蛋白质静电吸附平衡与动力学的研究
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摘要
鉴于非线性吸附平衡和吸附动力学的研究在蛋白质离子交换制备色谱过程的优化和放大中的重要性和不成熟性,本论文主要开展了以下几个方面的研究工作:
1.基于统计热力学理论,考虑了蛋白质与吸附剂间作用以及吸附相中蛋白质分子间作用,推导出描述蛋白质离子交换吸附平衡的统计热力学模型(ST模型);从溶液热力学的角度,考虑了吸附相中因蛋白质分子间作用的存在而使吸附相与蛋白质理想稀溶液的偏离,以及因蛋白质-蛋白质作用与蛋白质-反离子作用的差异产生的吸附相局部组成的非理想性,提出了蛋白质离子交换吸附平衡的非理想吸附相模型(NIAP模型)。利用单组分蛋白质BSA的离子交换吸附平衡数据考察了模型的适用性,研究了液相条件(pH、离子强度和缓冲液种类)对模型参数的影响。利用BSA和溶菌酶的双组分蛋白质离子交换吸附平衡数据考察了多组分ST模型的适用性,与Bosma和Wesselingh提出的有效面积模型(AA模型)相比,由于考虑了吸附相中蛋白质分子间的相互作用,ST模型可更好地描述双组分蛋白质的离子交换吸附平衡。
2.在蛋白质与吸附界面间只存在静电作用的前提下,利用分子平均场理论研究了蛋白质净电荷数、离子强度、缓冲液种类、蛋白质分子大小和吸附方向对单组分蛋白质吸附平衡的影响;考察了蛋白质净电荷数、离子强度和蛋白质分子大小对双组分蛋白质吸附平衡的影响,明确指出了各影响因素的具体作用机理
3.以化学势梯度作为扩散推动力的Maxwell-Stefan扩散通量方程(MS方程)取代以浓度梯度为扩散推动力的Fick定律,结合平行扩散模型,对BSA在阴离子吸附剂Q-Sepharose FF上的动态吸附数据进行了模拟,研究了溶液pH、盐浓度和溶液主体蛋白质初始浓度对蛋白质内扩散系数的影响。
Whereas the studies of nonlinear adsorption equilibrium and adsorption kineticsis imperfect and important in the optimization and scale-up of preparativeion-exchange chromatography process, the related studies are carried out in the thesis.The details in this work are summarized as follows.
1. Based on statistical thermodynamic theory, a statistical thermodynamic model(ST model) describing ion-exchange adsorption equilibrium was obtained withprotein-adsorbent and protein-protein interactions considered. Another ion-exchangeadsorption equilibrium model, non-ideal adsorption phase model (NIAP model), wasalso proposed through the approach of solution thermodynamics. The NIAP modeltakes into account the discrepancy of adsorption phase with ideal dilute solution ofprotein arising from interaction between adsorbed protein molecules and localcomposition non-ideality due to the difference between protein-protein andprotein-counterion interaction. Both ST model and the NIAP model fit well with theadsorption equilibrium data of a single-component protein (bovine serum albumin,BSA). The effects of pH, ionic strength and buffer type on model parameters arediscussed. In order to test the validity of multi-component ST model, binary-component adsorption equilibrium data of bovine serum albumin and lysozyme at thecation adsorbent was used. Compared with the available area model (AA model), theST model that takes into consideration of interactions between adsorbed proteinmolecules fit better with the experimental data of binary protein ion-exchangeadsorption equilibrium.
2. With the presupposition that only electrical interaction act between proteinmolecules and adsorption interface, molecular mean field theory was utilized toexplain the influences of protein net charge, ionic strength, buffer type, proteinmolecular size, adsorption orientation on single-component protein adsorptionequilibrium. Influences of protein net charge, ionic strength and protein molecularsize on binary protein adsorption equilibrium were also investigated. The actingmechanisms of these factors are clarified.
3. Giving up the Fick law, we adopted the Maxwell-Stefan equation (MS equation)that uses chemical potential gradient instead of concentration gradient as diffusiondriving force to reveal the effect of protein concentration. Simulation of the dynamic
uptake process of BSA on anion adsorbent Q-Sepharose FF was performed byemploying parallel diffusion model and MS equation. The influences of pH, ionicstrength and initial bulk protein concentration on protein diffusion coefficient werestudied.
