Protein Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, USA.
Anal Chem. 2009 Nov 1;81(21):8846-57. doi: 10.1021/ac901408j.
In the biotechnology industry, ion-exchange chromatography is widely used for profiling the charge heterogeneity of proteins, including monoclonal antibodies. Ionic strength based ion exchange separations, while having excellent resolving power and robustness, are product specific and time-consuming to develop. In the present work, a pH gradient based separation using a cation exchange column is described and shown to be a multiproduct charge sensitive separation method for monoclonal antibodies. Simple mixtures of defined buffer components were used to generate the pH-gradients that separate closely related antibody species. The form of the pH gradient was controlled and optimized by the pump as well as the buffer composition if necessary. During this work, the buffer compositions for the separation were optimized in parallel for several MAbs. The data shows that the multiproduct method is optimal for all of the MAbs studied. Operational aspects of the separation such as column chemistry, column length, and sample matrix indicate a very robust method. The pH gradient ion-exchange method is demonstrated to have significant resolving power and peak capacities far in excess of what we would expect for ionic strength elution ion-exchange. Data obtained demonstrates that the separation is relatively insensitive to column length. Direct analysis (no buffer exchange) of samples in matrixes consistent with in-process manufacturing pools is demonstrated. Such a capability is extremely useful for the high throughput evaluation of in-process and final product samples.
在生物技术行业中，离子交换色谱广泛用于分析蛋白质（包括单克隆抗体）的电荷异质性。基于离子强度的离子交换分离虽然具有出色的分辨率和稳健性，但具有产品特异性，开发过程耗时。在本工作中，描述了一种基于 pH 梯度的阳离子交换柱分离方法，并证明其是一种用于单克隆抗体的多产品电荷敏感分离方法。使用定义的缓冲成分的简单混合物来产生分离密切相关抗体种类的 pH 梯度。通过泵以及必要时的缓冲组成来控制和优化 pH 梯度的形式。在这项工作中，同时针对几种 MAbs 对分离的缓冲组成进行了优化。数据表明，该多产品方法对于所有研究的 MAbs 都是最佳的。分离的操作方面，如柱化学、柱长和样品基质，表明该方法非常稳健。pH 梯度离子交换方法具有显著的分辨率和峰容量，远远超过我们预期的离子强度洗脱离子交换。所获得的数据表明，该分离对柱长相对不敏感。在与过程制造池一致的基质中直接分析（无需缓冲液交换）的样品已得到证明。这种能力对于高通量评估过程中和最终产品样品非常有用。
