Unraveling the Stereochemical Complexity of Phosphorothioate-Modified Oligonucleotides Using Analytical Technologies

Oligonucleotide therapeutics are emerging as a promising modality for targeting disease-associated RNAs. Phosphorothioate (PS)-containing oligonucleotides have gained prominence due to their enhanced stability and pharmacodynamic properties. However, current manufacturing practices afford a mixture of Rp and Sp stereoisomers, and this distribution has been linked to changes in product efficacy. Understanding the sensitivity of analytical methods to changes in this quality attribute has therefore become critically important. Here, we used a suite of analytical techniques-ultraviolet (UV) thermal denaturation, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy-to evaluate the PS diastereomer distribution using Tegsedi, a Food and Drug Administration-approved PS-containing antisense oligonucleotide, and with other synthetic inotersen samples having varied PS diastereomer distributions. While UV and CD techniques showed limited sensitivity, NMR excelled in detecting small changes in the PS diastereomer distribution. The univariate metric of ³¹P integration was shown to be insufficient for this quality metric evaluation; application of principal component analysis to both 1D ³¹P and 2D ¹H,¹³C spectra revealed distinct PS changes that arose from the different activators used during manufacturing. This comprehensive evaluation highlights the necessity of advanced analytical techniques in ensuring the quality and consistency of PS-containing oligonucleotide therapeutics.