Sub/supercritical Fluid Chromatography Purification and Desalting of a Cyclic Dinucleotide STING Agonist

•An “endotoxin-free” purification and desalting of a cyclic dinucleotide (CDN) was achieved.•The purification and desalting were scaled up to produce multigram quantities of pure target.•Water with buffer additive was essential to improving the peak shape and resolution.•High co-solvent percentage was needed to adequately elute the extremely hydrophilic target in both steps. An efficient and “endotoxin-free” purification of a cyclic dinucleotide (CDN) STING agonist was achieved to produce multigram quantities of pure BMT-390025, an active pharmaceutical ingredient (API), for toxicological studies. A two-step sub/supercritical fluid chromatography (SFC) procedure was developed for the achiral purification and desalting of the polar ionic CDN. A robust SFC process employing methanol-acetonitrile-water with ammonium acetate as co-solvent in CO2 on BEH 2-ethylpyridine was established and scaled up as the first step to achieve a successful purification. The desalting/salt-switching (i.e. removing acetate and acetamide) was conducted using methanol-water with ammonium hydroxide as co-solvent on the same column in the second step to convert the final API to the ammonium salt. Water with additive was essential to eliminating salt precipitation and improving the peak shape and resolution. Due to the extreme hydrophilicity of BMT-390025, 65% of co-solvent was needed to adequately elute the target in both steps. More than 40 g of crude API was purified and desalted producing >20 g of pure BMT-390025 as the ammonium salt which was obtained with a chemical purity of >98.5% and met the endotoxin requirement of 80 g of its penultimate prior to the deprotection of the silyl group was purified at a high throughput of 6.3 g/h (0.42 g/day/g SP).