Comparison of protein A affinity sorbents
Comparison of protein A affinity sorbents
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DOI：
10.1016/s1570-0232(03)00092-8
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发表时间：
2003-06-25
影响因子：
3
通讯作者：
Jungbauer, A
中科院分区：
文献类型：
--
作者：
Hahn, R;Schlegel, R;Jungbauer, A
蛋白A是用于纯化单克隆和重组抗体的常用通用配体。研究了15种市售蛋白A培养基的性能。测定了人IgG的平衡和动态结合能力，并研究了从粗原料中捕获IgG。对于初始筛选，以小规模测定动态结合容量。具有良好性能的介质进一步用增加的柱高度测试。比较来自两个不同柱高度的数据，可以表明动态容量强烈地依赖于停留时间。基于琼脂糖的介质在停留时间长于3分钟时表现出更高的结合能力，而基于多孔玻璃的聚合物介质或介质表现出对流速和停留时间的较小依赖性。通过测定吸附等温线和用托马斯溶液拟合穿透曲线来定量描述这种行为。基于琼脂糖的介质具有较高的最大平衡结合容量和来自吸附等温线的解离常数较小。其他介质表现出较高的表观速率常数，表明更快的传质。这可以通过这些介质的较小颗粒直径来解释，并且可以假设由此更快地获得恒定图案条件。通过在标准化条件下进行抗体纯化来测试选择性。将含有2.5%胎牛血清的细胞培养上清液中的多克隆人IgG用作代表性原料。在所应用的条件下，几种吸附剂显示出非常紧密的IgG结合，并且在某些情况下，大部分样品保留在吸附剂上。该研究可为大规模净化过程的优化提供指导。(C)2002 Elsevier Science B.V.保留所有权利。
Protein A is a popular generic ligand for purification of monoclonal and recombinant antibodies. The performance of 15 commercially available protein A media was studied. Equilibrium and dynamic binding capacity for human IgG was determined and the capture of IgG from a crude feed-stock was investigated. For initial screening the dynamic binding capacity was determined at small scale. Media with good performance were further tested with increased column height. Comparing the data from the two different column heights it could be shown that the dynamic capacity strongly depends on the residence time. Agarose based media exhibited higher binding capacity at residence times longer than 3 min whereas polymeric media or media based on porous glass showed a lesser dependence on the flow velocity and the residence time. A quantitative description of this behavior was derived by determination of the adsorption isotherms and fitting the breakthrough profiles with the Thomas solution. Agarose based media exhibited higher maximum equilibrium binding capacities and the dissociation constants derived from adsorption isotherms were smaller. The other media exhibited higher apparent rate constants, indicating a faster mass transfer. This can be explained by the smaller particle diameter of these media and it can be assumed that constant pattern conditions are thereby obtained more quickly. Selectivity was tested by performing antibody purification under standardized conditions. Polyclonal human IgG in cell culture supernatant containing 2.5% fetal calf serum was used as a representative feed-stock. Under the applied conditions several sorbents showed very tight binding of IgG and in some cases most of the sample remained on the sorbent. The study can be useful as a guide for optimization of large-scale purification processes. (C) 2002 Elsevier Science B.V. All rights reserved.