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.