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

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 (Dm) of ONs is one of the key parameters in this context. In this work, Dm -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 Dm to viscosity and temperature were determined and subsequently applied to estimate Dm-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 Dm compared to measurements without IPRs, producing Dm-values very close to the theoretical Dm-values predicted by Young's equation. Additionally, similar Dm-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 Dm, with Dm-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.