蛋白A介质抗体吸附性能及耐碱性的量热学

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

对重组蛋白A(SpA)中参与抗体结合的Z结合域(SpA_Z),它的四串体重组配基SpA_(4Z)和耐碱性突变体SpA_(4M)等用于抗体纯化的亲和配基进行研究,将上述分子偶联于Sepharose CL-6B基质制得3种亲和介质。抗体吸附平衡实验表明抗体与上述色谱介质均具有较高的结合常数,且四串体重组配基的亲和性高于单一Z结合域。用等温滴定微量热仪(ITC)研究了抗体分别在SpA_Z-Sepharose和SpA_(4Z-Sepharose)色谱介质上结合的量热学变化,并与游离的SpA_Z和SpA_(4Z)进行比较,解析了焓变(ΔH)与熵变(ΔS)对抗体结合的贡献。结果显示,配基固定化导致结合常数降低,配基与抗体结合过程是由ΔH驱动的。使用微量差示扫描量热仪(DSC)检测了SpA_(4z)和SpA_(4m)的碱性稳定性,结果表明SpA_(4m)比SpA_(4z)具有更强的稳定性,色谱实验进一步验证了这一结果。

Three protein ligands, antibody binding domain Z （SpAz） , its four repeats of SpA4z （which currently used in some commercially available protein A adsorbents） and alkaline resistant mutant SpA4m were coupled onto Sepharose CL-6B to prepare affinity adsorbents： SpAZ-Sepharose, SpAaz.seph and SpA4m-Sepharese . Adsorption equilibria of antibody on SpAZ-Sepharose and SpA4Z-Sepharese indicates that anti- body 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 SpA4m was constructed to improve the SpA alkaline tolerance towards CIP treatment. The results of differential scanning calorimetry show that Spm4m has higher Tm value than SpA4z nd SpAz, indicating the novel mutant is more stable. The chromatographic results also reveal that SpA4m 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.