Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang Key Laboratory of Smart Biomaterials, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang Key Laboratory of Smart Biomaterials, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Oct 1;1208:123407. doi: 10.1016/j.jchromb.2022.123407. Epub 2022 Aug 6.
Short peptide biomimetic chromatography technology as a developing protein separation technology has huge potential for antibody purification. In this study, four tetrapeptide ligands (Ac-FYKH, Ac-YEHF, Ac-YFLH and Ac-FYHI) with high potential binding ability to antibody were selected for the optimal ligand to antibody purification. The results showed that Ac-YEHF-4FF resin had higher binding capacity and selectivity for hIgG among the four resins. And at pH 7.0 and 0.3 ml/min, the highest Q of Ac-YEHF-4FF resin was 26.2 mg/ml resin while its Q was just 2.2 mg/ml resin. Further, Ac-YEHF-4FF resin was used to purify protein mixtures. By binding at pH 7.0 and being eluted at pH 5.0 and pH 4.0, Ac-YEHF-4FF resin was well used to separate hIgG from BSA containing feedstock, HSA containing feedstock and human serum with the purity and yield both more than 95 %. And the screened resin could also separate mAb from CHO cell culture supernatant with purity 94.3 % and yield 97.5 %. The adsorption and separation results of Ac-YEHF-4FF resin indicated that the goal of getting the efficacy of critical residues from protein A to biomimetic its structure and function could be achieved, which had great significance to the establishment and improvement of tetrapeptide biomimetic chromatography, and also provided a new method for the field of antibody separation and purification.
短肽仿生层析技术作为一种新兴的蛋白质分离技术，在抗体纯化方面具有巨大的潜力。本研究选择了四种具有高潜在结合能力的四肽配体（Ac-FYKH、Ac-YEHF、Ac-YFLH 和 Ac-FYHI），用于优化抗体的亲和层析配基。结果表明，在四种亲和层析介质中，Ac-YEHF-4FF 树脂对 hIgG 的结合容量和选择性最高。在 pH 7.0 和 0.3 ml/min 条件下，Ac-YEHF-4FF 树脂的最大 Q 值为 26.2 mg/ml 树脂，而 Q 值仅为 2.2 mg/ml 树脂。进一步，将 Ac-YEHF-4FF 树脂用于蛋白质混合物的分离。通过在 pH 7.0 下结合，然后在 pH 5.0 和 pH 4.0 下洗脱，Ac-YEHF-4FF 树脂能够很好地从含 BSA 的进料、含 HSA 的进料和人血清中分离 hIgG，纯度和收率均超过 95%。筛选出的树脂还可以从 CHO 细胞培养上清液中分离 mAb，纯度为 94.3%，收率为 97.5%。Ac-YEHF-4FF 树脂的吸附和分离结果表明，从蛋白 A 获得关键残基的功效并仿生其结构和功能的目标是可以实现的，这对建立和完善四肽仿生层析技术具有重要意义，也为抗体分离和纯化领域提供了新的方法。
