Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances.

Highlights • Generic ion-exchange method for analytical and preparative separation of nucleotides. • Chromatographic resolution of over 20 nucleotides in a single experimental run. • The use of a cost-effective resin available in bulk format for preparative separation is demonstrated. • Aqueous ammonium bicarbonate-based eluent profile allows convenient drying process. • Fully implemented for preparative separation of nucleotides in multicomponent reaction mixtures. Abstract Nucleotides are among the most frequently used chemical building blocks in the research, development and manufacture of drug substances. They are composed of three highly polar subunit molecules (a nucleobase, a sugar, and at least one phosphate group), which makes their separation and analysis very challenging by conventional liquid chromatography techniques. Herein, we describe a simple, efficient, and cost-effective ion-exchange chromatography (IEC) method for the separation and purification of over 20 nucleotides. This method combines the use of a Tosoh TSKgel SuperQ-5P W resin in conjunction with a fully aqueous eluent profile (ammonium bicarbonate-based) that allows for a straightforward scale-up transition and convenient drying process with minimal environmental impact. This generic method was optimized using chromatography simulation software (ACD Labs/LC Simulator) and successfully applied to the preparative purification of multicomponent nucleotide mixtures using readily available Fast Protein Liquid Chromatography (FPLC) instrumentation. These IEC method conditions can be effectively applied as the starting point for method development and isolation of other highly polar nucleotide species beyond those investigated in this study. [ABSTRACT FROM AUTHOR]