Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
J Biotechnol. 2013 Jan 10;163(1):10-6. doi: 10.1016/j.jbiotec.2012.10.007. Epub 2012 Oct 23.
The increasing demand of monoclonal antibodies for therapeutic applications along with the high manufacturing cost have made it necessary to evaluate better process options and technologies for their purification. Affinity precipitation is an attractive alternative to traditional chromatographic methods by affording effective purification using a simple environmental trigger. The feature of elastin-like-protein (ELP) fused with antibody binding domains has already been explored for the purification of antibodies. However, ELP when fused with the bulkier domains such as Protein A, resulted in lower protein production. In this study, ELP was fused to smaller synthetic IgG binding domains such as the z or zz domain, resulting in up to 10-fold higher level of production. Both ELP-z and ELP-zz bind tightly to human immunoglobulin (HIgG) with a dissociation constant of 768±142 nM and 68±23 nM, respectively. Owing to the higher binding affinity, the use of ELP-zz resulted in more than 99% recovery of HIgG in four repeated binding and elution cycles with no observable decrease in the purification performance. The same binding and elution cycle was successfully implemented for the purification of monoclonal antibodies from hybridoma culture supernatant with close to 100% recovery.
随着治疗性单克隆抗体需求的增加和高制造成本，有必要评估更好的工艺选择和技术来进行其纯化。亲和沉淀通过使用简单的环境触发提供有效的纯化，是传统色谱方法的一种有吸引力的替代方法。融合了抗体结合结构域的弹性蛋白样蛋白 (ELP) 已经被探索用于抗体的纯化。然而，当 ELP 与体积较大的结构域（如蛋白 A）融合时，会导致蛋白质产量降低。在这项研究中，ELP 被融合到更小的合成 IgG 结合结构域，如 z 或 zz 结构域，从而使产量提高了 10 倍。ELP-z 和 ELP-zz 与人免疫球蛋白（HIgG）的结合非常紧密，解离常数分别为 768±142 nM 和 68±23 nM。由于更高的结合亲和力，在重复进行 4 次结合和洗脱循环后，使用 ELP-zz 可回收超过 99%的 HIgG，而纯化性能没有明显下降。同样的结合和洗脱循环也成功地用于从杂交瘤培养上清液中纯化单克隆抗体，回收率接近 100%。
