Isolation and purification of Human Milk Oligosaccharides by two‐dimensional chromatography for in vitro and in vivo studies

Abstract

Human Milk Oligosaccharides (HMO) are a structurally divers group of complex glycans that are highly abundant in human milk but not in infant formula. The structural diversity of HMO, however, makes it difficult to study the molecular mechanisms underlying their potential benefits for the breast‐fed infant. More than 150 different HMO have been described ranging in size from low molecular weight trisaccharides to high molecular weight oligosaccharides with more than 30 monosaccharide monomers and ranging from being neutral to carrying several negative charges. Not all HMO may trigger the same effects. Here, we describe the isolation and purification of HMO subfractions and even individual HMO by two‐dimensional chromatography. In the first dimension we used anion‐exchange chromatography (QAE) to separate pooled HMO in a neutral and 5 negatively charged fractions containing 0 to 5 sialic acids per HMO molecule, respectively. In the second dimension we used automated size exclusion chromatography to separate HMO based on their molecular weight. Structural composition of the generated HMO fractions was analyzed by HPLC and mass spectrometry. Our approach generated structurally defined HMO subfractions and even individual HMO in quantities sufficient for in vitro and in vivo experiments aimed to elucidate HMO structure‐function relationships. (Supported by NIH K99/R00DK078668 to LB)