Center for Molecular and Engineering Thermodynamics, Department of Chemical and Biomolecular Engineering, University of Delaware , 150 Academy Street, Newark, Delaware 19716, United States.
Langmuir. 2018 Jan 16;34(2):673-684. doi: 10.1021/acs.langmuir.7b03289. Epub 2017 Dec 29.
Knowledge of the nanoscale distribution of proteins in chromatographic resins is critical to our mechanistic understanding of separations performance. However, the nano- to mesoscale architecture of these materials is challenging to characterize using conventional techniques. Small-angle neutron scattering was used to probe (1) the nano- to mesoscale structure of chromatographic media and (2) protein sorption in these media in situ with protein-scale resolution. In particular, we characterize the effect of the architecture of cellulose-based and traditional and dextran-modified agarose-based ion-exchange resins on the nanoscale distribution of a relatively small protein (lysozyme) and two larger proteins (lactoferrin and a monoclonal antibody) at different protein loadings. Traditional agarose-based resins (SP Sepharose FF) can be envisioned as comprising long, thin strands of helical resin material around which the proteins adsorb, while higher static capacities are achieved in dextran-modified resins (SP Sepharose XL and Capto S) due to protein partitioning into the increased effective binding volume provided by the dextran. While protein size is shown not to affect the underlying sorption behavior in agarose-based resins such as SP Sepharose FF and XL, it plays an important role in the cellulose-based S HyperCel and the more highly cross-linked agarose-based Capto S, where size-exclusion effects prevent larger proteins from binding to the base matrix resin strands. Based on the data, we propose that entropic partitioning effects such as depletion forces may drive the observed protein crowding. In general, these observations elucidate the structure and point to the mechanism of protein partitioning in different classes of chromatographic materials, providing guidance for optimizing their performance.
了解蛋白质在色谱树脂中的纳米级分布对我们理解分离性能的机理至关重要。然而，使用传统技术来表征这些材料的纳米到介观结构具有挑战性。小角中子散射用于探测（1）色谱介质的纳米到介观结构和（2）这些介质中蛋白质的原位吸附，具有蛋白质尺度的分辨率。特别是，我们描述了纤维素基和传统以及葡聚糖修饰的琼脂糖基离子交换树脂的结构对相对较小的蛋白质（溶菌酶）和两种较大的蛋白质（乳铁蛋白和单克隆抗体）在不同蛋白质负载下的纳米级分布的影响。传统的琼脂糖基树脂（SP Sepharose FF）可以被想象为由螺旋状树脂材料组成的长而细的链，蛋白质在这些链上吸附，而在葡聚糖修饰的树脂（SP Sepharose XL 和 Capto S）中实现了更高的静态容量，这是由于蛋白质分配到由葡聚糖提供的增加的有效结合体积中。虽然蛋白质的大小不会影响 SP Sepharose FF 和 XL 等琼脂糖基树脂中的基本吸附行为，但它在纤维素基 S HyperCel 和交联程度更高的琼脂糖基 Capto S 中起着重要作用，其中尺寸排阻效应阻止较大的蛋白质与基本基质树脂链结合。基于这些数据，我们提出了熵分配效应（如耗散力）可能驱动观察到的蛋白质拥挤。一般来说，这些观察结果阐明了不同类别的色谱材料中蛋白质分配的结构和机制，为优化其性能提供了指导。
