Department of Chemistry, University of Puerto Rico at Humacao, CUH Station, Humacao, PR 00792, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Jul 1;930:13-21. doi: 10.1016/j.jchromb.2013.04.025. Epub 2013 Apr 24.
Despite membrane-based separations offering superior alternative to packed bed chromatographic processes, there has been a substantial lacuna in their actual application to separation processes. One of the major reasons behind this is the lack of availability of appropriately modified or end-group modifiable membranes. In this paper, an affinity membrane was developed using a commercially available serine protease inhibitor, para-aminobenzamidine (pABA). The membrane modification was optimized for protein binding capacity by varying: (i) the length of the spacer arm (SA; 5-atoms, 7-atoms, and 14-atoms) linking the ligand to membrane surface; (ii) the affinity ligand (pABA) density on membrane surface (5-25nmol/cm(2)). Resulting membranes were tested for their ability to bind plasminogen activators (PAs) from mono- and multi-component systems in batch mode. The membrane containing pABA linked through 7-atoms SA but similar ligand density as in the case of 5- or 14-atoms long SA was found to bind up to 1.6-times higher amounts of PA per nmoles of immobilized ligand from conditioned HeLa cell culture media. However, membranes with similar ligand densities but different lengths of SA, showed comparable binding capacities in mono-component system. In addition, the length of SA did not affect the selectivity of the ligand for PA. A clear inverse linear correlation was observed between ligand density and binding capacity until the point of PA binding optima was reached (11±1.0nmol/cm(2)) in mono- and multi-component systems for 7- as well as 14-atoms SA. Up to 200-fold purification was achieved in a single step separation of PA from HeLa conditioned media using these affinity membranes. The issues of ligand leaching and reuse of the membranes were also investigated. An extensive regeneration procedure allowed the preservation of approximately 95% of the PA binding capacity of the membranes even after five cycles of use.
尽管基于膜的分离为填充床色谱过程提供了优越的替代方案，但它们在实际分离过程中的应用仍然存在很大的差距。造成这种情况的一个主要原因是缺乏适当修饰或端基可修饰的膜。在本文中，使用一种市售的丝氨酸蛋白酶抑制剂对氨基苯甲脒（pABA）开发了一种亲和膜。通过改变连接配体和膜表面的间隔臂（SA；5 个原子、7 个原子和 14 个原子）的长度和膜表面的亲和配体（pABA）密度（5-25nmol/cm(2)）优化了膜的蛋白结合能力。以批处理模式测试了所得膜结合单组分和多组分系统中纤溶酶原激活剂（PA）的能力。发现含有通过 7 个原子 SA 连接的 pABA 的膜，但与 5 或 14 个原子长的 SA 相比，配体密度相似的膜能够结合条件化 HeLa 细胞培养基中每毫摩尔固定化配体高达 1.6 倍的 PA。然而，具有相似配体密度但 SA 长度不同的膜在单组分系统中表现出相似的结合能力。此外，SA 的长度不影响配体对 PA 的选择性。在单组分和多组分系统中，观察到配体密度与结合容量之间存在明显的线性反比关系，直到达到 PA 结合最佳点（11±1.0nmol/cm(2)），7-和 14-原子 SA 都是如此。使用这些亲和膜，在单步分离中可以从 HeLa 条件培养基中实现高达 200 倍的 PA 纯化。还研究了配体浸出和膜再利用的问题。广泛的再生程序允许即使在使用五个循环后，膜的 PA 结合能力也能保留约 95%。
