Development and validation of a novel ion-pair hydrophilic interaction liquid chromatography method for the separation and quantification of oligonucleotide therapeutics

Background: This study developed and systematically optimized an ion-pair hydrophilic interaction liquid chromatography (IP-HILIC) system for oligonucleotide medicine analysis, aiming to the issues of insufficient resolution and prolonged retention times commonly encountered in traditional hydrophilic interaction liquid chromatography (HILIC) and ion-pair reversed-phase liquid chromatography (IP-RPLC). Therefore, this study aimed to optimize and validate a TEAA-based IP-HILIC analytical system for both the effective separation and quantitative analysis of oligonucleotides, including evaluations of linearity, sensitivity, precision, and robustness. Methods: By synthesizing an amino/hexadecyl-bifunctionalized silica matrix and systematically optimizing mobile phase parameters (including ion-pair reagent type and concentration, organic solvent, and buffer pH), a highly efficient separation system was established. To validate the separation efficiency, a 21-mer oligonucleotide Fomivirsen and its truncated impurities (5′-21–1 and 3′-21–1) were synthesized, along with Food and Drug Administration(FDA)-approved Kynamro (20-mer), Macugen (28-mer), and Exondys 51 (30-mer) for method evaluation. Results: Experimental results demonstrated that under pH 8.0 conditions, a mobile phase system composed of 50 mM triethylamine acetate (TEAA) and acetonitrile enabled baseline separation of 20–30-mer oligonucleotides with a resolution ≥1.5. Using Fomivirsen impurities (e.g., 5′-21–1 and 5′-21–3) as examples, the retention times were optimized to between 3.76 and 6.76 min, achieving a resolution of 3.82. Acetonitrile shortened the analysis time by 34% compared with methanol, while 50 mM TEAA maintained resolution in the range of 2.58–3.71 and avoided the excessive retention seen at 100 mM, where the peak time extended to 7.57 min.Method validation demonstrated that the method exhibited good linearity (R²>0.999) over the concentration range of 0.05–10 μg/mL, with a limit of detection (LOD) of 0.2 μM and a limit of quantitation (LOQ) of 0.6 μM. The method also showed satisfactory precision (intra-day RSD < 1.5%, inter-day RSD < 2.1%) and was robust against minor variations in mobile phase composition, column temperature, and flow rate. Compared to traditional methods, IP-HILIC demonstrated significantly superior resolution (1.50–2.09) for long-chain oligonucleotides (e.g., 30-mer Exondys 51) over IP-RPLC (1.30) and HILIC (1.20), with better peak symmetry (tailing factor <1.2). Conclusion: This study provides an systematically optimized and validated analytical platform for the quality control of oligonucleotide medicines, particularly showing significant advantages in the separation of key impurities (e.g., n-1 sequences) for long-chain oligonucleotides (20–30 mer), laying a technical foundation for the precise analysis of hydrophilic biopolymers.