School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
J Chromatogr A. 2021 Jan 4;1635:461743. doi: 10.1016/j.chroma.2020.461743. Epub 2020 Nov 23.
In the present work, we describe the fundamental and practical advantages of a new strategy to improve the resolution of very closely eluting peaks within therapeutic protein samples. This approach involves the use of multiple isocratic steps, together with the addition of a steep negative gradient segment (with a decrease in mobile phase strength) to "park" a slightly more retained peak somewhere along the column (at a given migration distance), while a slightly less retained compound can be eluted. First, some model calculations were performed to highlight the potential of this innovative approach. For this purpose, the retention parameters (logk and S) for two case studies were considered, namely the analysis of a mixture of two therapeutic mAbs (simple to resolve sample) and separation of a therapeutic mAb from its main variant (challenging to resolve sample). The results confirm that the insertion of a negative segment into a multi-isocratic elution program can be a good tool to improve selectivity between critical peak pairs. However, it is also important to keep in mind that this approach only works with large solutes, which more or less follow an "on-off" type elution behavior. Two real applications were successfully developed to illustrate the practical advantage of this new approach, including the separation of a therapeutic mAb from its main variant possessing very close elution behavior, and the separation of a carrier protein from an intact mAb as might be encountered in a quantitative bioanalysis assay. These two examples demonstrate that improved selectivity can be achieved for protein RPLC through the inclusion of a negative gradient slope that selectively bifurcates the elution of two or more peaks of interest.
在本工作中，我们描述了一种新策略的基本和实际优势，该策略可提高治疗性蛋白样品中非常接近的洗脱峰的分辨率。该方法涉及使用多个等度步骤，并添加陡峭的负梯度段（降低流动相强度），以使稍微保留的峰在柱上的某个位置“停放”（在给定的迁移距离处），而稍微保留的化合物可以被洗脱。首先，进行了一些模型计算以突出该创新方法的潜力。为此，考虑了两个案例研究的保留参数（logk 和 S），即两种治疗性 mAb 的混合物的分析（简单的可分辨样品）和治疗性 mAb 与其主要变体的分离（难以分辨的样品）。结果证实，在多等度洗脱程序中插入负段可以是改善关键峰对之间选择性的有效工具。但是，还必须牢记，这种方法仅适用于或多或少遵循“开-关”类型洗脱行为的大溶质。成功开发了两个实际应用来举例说明这种新方法的实际优势，包括治疗性 mAb 与其主要变体的分离，该变体具有非常接近的洗脱行为，以及载体蛋白与完整 mAb 的分离，这种情况可能在定量生物分析测定中遇到。这两个示例表明，通过包含选择性分叉两个或更多感兴趣的峰洗脱的负梯度斜率，可以提高蛋白质 RPLC 的选择性。