1.基于统计热力学理论,考虑了蛋白质与吸附剂间作用以及吸附相中蛋白质分子间作用,推导出描述蛋白质离子交换吸附平衡的统计热力学模型(ST模型);从溶液热力学的角度,考虑了吸附相中因蛋白质分子间作用的存在而使吸附相与蛋白质理想稀溶液的偏离,以及因蛋白质-蛋白质作用与蛋白质-反离子作用的差异产生的吸附相局部组成的非理想性,提出了蛋白质离子交换吸附平衡的非理想吸附相模型(NIAP模型)。利用单组分蛋白质BSA的离子交换吸附平衡数据考察了模型的适用性,研究了液相条件(pH、离子强度和缓冲液种类)对模型参数的影响。利用BSA和溶菌酶的双组分蛋白质离子交换吸附平衡数据考察了多组分ST模型的适用性,与Bosma和Wesselingh提出的有效面积模型(AA模型)相比,由于考虑了吸附相中蛋白质分子间的相互作用,ST模型可更好地描述双组分蛋白质的离子交换吸附平衡。
2.在蛋白质与吸附界面间只存在静电作用的前提下,利用分子平均场理论研究了蛋白质净电荷数、离子强度、缓冲液种类、蛋白质分子大小和吸附方向对单组分蛋白质吸附平衡的影响;考察了蛋白质净电荷数、离子强度和蛋白质分子大小对双组分蛋白质吸附平衡的影响,明确指出了各影响因素的具体作用机理
3.以化学势梯度作为扩散推动力的Maxwell-Stefan扩散通量方程(MS方程)取代以浓度梯度为扩散推动力的Fick定律,结合平行扩散模型,对BSA在阴离子吸附剂Q-Sepharose FF上的动态吸附数据进行了模拟,研究了溶液pH、盐浓度和溶液主体蛋白质初始浓度对蛋白质内扩散系数的影响。
Whereas the studies of nonlinear adsorption equilibrium and adsorption kineticsis imperfect and important in the optimization and scale-up of preparativeion-exchange chromatography process, the related studies are carried out in the thesis.The details in this work are summarized as follows.
1. Based on statistical thermodynamic theory, a statistical thermodynamic model(ST model) describing ion-exchange adsorption equilibrium was obtained withprotein-adsorbent and protein-protein interactions considered. Another ion-exchangeadsorption equilibrium model, non-ideal adsorption phase model (NIAP model), wasalso proposed through the approach of solution thermodynamics. The NIAP modeltakes into account the discrepancy of adsorption phase with ideal dilute solution ofprotein arising from interaction between adsorbed protein molecules and localcomposition non-ideality due to the difference between protein-protein andprotein-counterion interaction. Both ST model and the NIAP model fit well with theadsorption equilibrium data of a single-component protein (bovine serum albumin,BSA). The effects of pH, ionic strength and buffer type on model parameters arediscussed. In order to test the validity of multi-component ST model, binary-component adsorption equilibrium data of bovine serum albumin and lysozyme at thecation adsorbent was used. Compared with the available area model (AA model), theST model that takes into consideration of interactions between adsorbed proteinmolecules fit better with the experimental data of binary protein ion-exchangeadsorption equilibrium.
2. With the presupposition that only electrical interaction act between proteinmolecules and adsorption interface, molecular mean field theory was utilized toexplain the influences of protein net charge, ionic strength, buffer type, proteinmolecular size, adsorption orientation on single-component protein adsorptionequilibrium. Influences of protein net charge, ionic strength and protein molecularsize on binary protein adsorption equilibrium were also investigated. The actingmechanisms of these factors are clarified.
3. Giving up the Fick law, we adopted the Maxwell-Stefan equation (MS equation)that uses chemical potential gradient instead of concentration gradient as diffusiondriving force to reveal the effect of protein concentration. Simulation of the dynamic
uptake process of BSA on anion adsorbent Q-Sepharose FF was performed byemploying parallel diffusion model and MS equation. The influences of pH, ionicstrength and initial bulk protein concentration on protein diffusion coefficient werestudied.
引文
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