Lienqueo María Elena, Shene Carolina, Asenjo Juan
Department of Chemical Engineering and Biotechnology, Institute for Cell Dynamics and Biotechnology, University of Chile, Beauchef 861, Santiago, Chile.
J Mol Recognit. 2009 Mar-Apr;22(2):110-20. doi: 10.1002/jmr.927.
This paper describes a methodology for optimizing performance of hydrophobic interaction chromatography (HIC) for protein mixtures in which Rate Model simulations and evaluation of cost function are used. The system under study was HIC of a two-protein mixture (alpha-chymotrypsin and alpha-amylase), carried out with different conditions (gradient steepness, salt, concentration, volume of sample, pH, type of matrix, and flow rates). Parameters in the rate model were obtained from different sources. Mass transfer parameters (Reynolds, Peclet, and Biot numbers) were calculated using empirical correlations. Under the experimental conditions Re number was small (0.23) and axial dispersion negligible (PeL>300). Mass transfer was controlled by intraparticle diffusion (Bi>50). The model assumes that equilibrium constant (b) in the Langmuir isotherm was salt concentration (I) dependent [b(I)]. Parameters in the relationship for b(I) were estimated from experimental single protein elution curves and used to simulate protein mixtures. Rate model simulations showed that if protein sample load to the column was below 1 mg, displacement effects were negligible; in other cases protein interactions would limit the proposed mathematical description of HIC. The calibrated Rate Model successfully predicted elution curves of the protein mixture with deviations lower than 6x10(-4) absorbance units. The model was also able to predict that the Butyl Sepharose--NaCl 4 M system allowed to obtain the highest resolution (>1) for the two-protein mixture evaluated. The cost function built for optimizing the performance of HIC considers yield, purity, concentration, and the time needed to accomplish the separation. This function contains two types of parameters that have to be determined. The ones dependent on the HIC system and process conditions were obtained from simulations of elution curves of the two-protein mixture, by the calibrated Rate Model. The other parameters are dependent on characteristic and quality of the protein product; these were assumed for illustration purpose. Minimization of the cost function allows determination of flow rate, gradient steepness, and the fraction of eluted peak that has to be collected. Novelty of the present work is in showing how parameters in the fundamentally based Rate Model for HIC can be calibrated and how simulations can be used for the optimization of process conditions for the separation of a protein mixture.
本文描述了一种用于优化蛋白质混合物疏水相互作用色谱（HIC）性能的方法，该方法使用速率模型模拟和成本函数评估。所研究的系统是两种蛋白质混合物（α-胰凝乳蛋白酶和α-淀粉酶）的HIC，在不同条件下（梯度陡度、盐浓度、样品体积、pH值、基质类型和流速）进行。速率模型中的参数来自不同来源。传质参数（雷诺数、佩克莱数和毕奥数）使用经验关联式计算。在实验条件下，雷诺数较小（0.23），轴向扩散可忽略不计（PeL>300）。传质由颗粒内扩散控制（Bi>50）。该模型假设朗缪尔等温线中的平衡常数（b）与盐浓度（I）有关[b(I)]。b(I)关系中的参数通过实验单蛋白洗脱曲线估计，并用于模拟蛋白质混合物。速率模型模拟表明，如果柱上的蛋白质样品负载低于1mg，置换效应可忽略不计；在其他情况下，蛋白质相互作用会限制所提出的HIC数学描述。校准后的速率模型成功预测了蛋白质混合物的洗脱曲线，偏差低于6×10(-4)吸光度单位。该模型还能够预测丁基琼脂糖-NaCl 4M系统对于所评估的两种蛋白质混合物可获得最高分辨率（>1）。为优化HIC性能而构建的成本函数考虑了产率、纯度、浓度以及完成分离所需的时间。该函数包含两类必须确定的参数。依赖于HIC系统和工艺条件的参数通过校准后的速率模型对两种蛋白质混合物洗脱曲线的模拟获得。其他参数依赖于蛋白质产品的特性和质量；为说明目的而假定了这些参数。成本函数的最小化允许确定流速、梯度陡度以及必须收集的洗脱峰部分。本工作的新颖之处在于展示了如何校准基于HIC基本原理的速率模型中的参数，以及如何使用模拟来优化蛋白质混合物分离的工艺条件。
