High capacity temperature-responsive affinity chromatography designed for antibody separation

Abstract

Antibody separation and purification methodologies with high separation efficiency, large adsorption capacity and high biological activity are challenging. In this study, a novel high capacity temperature-responsive affinity chromatography (TRAC) method was developed for antibody separation. With temperature-responsive dendrimer polyamidoamine-poly(N-isopropylacrylamide) (PAMAM-PNIPAAm) as the spacer arms and 4-mercapto ethyl pyridine (MEP) as the ligand, TRAC can not only be used to separate the antibody directly in response to temperature changes from 40 °C to 5 °C in an aqueous mobile phase at pH 8.0, but also at least two-fold higher adsorption capacity of the antibody can be achieved compared with linear spacer arms. BSA and γ -globulins can be separated completely using TRAC by performing adsorption at 40 °C and elution at 5 °C. The effect of temperature and the structure of TRAC stationary phases on the antibody separation were investigated, and the mechanism of antibody separation by TRAC was discussed in detail. This new technology was employed for the separation of IgG and IgY from human serum and egg yolk, and both the mass recoveries were found to above 90% with 98% purity. This novel methodology can overcome the previous negative effect of deactivation of the antibody by traditional harsh elution with low pH, low adsorption capacity. It can preserve its biological activity, and exhibit considerable potential for antibody separation in antibody drugs production.