Separation of Glycoproteins using Shielding Boronate Affinity Chromatography

University dissertation from Biotechnology (LTH)

Abstract: Glycoproteins, a class of biomolecules, exhibit a variety of biological activities and therefore are of importance in modern biotechnology and pharmaceutical industry. Pure glycoproteins are needed for structural and functional studies. However, the separation of glycoproteins has been a problem until now due to their structural complexity. Efficient separation techniques are thus required. This thesis presents a novel technique, shielding boronate affinity chromatography (SBAC), which eliminates non-specific boronate–protein interactions via introducing a so-called shielding reagent into the mobile phase. To be a suitable shielding reagent, the following requirements need to be fulfilled: (1) the shielding reagent should have an affinity to the boronate ligand and should not interact with the sample molecules to be purified; (2) the boronate–shielding reagent interactions must be stronger than non-specific boronate–protein interactions, while weaker than specific boronate-glycoprotein interactions; and (3) the shielding reagent should be non-toxic and chemi-cally stable in order to be applied to pharmaceutical industry. The shielding efficiency of a shiel-ding reagent is correlated to its chemical structure. Low-molecular-mass polyhydroxyl compounds with a conformation that allows the formation of tridentate complexes with the boronate anion exhibit the highest shielding efficiencies. Such compounds can also function as eluting agents when used at high concentrations. The feasibility of using SBAC to the separation of glycoproteins is demonstrated through several examples, such as (1) separation of a neoglycoprotein, maltose-modified Cht (Cht-Mal), from non-glycosylated Cht; (2) separation of Cht-Mal into individual fractions with different degrees of glycosylation; (3) separation of two mistletoe lectins from each other based on their degree of glycosylation; and (4) separation of glycated haemoglobin (GHb) from non-glycated Hb species as well as separation of GHb into two fractions that exhibit different glycation patterns. The study shows that SBAC has higher separation resolution than conventional boronate affinity chromatography.

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