Characterizing Oligomers Using a 2D-LC–MS Workflow

Sarapeta Therapeutics researchers based in Cambridge, Massachusetts developed a new method for analyzing phosphorodiamidate morpholino oligomers (PMOs) using two-dimensional liquid chromatography–mass spectrometry (2D-LC/MS). Their findings were published in the Journal of Chromatography A (1).

Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that can inhibit gene expression in a sequence-dependent manner (2). They are distinguished from natural nucleic acids and other oligonucleotide (ON) therapeutic platforms by the attachment of nucleobases to six-membered morpholino rings, as opposed to the 5-membered ribose rings found in RNA. The morpholine rings are connected through uncharged phosphorodiamidate linkages, which enhance resistance to enzymatic degradation and improve safety compared to earlier ON-modified backbone chemistries. With these unique features, PMO has become an increasingly important class of antisense oligonucleotide (ASO) therapeutics to treat various infectious diseases, such as Ebola and Duchenne muscular dystrophy (DMD). Four PMO therapeutics are approved by the United States Food and Drug Administration (U.S. FDA) for DMD including eteplirsen, golodirsen, casimersen, and viltolarsen.

In this study, a sensitive two-dimensional liquid chromatography/mass spectrometry (2D-LC/MS) workflow was developed to characterize PMOs. This workflow employed strong cation exchange chromatography (SCX) as the first dimension (¹D), and hydrophilic interaction chromatography (HILIC) as the second dimension (²D); in both dimensions, ammonium formate was used as the MS-compatible salt. Peaks of interest were collected using multiple heart cutting (MHC) and transferred to ²D HILIC for online desalting and orthogonal separation prior to high resolution MS (HR-MS) analysis for impurity identification. Active solvent modulation (ASM) was used to address the elution strength incompatibility between ¹D SCX and ²D HILIC. This allowed for improved peak shapes, leading to enhanced resolution and increased peak capacity with the 2D-LC approach.

To demonstrate the workflow, the scientists analyzed a 30-mer PMO spiked with n-1 deletion impurity markers. Subsequently, these were applied to effectively elucidate the impurity profiles of three PMO drug substances (ranging from 22-mer and 30-mer), thus affording impurity identification at low levels. The impurity profiles suggested common impurity classes were formed in PMOs, despite varying lengths or sequences, and were structurally related to their parent PMOs.

According to the researchers, the study presents the first application of a 2D-LC/MS workflow for PMO analysis, demonstrating improvements in resolution, impurity profiling, and MS sensitivity. Integrating of an automated online desalting step further streamlines the process, enabling efficient impurity analysis with accelerated data turnaround. The developed method offers significant value throughout the drug development lifecycle, from early-phase characterization to aid in process development and understanding, to comparability assessments following process changes or manufacturing site transfers.

Despite the second-dimension HILIC separation was primarily employed as a sample desalting tool, it also made notable contributions to improved resolution. Future research will focus on optimizing the HILIC dimension to enhance chromatographic performance while extending the method’s applicability to other types of ONs, which could require different separation strategies. Overall, the versatile 2D-LC/MS platform shows potential as a robust analytical tool for comprehensive impurity characterization and control in ON therapeutics.

References

(1) Wei, T.; Wang, M.; Cai, B. Z. Development of a Two-Dimensional Liquid Chromatography/Mass Spectrometry Workflow for Characterization of Phosphorodiamidate Morpholino Oligomers. J. Chromatogr. A 2025, 1757, 466129. DOI: 10.1016/j.chroma.2025.466129

(2) Deere, J.; Iversen, P.; Geller, B. L. Antisense Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition in Escherichia coli. Antimicrob. Agents Chemother. 2005, 49 (1), 249–255. DOI: 10.1128/AAC.49.1.249-255.2005