A comparative evaluation of random and site-specific immobilization techniques for the preparation of antibody-based chiral stationary phases.
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.
Franco
Elliott J
EJ
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115-2862, USA.
Hofstetter
Heike
H
Hofstetter
Oliver
O
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D008958:Models, Molecular; D015394:Molecular Structure; D012822:Silicon Dioxide / Q000737:chemistry; D013237:Stereoisomerism; D019809:Streptavidin / Q000737:chemistry
D000911:Antibodies, Monoclonal; D001710:Biotin; D012822:Silicon Dioxide; D019809:Streptavidin