Antibody Adsorption And Ligand Stability Of Protein A Adsorbents

Protein A chromatography has become one of the core techniques in antibody production,and a deep understanding of the interaction mechanism between Protein A and antibody is considered as the important prerequisite for the development of high-performance Protein A chromatography.In this work,two protein ligands,antibody binding domain Z and its four repeats of SpA4z were coupled onto Sepharose CL-6B to prepare affinity adsorbents:Z-Sepharose and SpA4z-Sepharose.Adsorption equilibria of antibody on two beads indicates that antibody had much high affinity to both beads,and the affinity increased significantly with an increment of domain Z in the ligands.Furthermore,calorimetric measurement of antibody and affinity beads was carried out in an isothermal titration calorimeter to analyze the thermal behavior of antibody adsorption.The results indicate that the immobilization of the ligand led to a decrease of the affinity,and it was driven by enthalpy.The research provided an insight into the interaction mechanism between protein A ligand and antibody.NaOH is the most commonly used chromatographic clean-in-place(CIP)reagent.In order to overcome fragile tolerance of Protein A ligand in alkaline condition,a mutant Protein A,SpA4mz was constructed to improve the SpA alkaline tolerance towards CIP treatment.The results of differential scanning calorimetry show that SpA4mz had higher Tm value than SpA4z and SpAz,indicating the novel mutant was more stable.Finally,the chromatographic results also reveal that SpA4m,had greater tolerance to alkaline condition.Therefore,the research provides insight into the interaction mechanism between SpA ligand and antibody and tolerance of the ligand to alkaline condition.