Institute for Biochemistry, University of Applied Sciences Mannheim, Germany.
J Chromatogr A. 2011 Aug 5;1218(31):5136-45. doi: 10.1016/j.chroma.2011.05.085. Epub 2011 May 30.
A large number of different stationary phases for ion-exchange chromatography from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (lysozyme, cytochrome c and two monoclonal antibodies) were determined for nine commercially available cation-exchange adsorbents. The linear gradient elution model in combination with a thermodynamic approach was used to analyse the characteristic parameters of the protein-stationary phase-interactions. Based on the pH dependency of the characteristic charge and the equilibrium constant for binding the differences between the standard Gibbs energies in the adsorbed and the solute state for the protein ΔG(P)° and the salt ΔG(S)° were calculated. The characteristic charge B of the proteins strongly depends on the molecular mass of the protein. For small proteins like lysozyme there is almost no influence of the stationary phase chemistry on B, while for the Mabs the surface modification strongly influences the B value. Surface extenders or tentacles usually increase the B values. The variation of the characteristic charge of the MABs is more pronounced the lower the pH value of the mobile phase is, i.e. the higher the negative net charge of the protein is. The standard Gibbs energy changes for the proteins ΔG(P)° are higher for the Mabs compared to lysozyme and more strongly depend on the stationary phase properties. Surface modified resins usually show higher ΔG(P)° and higher B values. A correlation between ΔG(P)° and B is not observed, indicating that non-electrostatic interactions as well as entropic factors are important for ΔG(P)° while for the B values the accessibility of binding sites on the protein surface is most important.
有大量来自不同制造商的不同离子交换色谱固定相可供选择，它们在许多化学和物理性质上有很大的差异。因此，结合机制也可能不同。在本报告的工作中，测定了模型蛋白（溶菌酶、细胞色素 c 和两种单克隆抗体）在九种市售阳离子交换吸附剂上的保留数据。线性梯度洗脱模型与热力学方法相结合，用于分析蛋白质-固定相相互作用的特征参数。基于特征电荷的 pH 依赖性和结合平衡常数，计算了蛋白质-吸附态和溶质态之间标准吉布斯自由能差ΔG(P)°和盐ΔG(S)°的差异。蛋白质的特征电荷 B 强烈依赖于蛋白质的分子量。对于像溶菌酶这样的小蛋白，固定相化学对 B 的影响几乎可以忽略不计，而对于 Mabs，表面修饰强烈影响 B 值。表面扩展剂或触角通常会增加 B 值。Mab 的特征电荷 B 的变化更为明显，即流动相的 pH 值越低，蛋白质的负净电荷越高。与溶菌酶相比，蛋白质的标准吉布斯自由能变化ΔG(P)°对于 Mab 更高，并且更强烈地依赖于固定相的性质。表面修饰的树脂通常显示出更高的ΔG(P)°和更高的 B 值。ΔG(P)°和 B 值之间没有相关性，表明非静电相互作用和熵因素对ΔG(P)°很重要，而对于 B 值，蛋白质表面结合位点的可及性是最重要的。
