In ionexchange chromatography, the pH gradient mode becomes more and more popular today for the analysis of therapeutic proteins as this mode can provide higher or alternative selectivity to the commonly used salt gradient mode. Ideally, a linear pH response is expected when performing linear gradients. However up to now, only a very few buffer systems have been developed and are commercially available which can perform nearly linear pH responses when flowing through a given column. It is also known that a selected buffer system (mobile phase) can work well on one column but can fail on other column. The goal of this study was to practically evaluate the effects that ionexchange columns (weak and strong exchangers) might have on effluent pH, when performing linear pH gradient separations of therapeutic monoclonal antibodies. To attain this objective, the pH was monitored on-line at the column outlet using a specific setup. To make comprehensive observations of the phenomenon, four different mobile phase conditions and five cation exchange columns (weak and strong exchangers) were employed. The obtained pH responses were systematically compared to responses measured in the absence of the columns.

From this work, it has become clear that both the column and mobile phase can have significant effects on pH gradient chromatography and that their combination must be considered when developing a new method. Phase systems (column + mobile phase) providing linear pH responses are indeed the most suitable for separating mAbs with different isoelectric points and, with them, it is possible to elute mAbs across wide retention time ranges and with high selectivity.

在离子交换色谱中，pH梯度模式如今对于分析治疗性蛋白质越来越受欢迎，因为该模式可以提供比常用的盐梯度模式更高或更高的选择性。理想情况下，执行线性梯度时，线性pH响应是可以预期的。然而，到目前为止，只有很少的缓冲系统已经开发出来，并且在市场上可以买到，当流过给定的色谱柱时，该系统可以执行几乎线性的pH响应。还已知选定的缓冲液系统（流动相）在一根色谱柱上可以很好地工作，而在另一根色谱柱上可能会失败。这项研究的目的是，在对治疗性单克隆抗体进行线性pH梯度分离时，实际评估离子交换柱（弱交换器和强交换剂）对流出物pH的影响。为了达到此目的，使用特定的设置在色谱柱出口在线监测pH。为了对该现象进行全面观察，使用了四个不同的流动相条件和五个阳离子交换柱（弱而强的交换剂）。系统地将获得的pH响应与没有色谱柱时测得的响应进行比较。

从这项工作中，可以清楚地看到，色谱柱和流动相都可以对pH梯度色谱产生重大影响，开发新方法时必须考虑它们的组合。提供线性pH响应的相系统（色谱柱+流动相）确实最适合分离具有不同等电点的mAb，并且通过它们，可以在宽的保留时间范围内以高选择性洗脱mAb。