Determination of binding constants between one protein and multiple carbohydrates by affinity chromatography on a microchip

Development of rapid, reliable and high throughput methods for evaluating the interactions between different carbohydrates and a same protein is critical to carbohydrate drug development. In this study, we develop a novel strategy based on an affinity chromatography for quickly determining the binding constants of different carbohydrates to a same protein. The core of our method is the inversely proportional relationship between the binding constant and a new termed parameter, critical elution concentration (CMC). CMC is defined as the lowest concentration of displacing reagent, a series of carbohydrates herein, at which the protein specifically bond to the affinity column can be eluted off as an intact peak by the carbohydrate solution in a certain time. The interactions between a series of sulfate polysaccharides and granulocyte colony-stimulating factor (G-CSF) are selected as model. Through a 200 μm long heparin affinity column microfabricated inside a channel of 50 μm width and 20 μm height, the binding constant of each G-CSF–polysaccharide binding pair can be obtained within 1 h, around one sixth of time needed by traditional capillary electrophoresis based method.