Improvements to in-Line Desalting of Oligosaccharides Separated by High-pH Anion Exchange Chromatography with Pulsed Amperometric Detection

Abstract

High-pH anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD) (1) is routinely used to separate neutral and charged oligosaccharides differing by branch, linkage, and positional isomerism. Oligosaccharides are eluted in 0.1 M NaOH with gradients of sodium acetate (up to 0.25 M). Analyses of HPAEC/PAD-purified oligosaccharides generally require neutralization and removal of eluent salts. To facilitate the process, we designed and produced a cation-exchange system to remove sodium ions (Na+) from the eluent after oligosaccharide detection [the Carbohydrate Membrane Desalter (CMD), with a volatile regenerant]. Exchange of >99.5% of eluent Na+for hydronium ions (H3O+) within the CMD generates dilute acetic acid (removable by vacuum evaporation). The exchange process desalts up to0.35 M Na+at 1.0 ml/min. Oligosaccharides collected after on-line desalting, evaporated and resuspended in their original volume of deionized water contained ≤350 μM residual Na+when the eluting sodium concentration was 300 mM. This represents a desalting efficiency of >99.8%. Recovery of neutral and sialylated oligosaccharides under these conditions ranged from 75 to 100%. With the CMD system and postcollection evaporation, HPAEC/PAD can purify oligosaccharides ready for further characterization. As a proof test, oligosaccharides from a human monoclonal antibody were separated by HPAEC/PAD, desalted with the CMD system, dried, and analyzed by matrix-assisted laser desorption–ionization, time-of-flight mass spectrometry.