Frontal Affinity Chromatography Coupled to Mass Spectrometry: An Effective Method for Kd Determination and Screening of α‐Gal Derivatives Binding to Anti‐Gal Antibodies (IgG)

Frontal affinity chromatography with mass spectrometric detection (FAC/MS) was developed as an effective method for rapid determination of Kd values for α‐Gal derivatives binding to human anti‐Gal IgG antibodies. Using this method, Kd values for 23 α‐Gal compounds were determined for the first time, including an α‐Gal terminated N‐linked oligosaccharide which mimics a single N‐glycoform present on the surface of animal cells. A mixture of eight α‐Gal derivatives, a model for an α‐Gal compound library, was successfully screened against this anti‐Gal IgG using FAC/MS. The analyte breakthrough sequence, indicated by the ion chromatogram, reflected the magnitude of the Kd values, confirming its potential application in the screening of new α‐Gal derivatives and mimetics. Ten α‐Gal derivatives were designed and synthesized chemically or enzymatically. Among the compounds analyzed, trivalent compound 26 demonstrated the strongest binding affinity to anti‐Gal IgG with a Kd value of 3.1 µM. The α‐Gal terminated N...