Separation of neoglycoproteins with different degrees of glycosylation by boronate chromatography

The separation of glycoproteins as glycoforms with specific degrees of glyc osylation has been a problem until now. A technique involving boronate affi nity chromatography has been developed to separate a heterogeneous sample o f neoglycoprotein, chymotrypsin modified with maltose via reductive aminati on, into individual fractions with different degrees of glycosylation. Low- molecular-mass polyhydroxyl compounds, such as tris(hydroxymethyl)aminometh ane (Tris), pentaerythritol, and triethanolamine proved efficient eluents d ue to their ability to form strong tridentate complexes with the boronate l igand. Compounds leading to either: too strong interaction with the boronat e ligand (e.g. D-sorbitol and polyvinly alcohol), or too weak or no interac tion (e.g. dextran) were not suitable eluents. The study provided the oppor tunity to probe further into the effect of glycosylation on the function of glycoproteins.