Department of Chemical Engineering, University of Delaware, Newark, DE 19716, USA.
J Chromatogr A. 2011 Jul 22;1218(29):4698-708. doi: 10.1016/j.chroma.2011.05.054. Epub 2011 May 25.
Protein transport behavior was compared for the traditional SP Sepharose Fast Flow and the dextran-modified SP Sepharose XL and Capto S resins. Examination of the dynamic binding capacities (DBCs) revealed a fundamental difference in the balance between transport and equilibrium capacity limitations when comparing the two resin classes, as reflected by differences in the locations of the maximum DBCs as a function of salt. In order to quantitatively compare transport behavior, confocal microscopy and batch uptake experiments were used to obtain estimates of intraparticle protein diffusivities. For the traditional particle, such diffusivity estimates could be used to predict column breakthrough behavior accurately. However, for the dextran-modified media, neither the pore- nor the homogeneous-diffusion model was adequate, as experimental dynamic binding capacities were consistently lower than predicted. In examining the shapes of breakthrough curves, it was apparent that the model predictions failed to capture two features observed for the dextran-modified media, but never seen for the traditional resin. Comparison of estimated effective pore diffusivities from confocal microscopy and batch uptake experiments revealed a discrepancy that led to the hypothesis that protein uptake in the dextran-modified resins could occur with a shrinking-core-like sharp uptake front, but with incomplete saturation. The reason for the incomplete saturation is speculated to be that protein initially fills the dextran layer with inefficient packing, but can rearrange over time to accommodate more protein. A conceptual model was developed to account for the partial shrinking-core uptake to test whether the physical intuition led to predictions consistent with experimental behavior. The model could correctly reproduce the two unique features of the breakthrough curves and, in sample applications, parameters found from the fit of one breakthrough curve could be used to adequately match breakthrough at a different flow rate or batch uptake behavior.
比较了传统的 SP Sepharose Fast Flow 和葡聚糖修饰的 SP Sepharose XL 和 Capto S 树脂的蛋白质传输行为。动态结合容量（DBC）的检测表明，当比较两种树脂类别时，传输和平衡能力限制之间的平衡存在根本差异，这反映在盐的函数的最大 DBC 位置的差异。为了定量比较传输行为，使用共聚焦显微镜和批量吸收实验来获得颗粒内蛋白质扩散率的估计值。对于传统颗粒，可以使用这种扩散率估计值准确预测柱突破行为。然而，对于葡聚糖修饰的介质，无论是孔扩散模型还是均相扩散模型都不充分，因为实验动态结合容量始终低于预测值。在检查突破曲线的形状时，很明显模型预测未能捕捉到两个在葡聚糖修饰的介质中观察到的特征，但在传统树脂中从未见过。比较共聚焦显微镜和批量吸收实验估计的有效孔扩散率揭示了一个差异，这导致了一个假设，即葡聚糖修饰的树脂中的蛋白质摄取可能发生在具有收缩核样锐利摄取前沿的情况下，但不完全饱和。不完全饱和的原因推测是蛋白质最初以低效包装填充葡聚糖层，但随着时间的推移可以重新排列以容纳更多的蛋白质。开发了一个概念模型来解释部分收缩核摄取，以测试物理直觉是否导致与实验行为一致的预测。该模型可以正确再现突破曲线的两个独特特征，并且在样品应用中，从一个突破曲线拟合中找到的参数可用于充分匹配不同流速或批量吸收行为下的突破。
