Microcalorimetric Analysis on Antibody Adsorption and Alkali Resistance of Protein A Adsorbents

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Abstract

Three protein ligands, antibody binding domain Z (SpA_Z), its four repeats of SpA_4z (which currently used in some commercially available protein A adsorbents) and alkaline resistant mutant SpA_4m were coupled onto Sepharose CL-6B to prepare affinity adsorbents: SpAZ-Sepharose, SpA_4Z-Sepharose and SpA_4m-Sepharose. Adsorption equilibria of antibody on SpAZ-Sepharose and SpA_4Z-Sepharose indicates that antibody has 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 lead to a decrease of the affinity, and it is 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. Mutant SpA_4m was constructed to improve the SpA alkaline tolerance towards CIP treatment. The results of differential scanning calorimetry show that SpA_4m has higher T_m value than SpA_4z nd SpA_Z, indicating the novel mutant is more stable. The chromatographic results also reveal that SpA_4m has 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.

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	Sheng Zhi
	Shi Qinghong
	Bai Shu

Keywords： antibody； chromatography； bioseparation； isothermal titration calorimetry； differential scanning calorimetry

Received 2015-09-28;

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Sheng Zhi, Shi Qinghong, Bai Shu.Microcalorimetric Analysis on Antibody Adsorption and Alkali Resistance of Protein A Adsorbents[J] Chemcial Industry and Engineering, 2017,V34(4): 75-82

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