Harmonization of a Physicochemical Profiling Platform of Therapeutic Oligonucleotides

Although various physicochemical characterization methods are used for therapeutic oligonucleotides, no established profiling protocols are available in pharmaceutical industry, as done for small molecule drugs. Especially, higher-order structure of oligonucleotides requires more attention, as it is related to formulation properties such as viscosity, solubility and efficacy. In the profiling process in pharmaceutical industry, in addition to the scientific evaluation, business aspects such as the amount of available oligonucleotides and the timing of the evaluation must also be considered.
In this presentation, I will introduce collaborative work with five Japanese pharmaceutical companies to establish profiling protocol of physicochemical properties of therapeutic oligonucleotides. We have first evaluated thrombin aptamer, which is known to form strong higher-order structure, and its modified ones, as model compounds to establish physicochemical profiling platform of oligonucleotides (Mol. Pharmaceutics 21, 3471-3484 (2024)), where UV, CD, DSC, FT-IR, and NMR were used for the analysis. Focus was made on required amount of oligonucleotides and timing of evaluation as well as impact of base sequence on formation of the higher-order structure. Then, formulation properties including solubility and viscosity of highly concentrated solutions were evaluated to find relevance between those properties and the higher-order structure. Also, an attempt was made to predict the higher order structure of concentrated solution from low concentration data by reducing activity of water. Malat 1, which forms only weak higher-order structure, and its modified forms were next evaluated on the same evaluation platform for confirming applicability of the same characterization protocol. Limitation and complementary roles of each evaluation method will also be discussed.