Category: Formulation and Quality
Purpose: Antisense Oligonucleotides (ASO) are typically formulated in buffered solution (e.g. artificial cerebrospinal fluid or phosphate buffered solutions). The amounts of the inorganic ions (e.g. sodium, potassium, magnesium, calcium, chloride, and phosphate) need to be controlled in order to meet the final target formulation. During process development, these ions are measured to ensure the solution composition does not change due to the ultrafiltration/diafiltration (UF/DF) process and to ensure the final targets are met in the drug product (DP). Currently, inductively coupled plasma optical emission spectrometry (ICP-OES) is used, but only for cation analysis. The goal was to develop a QC-friendly quantitative method for both cation and anion analysis of formulated ASO drugs with a simple liquid chromatography (LC) approach.
Methods: A mixed-mode HILIC LC column, with anion-exchange and cation-exchange functionalities, was used in conjunction with a charged aerosol detector (CAD). Water and ammonium formate buffer were selected as the mobile phases.
Results: LC conditions and CAD parameters, such as injection volume (Figure 1), pH (Figure 2), gradient program, evaporation temperature and data filter constant, were optimized to obtain separation of six ions with suitable sensitivity (Figure 3). This method allows for analyzing monovalent and multivalent cation and anions ions simultaneously. ASOs were spiked in the solution to show the retention times and peak shapes are not impacted by the matrix. This method was also shown be to quantitative.
Conclusion: A quantitative method for inorganic ion analysis in ASO drugs was successfully developed. Multiple cations and anions are separated with adequate resolution with and without the presence of ASO. The column washing procedure, sample preparation, and run time are currently being optimized. With this LC-CAD approach, ion analysis for ASO drugs can be performed in most analytical labs to support drug development.