(T1030-01-01) Development of Physicochemical Profiling Platform for Therapeutic Oligonucleotides

Purpose: Oligonucleotide, as a novel class of therapeutic modality, usually has been formulated as a highly concentrated solution. Highly concentrated solution may have problems including aggregation, liquid-liquid phase separation, and increase in viscosity, which may be caused by intra- and/or intermolecular interactions. This study aims to understand on their higher-order structure and its relationship with physiochemical properties using thrombin-binding aptamer (TBA), which forms guanine quadruplex (G4) structure, and four modified TBAs as model oligonucleotides for efficient formulation development. In addition, we attempted the establishment for comprehensive physicochemical evaluation platform of oligonucleotides.

Methods: TBA and four modified TBAs (T1 to T4) that have similar sequences but offer different intra/intermolecular interactions were synthesized. PBS_in and PBS_out, having similar aqueous environment of inside and outside of cells, respectively, were used (Table 1). TBA and four modified TBAs were evaluated using ultraviolet spectroscopy (UV), circular dichroism (CD), differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) and capillary electrophoresis (CE). In addition, the solution properties including solubility, viscosity, and stability and the antithrombin activity were investigated. Detailed analytical methods and conditions were shown in Table1.

Results: UV melting curves during the heating process are shown in Fig. 1. The first and second heating curves agreed well in both PBS_in and PBS_out. The analysis by UV revealed improved thermal stability for T1 compared to TBA presumably due to the formation of larger number of the G4 quartets, whereas no obvious melting temperature was found for T4 as expected based on its sequence (Fig. 1 and Table 1). The melting temperature (T_m) in PBS_in was higher than those in PBS_out for all samples except T4, indicating potassium ion played an important role for stabilizing the higher-order structure. The same melting behaviors were observed by DSC. Through CD spectrum, parallel G4 structure was observed only for T1. TBA, T2, and T3 mainly formed antiparallel G4 structure, and T4 did not form a high-order structure. However, NMR proved existence of multiple topologies for these oligonucleotides. We compared the physicochemical properties such as solubility, viscosity, and stability focusing on T1 and T4, because of their large difference in the higher-order structure. The solubility was much lower and the viscosity was much higher for T4 relative to T1 (Table 1). In addition, the shear rate dependence of the viscosity of T4 solution showed that T4 solution has the non-Newtonian behavior. These investigations revealed importance to form the higher-order structure to increase solubility and decrease viscosity of the solution. All samples exhibited higher stability in PBS_in, suggesting influence of the higher-order structure on the chemical stability (Fig. 2). It is also supported by the fact that T1 and T4 were the most stable and the most unstable, respectively, among all the samples tested. In PBS_in, the degradation appeared to be faster than expectation from the Arrhenius fit for T2, T3, and T4 at 60 °C. As T_m for T2 and T3 was found near 50 °C, the higher-order structure was likely to be responsible for chemical stabilization. Similar tendency could be found for PBS_out. The anticoagulant activity was also very sensitive to the higher-order structure.

Conclusion: We identified the intra- and intermolecular interaction and high-order structure of modified TBAs including TBA with complemental analytical techniques and clarified its influence on their physicochemical properties. These results suggested importance of elucidation of higher-order structure for development of therapeutic oligonucleotides. From a practical point of view, all these evaluation methods can be applied to only a few candidates which survived for the developability assessment and the chemistry manufacturing controls (CMC) development, because of limitation in the sample amount. These knowledges should be fruitful for deciding priority and timing of each evaluation item during developmental studies of oligonucleotides.

Acknowledgements: This study was conducted as part of Center of Excellence for Pharmaceutical Materials Science (Materials Open Platform for Pharmaceutical Science) led by National Institute for Materials Science. Conflict of Interest: All authors are employees of each affiliation.