The use of multiple liquid chromatography methods augmented by phosphorus-31 nuclear magnetic resonance to characterize the diastereomer composition in synthetic oligonucleotides

Synthetic oligonucleotide therapeutics represent a rapidly advancing class of drugs that target RNA to modulate gene expression. The phosphorothioate modification to the nucleic acid backbone is routinely used to increase resistance to enzymatic degradation and improve the pharmacological profile. Each phosphorothioate modification creates a stereogenic center, leading to the formation of diastereomers at every modified linkage. This results in a final drug product containing a complex and heterogenous mixture of diastereomers. With the increasing regulatory approval of synthetic oligonucleotide drug products, there is a pressing need to develop analytical methods that can characterize their diastereomer composition. We detail the use of four liquid chromatography methods complemented by ³¹P NMR to characterize the diastereomer composition of fully phosphorothioated short (2- and 5-mers) and full-length (20-mers) oligonucleotide sequences. We also assessed the effect of how specific chemical activators influenced the diastereomer composition in these synthetic sequences. Our data showed that the use of multiple liquid chromatography methods augmented by ³¹P NMR provided complementary and additional insight into the diastereomer composition of phosphorothioate-linked oligonucleotides. Further, our data also indicated that the chemical activator substantially influenced the diastereomer content but other important factors including the overall chemical synthesis process and sequence specific nucleobases and modifications play an important role as well.