Effect of pore size on plate height for 23-mer oligonucleotides separated using fully porous particles and ion-pairing reversed-phase liquid chromatography.

Identification and quantification of impurities in therapeutic oligonucleotides (ONs) by liquid chromatography is a critical component of control strategies used in pharmaceutical manufacturing. While ion-pairing reversed-phase liquid chromatography (IPRP-LC) remains the gold standard for these separations, the fundamental influence of stationary phase physical parameters on the kinetic performance of ON separations remains under-characterized compared to the knowledge base that exists for small molecule separations. In this work we have investigated the effect of stationary phase pore size and particle size on plate height for a 23-mer surrogate therapeutic oligonucleotide under IPRP conditions. Plate heights (H) were determined under isocratic conditions for eight fully porous stationary phases, including polymeric and silica-based materials, with pore sizes ranging from 80 to 4000 Å and particle sizes from 1.8 to 5.0 µm. To enable comparability across different materials, mobile phase conditions were carefully adjusted to obtain plate height data at a constant retention factor of 2. The results show that intraparticle pore size is a stronger determinant of plate height than particle size for the ON studied here. For silica-based materials, increasing the pore size from 80 Å to 300 Å resulted in significantly lower plate heights and a weaker dependence of efficiency on mobile phase velocity. Remarkably, a column packed with 3.5 µm, 300 Å particles yielded a three-fold lower plate height than a 1.8 µm, 80 Å column at practically relevant flow rates. Moreover, we found that the observed trends in isocratic plate heights translated well to solvent gradient elution conditions that are more relevant to practical separations of ON mixtures. Finally, results from a brief set of pore penetration experiments using poly-thymidine standards suggest that diffusion of ONs larger than five bases through the intraparticle pores of conventional stationary phase materials (80-120 Å) is probably highly hindered. These findings underscore the importance of using wider-pore (e.g., >200 Å) stationary phases to maximize resolution in the analysis of therapeutic ONs.