A comparative evaluation of random and site‐specific immobilization techniques for the preparation of antibody‐based chiral stationary phases

Abstract

In this study, one random and four site‐directed conjugation strategies were applied to immobilize an mAb, which stereoselectively binds to L‐amino acids, onto silica particles. The resulting chiral stationary phases (CSPs) were used for enantiomer separation of the model‐analyte D,L‐phenylalanine and further examined in frontal affinity chromatography. Although random immobilization of the antibody onto discuccinimidyl carbonate‐activated silica resulted in a CSP that enabled baseline separation of the enantiomers of D,L‐phenylalanine, the amount of available binding sites was considerably lower compared to the CSPs prepared by site‐directed strategies. Immobilization of antibody via its carbohydrate chains, either directly via hydrazone bonds between the support and the protein or indirectly via binding carbohydrate‐biotinylated antibody to streptavidin‐derivatized silica, resulted in medium column efficiencies. Higher amounts of available active sites were obtained by immobilizing the antibody indirectly through the “crystallizable fragment (Fc)” receptor protein A/G. The best results with regard to amount of available binding sites and column efficiency were obtained by first biotinylating the antibody specifically at its C‐termini using carboxypeptidase Y and immobilizing the biotinylated antibody on streptavidin‐derivatized silica.