Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH
Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH
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DOI：
10.1016/j.jpba.2010.01.014
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发表时间：
2010-06-05
影响因子：
3.4
通讯作者：
Jiskoot, Wim
中科院分区：
文献类型：
--
作者：
Tantipolphan, Ruedeeporn;Romeijn, Stefan;Jiskoot, Wim
使用酸性非生理洗脱剂的胰岛素HP-SEC药典规程不代表胰岛素在配方中的缔合状态。本研究旨在利用在线紫外光吸收和多角度激光光散射检测技术，评价胰岛素在生理(含水、中性pH)洗脱液中在HP-SEC中的洗脱行为。考察了配方中胰岛素浓度和缔合状态(圆二色谱监测)和洗脱液组成(锌离子、精氨酸)对其洗脱行为的影响。我们发现，胰岛素在“生理性”HP-SEC中的洗脱行为既受胰岛素分子的动态缔合-解离作用的影响，也受胰岛素-柱相互作用的影响。胰岛素分子在HP-SEC洗脱液中重新平衡，使其洗脱行为实际上对配方中胰岛素的缔合状态不敏感。洗脱液中的锌离子促进了胰岛素与六聚体的结合，而精氨酸抑制了锌离子的作用，并诱导了胰岛素与二聚体和单体的柱上解离。“生理学”和棉仁式HP-SEC的联合结果显示，与单一方法中的任何一种方法相比，可以更好地观察热应激胰岛素的聚集状态。通过这项研究获得的洞察力对于正确评估胰岛素的HP-SEC数据至关重要。(C)2010爱思唯尔B.V.保留所有权利。
The pharmacopoeia protocol for HP-SEC of insulin, using an acidic non-physiological eluent, does not represent insulin's association state in the formulation. This study aimed to evaluate insulin's elution behavior in HP-SEC in a "physiological" (aqueous, neutral pH) eluent, using on-line UV absorption and multi-angle laser light scattering detection. The effect of insulin concentration and association state in the formulation (monitored by circular dichroism) and eluent composition (zinc ion, arginine) on its elution behavior was assessed. We showed that the elution behavior of insulin in "physiological" HP-SEC is affected by both dynamic association-dissociation of insulin molecules and insulin-column interactions. Insulin molecules re-equilibrated in the HP-SEC eluent, making its elution behavior practically insensitive to the association state of insulin in the formulation. Zinc ions in the eluent promoted association of insulin to hexamers, whereas arginine overruled the effect of zinc ions and induced on-column dissociation of insulin to dimers and monomers. Combined results from "physiological" and cotnpendial HP-SEC were shown to provide a better view of the aggregation state of heat-stressed insulin than either of the single methods. The insights obtained with this study are crucial for a proper evaluation of HP-SEC data of insulin. (C) 2010 Elsevier B.V. All rights reserved.