Decoding Oligonucleotide Stereochemistry: Individual Phosphorothioate Configuration Ratios and Intact Sequence Diastereomeric Composition

Phosphorothioate (PS) modifications in oligonucleotide therapeutics introduce chiral centers that create complex diastereomeric mixtures. This complexity presents significant analytical challenges for stereochemical characterization, which is essential for drug development, regulatory evaluation, and generic product comparability studies. Current analytical approaches face substantial limitations in directly characterizing the stereochemistry of finished products at both the intact sequence level and the individual PS linkage level simultaneously. We present a high-resolution ion mobility mass spectrometry (HRIM-MS) methodology that enables unprecedented concurrent analysis at both stereochemical levels. Using a PS-modified 3-mer sequence as a proof-of-concept model, we demonstrated HRIM-enabled baseline separation of all four expected diastereomers (Rp-Rp, Rp-Sp, Sp-Rp, Sp-Sp). This separation enabled direct measurement of intact sequence diastereomeric composition. Through in-source fragmentation and analysis of the resulting 2-mer fragment ions, each containing a single defined PS linkage, we directly measured Rp/Sp configuration ratios at individual PS linkages. Intriguingly, our analysis revealed a discrepancy between directly measured intact sequence diastereomeric compositions and those predicted from directly measured individual PS configuration ratios in some samples. This finding demonstrates that the intact diastereomeric composition may deviate from simple statistical combinations of individual PS configuration ratios. This has potential implications for oligonucleotide therapeutics, as batches with similar individual PS configuration ratios may exhibit substantially different intact sequence diastereomeric profiles, potentially affecting therapeutic performance or equivalence to the reference listed drug. This study underscores the importance of comprehensive stereochemical characterization at multiple levels for establishing rigorous quality standards, ensuring manufacturing consistency, and enabling confident comparability assessments of oligonucleotide therapeutics.