Using Polybutylene Terephthalate-Based Stationary Phase to Analyze Oligonucleotides

As more nucleic acid-based pharmaceuticals enter the market, scientists are increasingly looking for ways to better analyze synthetic oligonucleotides.

As part of an effort to better analyze oligonucleotides, a team of scientists, led by Michael Lämmerhofer of the University of Tübingen, tested a polybutylene terephthalate (PBT)-based stationary phase under ion-pairing free RP mode for the liquid chromatography electrospray ionization mass spectrometry (LC–ESI-MS) analysis of siRNA, specifically Patisiran. Their findings were published in the Journal of Chromatography A (1).

Michael Lämmerhofer | Image Credit: © Michael Lämmerhofer

These methods “act by Watson–Crick base-pairing and, therefore, RNA can theoretically target any gene of interest by selecting a specific nucleotide sequence on the targeted RNA,” Lämmerhofer said. They are typically administered in lipid nanoparticles (LNPs), which deliver the nucleic acid drugs and consist of substances like cholesterol and phospholipids. Endocytosis incorporates these LNPs into target cells.

The team compared the resulting retention profile and chromatographic orthogonality to other LC-modes, such as hydrophilic-interaction chromatography (HILIC), ion-pairing RPLC, mixed-mode chromatography (MMC), all while factoring in normalized retention times. If both dimensions have similar retention times, coelution will likely occur in both separations, leading to no gain in selectivity and resolution with 2D-LC. Moreover, orthogonal retention mechanisms increase the chance that first-dimension coelutions will be resolved in the second dimension. Comparatively, while in an ion-pairing-free mode, the PBT-bonded RPLC column held superior results on both fronts, showing promise in the process they were hoping to use it for.

To accommodate the higher orthogonality of this method, the ion-pairing free PBT-bonded RPLC was marked as the first dimension in a selective and comprehensive 2D-LC setup, with the second dimension being HILIC. The resulting combination allowed for improved separation and resolution for comprehensively analyzing siRNA samples, enabling a more accurate process of detecting impurity peaks.

According to Lämmerhofer, “Analytical chemistry needs complementary methods for i) confirmation of the results from one method, ii) for having a proper overview on different molecular entities, iii) for resolving the full complexity of molecular systems.”

Reference

(1) Li, F.; Chen, S.; Studzińska, S.; Lämmerhofer, M. Polybutylene terephthalate-based stationary phase for ion-pair-free reversed-phase liquid chromatography of small interfering RNA. Part 2: Use for selective comprehensive two-dimensional liquid chromatography. J. Chromatogr. A 2023, 1701, 464069. DOI: https://doi.org/10.1016/j.chroma.2023.464069

About the Author

Aaron Acevedo is the Assistant Editor of LCGC and Spectroscopy. Direct correspondence to: aacevedo@mjhlifesciences.com