able to detect changes in R/S composition, the NP1/LC-MS method demonstrates superior sensitivity compared to the spectroscopic methods (CD and ³¹P NMR) and allows spatial localization of the changes.