Miniaturized antithrombin III affinity monolithic columns coupled to TOF-MS for the selective capture and release of fondaparinux a high affinity antithrombin III ligand

Abstract

This work explores the capability of antithrombin III-functionalized capillary monolithic columns (in-line coupled with MS detection) to selectively capture, release and detect high affinity binders of antithrombin III (AT III) from oligosaccharides mixtures. The in-situ characterization of home-made AT III affinity columns was done by frontal affinity chromatography coupled to MS detection using fondaparinux as model ligand. Three different preparation methods of miniaturized antithrombin III monolithic affinity columns were optimized and compared. Immobilization of antithrombin III onto Concanavalin A functionalized column is the simplest method but leads to lowest protein density. The two other methods, direct grafting on aldehyde preactivated monoliths and immobilization of biotinylated antithrombin III to streptavidin-functionalized columns, require the presence of fondaparinux to protect the heparin binding site during the grafting process. Up to 1.3 ± 0.3 pmol cm^-1 of antithrombin III were immobilized with both methods. The direct coupling of such miniaturized affinity columns to MS-detection was made possible by optimization of the elution step. Ammonia (0.1 M) was chosen as an efficient and MS compatible solvent to elute fondaparinux. Finally, the complete analytical workflow (capture/elution/detection) was demonstrated to allow the selective capture and elution of fondaparinux initially contained in a complex oligosaccharide mixture with a limit of detection of 1 pmol.

Keywords: Antithrombin III; Biofunctionalization; Frontal affinity chromatography; Miniaturization; Organic monolith; TOF-MS.