Protein Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
J Chromatogr A. 2009 Dec 18;1216(51):8904-9. doi: 10.1016/j.chroma.2009.10.045. Epub 2009 Oct 20.
Size exclusion chromatography is a widely performed analysis of monoclonal antibodies, primarily used to monitor the levels of higher weight molecular species such as aggregates. Owing to the subtleties of these separation mechanisms and frequently observed partial resolutions of components in these separations, many common methods for increasing the method throughput are not practical as they trade off resolution for speed. Short columns, high flow rates and smaller particles are examples of these approaches. In this paper a practical method is demonstrated for injecting samples onto the column in rapid succession and gating the detection window to monitor the elution of each sample individually. At any given instant approximately two samples are eluting through the column. By co-ordinating the injection and detection time windows the samples can be kept discrete and significant throughput enhancements achieved, up to nearly 2-fold improvements are demonstrated. A rudimentary theory is development to show that the throughput improvements can be predicted to approximation by simple column characteristics. Experimental results for a series of monoclonal antibodies demonstrate the equivalency of the method to a conventional injection approach, the throughput increase, and the robustness of the method.
体积排阻色谱法是一种广泛应用于单克隆抗体分析的方法，主要用于监测高分子量分子种类（如聚集物）的水平。由于这些分离机制的微妙性以及这些分离中经常观察到的部分组分分辨率，许多常见的提高方法通量的方法并不实用，因为它们是以牺牲分辨率为代价来提高速度。短柱、高流速和小颗粒就是这些方法的例子。本文介绍了一种实用的方法，可快速连续地将样品注入柱中，并对检测窗口进行门控，以单独监测每个样品的洗脱情况。在任何给定的时刻，大约有两个样品通过柱子洗脱。通过协调进样和检测时间窗口，可以使样品保持离散，从而实现显著的通量提高，可实现近 2 倍的提高。开发了一个基本理论来表明，通过简单的柱特性可以近似预测吞吐量的提高。一系列单克隆抗体的实验结果证明了该方法与传统进样方法的等效性、通量的提高以及该方法的稳健性。
