Effects of ionic strength and mobile phase pH on the binding orientation of lysozyme on different ion-exchange adsorbents
Effects of ionic strength and mobile phase pH on the binding orientation of lysozyme on different ion-exchange adsorbents
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DOI：
10.1016/j.chroma.2007.12.085
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发表时间：
2008-06-13
影响因子：
4.1
通讯作者：
Hubbuch, Juergen
中科院分区：
文献类型：
--
作者：
Dismer, Florian;Petzold, Martin;Hubbuch, Juergen
色谱法是生物技术工业中用于纯化生物药物的最广泛使用的技术。令人惊讶的是，工艺开发通常仍然基于经验研究或经验;最近采用高通量筛选站来最大限度地减少开发时间并提高筛选质量。尽管如此，实验工作仍然很高，更详细的了解吸附机制的分子水平上的色谱分离可能有助于在未来选择和设计的色谱步骤在电脑上。在这项研究中，我们专注于阐明蛋白质的方向吸附到色谱树脂上。我们确定了溶菌酶在SP Sepharose Fast Flow上的两个特征结合位点和溶菌酶与SP Sepharose XL的一个多点相互作用位点。增加离子强度并没有显着影响的结合，而在移动的相pH值的变化导致了重新定位的SP琼脂糖FF。这一现象与理论上的考虑一致，包括对表面电荷分布随pH值变化的详细描述和线性洗脱实验，给出了为什么蛋白质经常保留在离子交换材料上超过其等电点的想法。(C)2008 Elsevier B. V.保留所有权利。
Chromatography is the most widely used technique for the purification of biopharmaceuticals in the biotech industry. Surprisingly, process development is often still based on empirical studies or experience; recently high-throughput screening stations are employed to minimize development time and to improve screening quality. Still, experimental effort remains high and a more detailed understanding of adsorption mechanisms on a molecular level underlying chromatographic separation could help in the future to select and design chromatography steps in silico. In this study, we focused on the elucidation of protein orientation upon adsorption onto a chromatographic resin. We identified two characteristic binding sites of lysozyme on SP Sepharose Fast Flow and one multipoint interaction of lysozyme with SP Sepharose XL. Increasing ionic strength did not significantly influence the binding, whereas changes in the mobile phase pH led to a re-orientation on SP Sepharose FF. This phenomenon agrees well with theoretical considerations, including a detailed description of the surface charge distribution with changing pH and linear elution experiments, giving an idea why proteins are often retained on ion-exchange materials beyond their isoelectric point. (C) 2008 Elsevier B.V. All rights reserved.