At-line analysis of antisense oligonucleotide purified fractions using fast chromatographic methods

In the manufacturing of antisense oligonucleotides (ASOs), rapid analytical methods are crucial for efficient process control and quality assurance. However, current analytical techniques for ASO characterization are time-consuming, creating bottlenecks in the manufacturing process. To address this challenge, this study developed and implemented a fast chromatographic method for at-line analysis of ASO purified fractions, with a focus on integration with Multi-column Counter-current Solvent Gradient Purification (MCSGP). A fast 6.5-minute ion-pair liquid chromatography method with UV and MS detection was developed. Method parameters were optimised, including injection volume (2-4 µL) and sample concentration (0.1-0.2 mg/mL), to balance speed, sensitivity, and chromatographic performance. The method performance was comparable to a validated 40-minute LC-UV-MS method using various ASO samples, including conjugated crude and purified materials. The fast method demonstrated improved limits of detection and quantification compared to the conventional method, while maintaining linearity (R² > 0.99) and robust performance with high-salt MCSGP samples (up to 60 mM NaCl). Comparable accuracy was achieved for main components (UV purity, MS purity, % full length-n; %RSD < 2 %) across different sample types. The method's speed and compatibility with at-line and potential online analysis represent a significant advancement in ASO manufacturing process control.