Characterization of unstable ion-exchange chromatographic separation of proteins.
Characterization of unstable ion-exchange chromatographic separation of proteins.
复制标题
蛋白质不稳定离子交换色谱分离的表征。
DOI：
10.1016/s0021-9673(99)00592-0
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
O. Kaltenbrunner
中科院分区：
文献类型：
--
作者：
S. Yamamoto;P. Watler;D. Feng;O. Kaltenbrunner
通过逐步洗脱离子交换色谱法对蛋白质进行非常精细的分离通常是一个不稳定的过程。为了表征这种过程的不稳定性，洗脱体积的变化进行了检查的模型方程，其中包含的离子交换容量和吸附位点的数量。模型计算所需的数据由梯度洗脱实验获得。作为一个模型分离系统，选择了一个模型蛋白质（β-乳球蛋白）在等电点附近的弱阳离子交换色谱柱上的逐步洗脱。洗脱体积显著变化，离子交换容量变化较小。发现必须调节洗脱缓冲液的离子强度以补偿由于离子交换容量变化而引起的洗脱体积的变化。还发现洗脱缓冲液的离子强度和pH是影响洗脱体积的重要变量。在该模型分离系统中，表明pH值应在±0.1单位内，离子强度应在±0.002 mol/l内，以满足标准（±5%洗脱体积变化）。建议获得梯度洗脱数据，以预测逐步洗脱中洗脱体积的变化。通过使用梯度洗脱数据，可以执行过程诊断，并且可以获得关于过程稳定性的重要信息。
Very fine separation of proteins by stepwise elution ion-exchange chromatography is very often a unstable process. To characterize the unstability of such processes the elution volume variations were examined by the model equation which contained the ion-exchange capacity and the number of adsorption sites. The data needed for the model calculation were obtained from gradient elution experiments. As a model separation system stepwise elution of a model protein (β-lactoglobulin) near the isoelectric point on a weak cation-exchange chromatography column was chosen. The elution volume varied significantly with a small change in the ion-exchange capacity. It was found that the ionic strength of the elution buffer must be adjusted in order to compensate a change in the elution volume due to the ion-exchange capacity variations. The ionic strength and the pH of the elution buffer were also found to be important variables affecting the elution volume. In this model separation system, it was indicated that the pH should be within ±0.1 unit and the ionic strength within ±0.002 mol/l in order to meet the criteria (±5% elution volume variation). It is recommended that gradient elution data be obtained for predicting elution volume variations in stepwise elution. By using the gradient elution data the process diagnosis can be performed, and the important information on the process stability can be obtained.