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 (logk₀ 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.

在当前的工作中，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质样品中非常紧密洗脱的峰的分离度。

这种方法涉及使用多个等度步骤，以及添加一个陡峭的负梯度部分（流动相强度降低），以“保留”沿色谱柱某处（在给定的迁移距离处）保留的峰，保留下来的化合物稍为洗脱。

首先，进行了一些模型计算以突出这种创新方法的潜力。为此，考虑了两个案例研究的保留参数（log k ₀和S），即分析两种治疗性mAb的混合物（简单地分离样品）和将治疗性mAb从其主要变体中分离出来（挑战解析样本）。结果证实，将负片段插入多等度洗脱程序可以是提高关键峰对之间选择性的好工具。但是，请记住，此方法仅适用于大溶质，或多或少遵循“开-关”型洗脱行为，这一点也很重要。

已成功开发了两个实际应用，以说明该新方法的实际优势，包括将治疗性mAb从具有非常接近洗脱行为的主要变体中分离出来，以及从完整mAb中分离出载体蛋白的过程。定量生物分析测定。这两个例子表明，通过包含负梯度斜率可以选择性提高蛋白质RPLC的选择性，该斜率选择性地使两个或多个目标峰的洗脱物分叉。