Possibilities and limitations of computer assisted chiral HPLC method development for ozanimod on polysaccharide based chiral stationary phases

Abstract

Abstract In this study, a direct HPLC method was developed to determine the enantiomeric purity of the immunomodulatory drug, ozanimod. A systematic method development process was followed, incorporating risk assessment, identification of critical analytical procedure parameters, initial screening of stationary phases, and software-assisted optimization of method parameters. Eight different polysaccharide-based chiral columns were selected to assess chiral separation of enantiomers under polar organic elution mode. The most promising results were obtained using a methanol:2-propanol mixture on the amylose-based Chiralpak AD column. Following this, systematic modeling was conducted using DryLab software to optimize method conditions, including isocratic eluent composition, temperature, and flow rate. Baseline separation was achieved within fifteen minutes using the optimized parameters: Chiralpak AD column thermostated at 10 °C, and a mobile phase of methanol:2-propanol: diethylamine, 70:30:0.1 (v/v/v %), delivered at a flow rate of 0.8 mL/min. The developed method was validated according to current guidelines and in silico robustness testing was conducted to determine tolerance limits for critical separation parameters and their impact on enantioresolution. Our findings demonstrate the utility of DryLab, typically employed for reversed-phase achiral separations, in optimizing chiral methods even in polar organic mode. Limitations of the selected approach the development of chiral separation methods are also highlighted.

Keywords

• Ozanimod
• Chiral separation
• Polysaccharide-type chiral column
• AQbD
• DryLab
• Computer-assisted method development