Molecular diffusion of oligonucleotides: Influence of temperature, ion-pairing reagents, and chemical modifications...

Oligonucleotides (ONs) are an exciting and relatively novel class of therapeutics that selectively target DNA and RNA sequences to prevent the production of disease-related proteins. To ensure patient safety and therapeutic efficacy, robust analytical monitoring of oligonucleotides and their potential impurities is required. High-performance liquid chromatography (HPLC) and more specifically, ion-pairing (IP) HPLC, is considered the gold standard for this. However, the lack of a fundamental understanding of the band broadening mechanisms of ONs in HPLC currently hampers a more knowledge-driven column development. The molecular diffusion coefficient (D_m) of ONs is one of the key parameters in this context. In this work, D_m -values of ONs were systematically evaluated under a wide range of relevant chromatographic conditions using the Taylor-Aris method. Measurements of various poly-deoxythymidine (poly(dT)) ONs between 30 and 60 °C revealed a strong and consistent influence of temperature, with higher temperatures increasing ON diffusion. To further characterize this behaviour, constants relating D_m to viscosity and temperature were determined and subsequently applied to estimate D_m-values for diverse ONs under varying conditions, with maximum residual errors of 34% in the absence of ion-pairing reagents (IPRs) and 16% in the presence of IPRs. The presence of IPRs in the mobile phase consistently reduced D_m compared to measurements without IPRs, producing D_m-values very close to the theoretical D_m-values predicted by Young's equation. Additionally, similar D_m-values were obtained for ONs with IPRs of different hydrophobicity, indicating a limited influence of these IPR types on ON diffusion. Similarly, chemical modifications such as phosphorothioate linkages (PS), 2'-O-Methyl (2'-O-Me) and 2'-O-Methoxyethyl (2'-O-MOE) did not significantly affect ON diffusion coefficients. In contrast, locked nucleic acid (LNA)-modified ONs showed an important reduction in D_m, with D_m-values being >40% lower than those of the corresponding PS-modified ONs under identical conditions. This reduction may be attributed to the extra protruding cyclic ring which is expected to increase the hydrodynamic radius of LNA-modified ONs. Furthermore, ONs with different secondary structures showed diffusion behaviour comparable to poly(dT) ONs, both in the presence and absence of an IPR.