Hydrophilic Interaction Liquid Chromatography for Oligonucleotide Therapeutics: Method Consideration

Hydrophilic interaction liquid chromatography (HILIC) has emerged as a promising alternative to traditional ion-pair reversed phase liquid chromatography (IP-RPLC) methods for separating oligonucleotides (ON). This work investigates the application of HILIC to the separation of ON sequence and length variants, duplexes, and single-stranded components. Method variables, including ionic strength, temperature, mobile phase composition, and gradient optimization, are investigated in detail. A sequential injection procedure is also proposed to eliminate breakthrough effects and peak splitting. Some considerations for generic (platform) methods are also suggested.