Liaoning Key Laboratory of Molecular Recognition and Imaging, School of Bioengineering, Dalian University of Technology, Dalian 116023, PR China.
Liaoning Key Laboratory of Molecular Recognition and Imaging, School of Bioengineering, Dalian University of Technology, Dalian 116023, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Aug 1;1179:122652. doi: 10.1016/j.jchromb.2021.122652. Epub 2021 Mar 11.
Mixed-mode chromatography provides a promising strategy for industrial protein purification for its potential merit of balancing efficiency and cost-effectiveness. However, mixed-mode media with satisfactory selectivity and binding capacity towards antibody are still urgently needed. A new type of mixed-mode chromatography resin was prepared using benzotriazole-5-carboxylicas as ligand (BTA MM), and its application in antibody separation was explored. A typical pH-dependent protein binding was observed, and the neutral condition was favorable for antibody adsorption. Dynamic binding capacity of human immunoglobulin G (hIgG) was 57.7 mg/mL at pH 7.4 (10 mM phosphate buffer, containing 150 mM NaCl), while elution with acidic solutions (pH 3-4) could achieve a recovery of more than 85%. Protein adsorption on the resin showed a salt-independent manner, thus it could work under physiological solution conditions, with satisfied antibody selectivity. One-step purification of antibody components from human serum samples could obtain a product with the purity more than 84%. Satisfied performance was also observed when the adsorbent was used for purifying a IgG1-type monoclonal antibody (mAb) from cell culture supernatant. In addition, the benzotriazole adsorbent has been found stable enough to withstand autoclave sterilization and other harsh conditions, including 1 M NaOH, 1 M HCl, and 75% ethanol. The results proved the potential of this type of mixed-mode chromatography medium for industrial antibody purification.
混合模式色谱法为工业蛋白质纯化提供了一种有前途的策略，因为其具有平衡效率和成本效益的潜力。然而，仍然迫切需要具有令人满意的抗体选择性和结合能力的混合模式介质。使用苯并三唑-5-羧酸作为配体（BTA MM）制备了一种新型混合模式色谱树脂，并探索了其在抗体分离中的应用。观察到典型的 pH 依赖性蛋白质结合，中性条件有利于抗体吸附。在 pH 7.4（10 mM 磷酸盐缓冲液，含 150 mM NaCl）下，人免疫球蛋白 G（hIgG）的动态结合容量为 57.7 mg/mL，而用酸性溶液（pH 3-4）洗脱可实现超过 85%的回收率。蛋白质在树脂上的吸附呈盐无关方式，因此它可以在生理溶液条件下工作，具有令人满意的抗体选择性。从人血清样品中一步纯化抗体成分可获得纯度超过 84%的产物。当吸附剂用于从细胞培养上清液中纯化 IgG1 型单克隆抗体（mAb）时，也观察到了令人满意的性能。此外，苯并三唑吸附剂已被发现足够稳定，可以承受高压灭菌和其他苛刻条件，包括 1 M NaOH、1 M HCl 和 75%乙醇。结果证明了这种混合模式色谱介质在工业抗体纯化中的潜力。
