Scale-up of analytical IP-RP-HPLC methods for high-purity semi-preparative purification of oligonucleotides

Oligonucleotides (ONs) have a wide variety of applications in molecular biology, diagnostics and biotechnology. Liquid chromatographic techniques are often used for their characterization and purification. The process of scaling up chromatographic methods for ON purification is theoretically well-understood, yet practical examples and detailed considerations are often lacking. This study addresses this gap by focusing on the scale-up of analytical ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) methods to the semi-preparative scale. Key properties such as linear velocity, load and gradient profiles were investigated, to ensure consistency between analytical and preparative formats. Using identical resin across different columns and a single HPLC system with two different configurations, we successfully achieved matching separation profiles between analytical and preparative scales. The scaled-up methods allowed effective purification of studied ON samples, yielding HPLC purities of 92.9 % and higher, with recoveries ranging from 94.1 % to 99.3 %. Our results demonstrate the importance of maintaining kinetic and dynamic equivalence during the scale-up process to allow efficient purification while maintaining separation achieved on the analytical scale. This study offers a comprehensive framework for scaling analytical IP-RP-HPLC methods and collecting high purity fractions, allowing further characterization of full-length products and impurities of interest.