Why and How to Control P-Chirality in Phosphorothioated Therapeutic Oligonucleotides: Analytical Challenges Associated with Determination of Stereochemical Composition

A surge in the approval of therapeutic oligonucleotides since 2016 has made this class of chemical modalities an essential toolbox for addressing unmet medical needs in the realm of rare or difficult-to-treat diseases. Therapeutic oligonucleotides are complex drugs, primarily due to the active pharmaceutical ingredient, which presents numerous oligonucleotide-specific impurities that are challenging to address during the manufacturing process. A less common challenge is the control of the stereochemical composition of P-chiral morpholino and phosphorothioate therapeutic oligonucleotides. These are produced in a non-stereoselective manner, resulting in a mixture of different stereoisomers that collectively form the final drug substance. Thus, the control of stereochemical composition has an important role in the manufacturing process of therapeutic oligonucleotides, either as an in-process control or a final characterization test. In this Perspective, we first present the current status of oligonucleotide therapeutics, with a focus on phosphorothioate derivatives. We then highlight the regulatory framework for controlling the stereochemical composition of these drugs, provide brief insights into the biological significance of stereochemical composition for phosphorothioates, and discuss the origin of stereochemical composition in the synthetic and downstream processes. Subsequently, we provide an in-depth analysis of analytical methods used to determine the stereochemical composition of therapeutic oligonucleotides, including liquid chromatography approaches in various separation modes and emerging modern techniques like ion mobility spectrometry. Furthermore, capillary electrophoresis, duplex melting temperature, and NMR spectroscopy are also discussed in the context of stereochemical composition analysis of therapeutic oligonucleotides. Finally, we highlight novel enzymatic methods for cleaving therapeutic oligonucleotides into smaller fragments that can be analyzed for stereochemical composition using standard techniques.