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.

Optimization of antibody immobilization for on‐line or off‐line immunoaffinity chromatography

“…These methods routinely use 2 M Na 2 SO 4 during immobilization to get the antibodies to partially precipitate on the matrix surface, thus improving immobilization dramatically [9], but also making crosslinks between the antibodies themselves possible. Finally, site-directed immobilization of antibodies may be performed in a two-stage approach by using a Protein G-derivatized matrix [9,11] (method 4) followed by chemical crosslinking of antibody and Protein G. Site-directed methods appear generally to result in IA-matrices with increased capacities [25]. An overview of the methods is shown in Table 1.…”

Trends and Perspectives in Immunosensors

“…Strategies available for the covalent site-specific oriented immobilisation of Abs exist using the carbohydrate chains of Abs for immobilisation [17]. One simple oriented immobilisation strategy involves chemically coupling the Ab directly onto the transducer surface, e.g.…”

A practical method for the quantitative assessment of non-enantioselective versus enantioselective interactions encountered in liquid chromatography on brush-type chiral stationary phase

“…We believe that a detailed understanding of the nature and the origin of non-enantioselective interactions is indispensible for researchers concerned both with the development of CSPs or their use in analytical and preparative separation applications. Specifically, in the field of CSP development a sound understanding of nonselective interactions will provide researchers with useful guidance for the engineering of new surfaces with non-invasive properties, e.g., by appropriate choice of support materials, linker-functionalities, and efficient endcapping strategies [4,[18][19][20][21][22][23]. Information on the unfavourable contributions of non-enantioselective interactions is also important for the structure-based refinement of established and the design of new SOs via the amelioration of enantioselectivity by means of reducing the number and activity of the non-enantioselective sites in the CSP [4,22].…”