High-pH Anion-Exchange Chromatography (HPAEC) and Pulsed Amperometric Detection (PAD) for Carbohydrate Analysis

For over 30 years high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) has been an extensively used technique for carbohydrate analysis in the biotechnology industry. This article reviews the basic principles of HPAEC/PAD separation and detection technology and provides an up-to-date description of the chromatography columns, equipment, and accessories used to execute HPAEC/PAD methods. The major applications of HPAEC/PAD in the biotechnology industry and how those applications are used to characterize glycoprotein biopharmaceuticals, including monoclonal antibodies, are reviewed. These applications are monosaccharide and sialic acid analyses for determination of a glycoprotein's carbohydrate composition, and oligosaccharide (glycan) profiling. Coupling the later application to high-resolution accurate mass (HRAM) mass spectrometry is used to deduce a therapeutic glycoprotein's oligosaccharide structures. HPAEC/PAD oligosaccharide profiling is also demonstrated to be part of consistency and comparability analyses during the production of a therapeutic glycoprotein. Other topics reviewed include application of HPAEC/PADs to vaccine development and assay, to assay of aminoglycoside antibiotics, and human milk oligosaccharides. This article also discusses how HPAEC/PAD has been used in United States Pharmacopeia drug product and drug substance monographs, and general chapters.