蛋白A介质抗体吸附性能及耐碱性的量热学

摘要

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SpAZ-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。

	Service

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	Email Alert
	RSS
	作者相关文章
	盛志
	史清洪
	白姝

关键词：抗体色谱生物分离等温滴定量热微量示差扫描量热

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.

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

Received 2015-09-28;

Fund:

国家自然科学基金（21276189）；天津市应用基础与前沿技术研究计划（15JCYBJC48500）。

Corresponding Authors: 史清洪,E-mail:qhshi@tju.edu.cn。 Email: qhshi@tju.edu.cn

About author: 盛志(1988-),男,硕士,助理工程师,主要研究方向为生物分离。

引用本文:

盛志, 史清洪, 白姝.蛋白A介质抗体吸附性能及耐碱性的量热学[J]. 化学工业与工程, 2017,34(4): 75-82

Sheng Zhi, Shi Qinghong, Bai Shu.Microcalorimetric Analysis on Antibody Adsorption and Alkali Resistance of Protein A Adsorbents[J]. Chemcial Industry and Engineering, 2017,34(4): 75-82

[1] 崔加友, 李菁, 林东强, 等. 疏水性电荷诱导色谱分离抗HER2单克隆抗体[J]. 化工学报, 2014, 65(10): 3 931-3 937 Cui Jiayou, Li Jin, Lin Dongqiang, et al. Separation and purification of anti-HER2 monoclonal antibody with hydrophobic charge-induction chromatography[J]. CIESC Journal, 2014, 65(10): 3 931-3 937 (in Chinese)
[2] Linhult M, Gulich S, Hober S. Affinity ligands for industrial protein purification[J]. Protein and Peptide Letters, 2005, 12(4): 305-310
[3] Geiger T, Clarke S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides Succinimide-linked reactions that contribute to protein degradation[J]. The Journal of Biological Chemistry, 1987, 262(2): 785-794
[4] Nilsson B, Tomas M, Jansson B. A synthetic IgG-binding domain based on Staphylococcal protein A[J]. Protein Engineering, 1987, 1(2): 107-113
[5] Xia H, Wang S, Wu P, et al. Molecular modification of Protein A to improve the elution pH and alkali resistance in affinity chromatography[J]. Applied Biochemistry and Biotechnology, 2014, 172(8): 4 002-4 012
[6] Arouri A, Garidel P, Kliche W, et al. Hydrophobic interactions are the driving force for the binding of peptide mimotopes and Staphylococcal protein A to recombinant human IgG1[J]. European Biophysics Journal: EBJ, 2007, 36(6): 647-660
[7] D'Agostino B, Bellofiore P, De Martino T, et al. Affinity purification of IgG monoclonal antibodies using the D-PAM synthetic ligand: Chromatographic comparison with protein A and thermodynamic investigation of the D-PAM/IgG interaction[J]. Journal of Immunological Methods, 2008, 333(1/2): 126-138
[8] Lund L N, Christensen T, Toone E, et al. Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry[J]. Journal of Molecular Recognition:JMR, 2011, 24(6): 945-952
[9] Starovasnik M A, O'Connell M P, Fairbrother W J, et al. Antibody variable region binding by Staphylococcal protein A: Thermodynamic analysis and location of the Fv binding site on E-domain[J]. Protein Science: A Publication of the Protein Society, 1999, 8(7): 1 423-1 431
[10] Van Eldijk M B, Smits F C, Thies J C, et al. Thermodynamic investigation of Z33-antibody interaction leads to selective purification of human antibodies[J]. Journal of Biotechnology, 2014, 179: 32-41
[11] Shi Q, Shen F, Sun S. Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography[J]. Biotechnol Prog, 2010, 26(1): 134-141
[12] Niedziela-Majka A, Kan E, Weissburg P, et al. High-Throughput screening of formulations to optimize the thermal stability of a therapeutic monoclonal antibody[J]. Journal of Biomolecular Screening, 2014, 20(4): 552-559
[13] Ghose S, Hubbard B, Cramer S. Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials[J]. Biotechnology and Bioengineering, 2007, 96(4): 768-779