Development of multiple heartcutting two-dimensional liquid chromatography with ion-pairing reversed-phase and...

As the clinical applications of RNA-based medicines continue to grow, innovative analytical approaches are essential to assess quality attributes of these complex therapeutic molecules. Synthetic oligonucleotides (ONs) such as small interfering RNA and antisense oligonucleotides are an expanding sub-class of genetic medicines that typically consist of single- or double-stranded RNA or DNA molecules ranging from 20 to 30 nucleotides in length. Characterization of synthetic ONs presents considerable analytical challenges due to their relatively large molecular size, polyanionic nature, and the potential for numerous synthetic modifications and impurities. It is becoming increasingly clear that conventional chromatographic methodologies are not always sufficient to fully resolve the full impurity profile present in these samples. 2D-LC is a potentially effective solution, enabling rapid resolution of species that co-elute from the first dimension using a complementary second dimension separation. In this work we explore the potential utility of pairing HILIC separation in the second dimension as a complement to a first dimension ion-pairing reversed-phase separation. Using a 23-mer surrogate ON and several closely related structures representing potential impurities, we find that the selectivities of the IPRP and HILIC separations are highly complementary, and thus suitable for pairing in a 2D separation format. Moreover, the mass loadability of the two separations are similar under the conditions studied, which gives the user flexibility when choosing which separation to use in the first dimension. We then demonstrate that an iterative retention modeling approach can be used to systematically discover shallow second dimension gradients needed to resolve species that co-elute from the first dimension column. In an unexpected twist, we found that the interface conditions must be optimized when using Active Solvent Modulation, because the weakest diluent (i.e., high ACN for a HILIC separation) does not always yield the best second dimension separation. Finally, we developed IPRP-HILIC methods for two different ON sequences that demonstrate improved resolution of previously co-eluting impurities and sufficient sensitivity for detection at 0.1 % relative to the full length product.