A hybrid mode of hydrophobic interaction chromatography (HHIC) is an emerging analytical technique for the separation of biomolecules under non-denaturing conditions that combines elements of conventional hydrophobic interaction and reversed-phase chromatography. This article explores the impact of mobile phase composition such as salt concentration and organic modifier on the separation of therapeutic monoclonal antibodies and related large biomolecules using poly (alkyl aspartamide) silica HIC columns. The initial mobile phase salt concentration had a significant impact on the separation of a mixture of large biomolecules demonstrating that the relationship of elution and salt concentration was more complex than in conventional HIC. In general, the earlier eluting components exhibited greater retention at higher salt concentration as is typical of HIC separations. Conversely, the later eluting components showed greater retention at lower initial salt concentration. This differential is useful for improving the overall separation by widening the elution window for components of a mixture. In addition, no significant unfolding of the proteins was detected by intrinsic fluorescence or electrospray mass spectrometry. The impact of linear velocity and gradient steepness was also evaluated.

中文翻译：

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单克隆抗体和相关生物分子的疏水相互作用色谱的混合模式：洗脱条件对使用聚（烷基天冬酰胺）硅胶柱的色谱性能的影响。

疏水相互作用色谱法（HHIC）的混合模式是一种新兴的分析技术，用于在非变性条件下分离生物分子，该技术结合了常规疏水相互作用和反相色谱法的要素。本文探讨了使用聚（烷基天冬酰胺）硅胶HIC色谱柱对诸如盐浓度和有机改性剂等流动相成分对治疗性单克隆抗体和相关大生物分子分离的影响。初始流动相盐浓度对大生物分子混合物的分离有重大影响，表明洗脱和盐浓度的关系比常规HIC更复杂。一般来说，较早的洗脱组分在高盐浓度下表现出更大的保留，这是HIC分离的典型特征。相反，后来洗脱的组分在较低的初始盐浓度下显示出更大的保留。该差异可通过扩大混合物成分的洗脱窗口来改善总体分离。另外，通过固有荧光或电喷雾质谱未检测到蛋白质的显着展开。还评估了线速度和梯度陡度的影响。通过固有荧光或电喷雾质谱未检测到蛋白质的显着展开。还评估了线速度和梯度陡度的影响。通过固有荧光或电喷雾质谱未检测到蛋白质的显着展开。还评估了线速度和梯度陡度的影响。