Zhang Yuan, Cai Lingli, Wang Ying, Li Yifeng
Technology and Process Development (TPD), WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China.
Technology and Process Development (TPD), WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China.
Protein Expr Purif. 2019 Dec;164:105458. doi: 10.1016/j.pep.2019.105458. Epub 2019 Jul 22.
We previously developed a method that can significantly improve Protein A chromatography's capability of removing antibody aggregates. That particular method requires polyethylene glycol (PEG) and 400 mM or more of calcium chloride/sodium chloride to be added to wash and elution buffers. Consequently, Protein A chromatography performed using this method has relatively high concentration of salt in its eluate. The high salt content prevents the neutralized eluate from binding to ion exchange columns without conductivity adjustment. In the current study we demonstrated that mixed-mode chromatography can be used as a subsequent step to Protein A chromatography with high-salt-containing eluate. As mixed-mode ligand mediates salt-tolerant adsorption, it allows the neutralized Protein A eluate to be directly loaded without the need of conductivity adjustment, and thus enables a smooth and convenient connection between capture and polishing steps. In this work we also showed that the mixed-mode chromatography, performed in bind-elute mode, removed most of PEG in the Protein A eluate.
我们之前开发了一种方法，该方法可以显著提高蛋白A层析去除抗体聚集体的能力。该特定方法要求在洗涤和洗脱缓冲液中添加聚乙二醇（PEG）以及400 mM或更高浓度的氯化钙/氯化钠。因此，使用该方法进行的蛋白A层析在其洗脱液中的盐浓度相对较高。高盐含量使得未经电导率调整的中和洗脱液无法与离子交换柱结合。在当前研究中，我们证明混合模式层析可作为含高盐洗脱液的蛋白A层析的后续步骤。由于混合模式配体介导耐盐吸附，它允许直接加载中和后的蛋白A洗脱液而无需进行电导率调整，从而实现捕获和精制步骤之间的顺畅便捷连接。在这项工作中，我们还表明，以结合-洗脱模式进行的混合模式层析去除了蛋白A洗脱液中的大部分PEG。
