Binary protein adsorption to DEAE sepharose FF

Equilibrium between ion exchangers and proteins is one of the most important factors in ion exchange chromatography. A model system was used to simulate the adsorption of human serum albumin (HSA) and ovalbumin (OVA) as a binary protein system to the DEAE Sepharose FF as an anion exchanger. Two models, one based on a competition between adsorbing molecules and the other a non competitive model have been compared to experimental results. Competitive adsorption was seen in experiments in which breakthrough curves and the profiles of adsorbed proteins in packed beds were determined. However, although the results for packed bed experiments were more closely predicted by the competitive model, some discrepancies were found, suggesting that when considering multicomponent protein adsorption to ion exchangers it may also be necessary to take account of factors such as the molecular size of adsorbing proteins and any potential inter protein interaction, which may hinder the development of a general model of multicomponent protein adsorption to ion exchangers.     

蛋白质在膨胀床吸附层析剂的静态和动态吸附性能

以牛血清白蛋白 (BSA)为目标蛋白 ,考察膨胀床用离子交换树脂StreamlineDEAE的静态和动态吸附性能 ,并和离子交换树脂DEAESepharoseFF进行比较 ,实验发现两者的静态吸附性能相似 ,而动态吸附性能差别较大。根据动态吸附数据计算出液膜扩散系数和孔内扩散系数。     

Adsorption Performance of Proteins to CM Sepharose FF and DEAE Sepharose FF Adsorbents

Adsorption equilibrium and kinetics were studied for the binding of proteins to CM Sepharose FF and DEAE Sepharose FF. The influence of temperature, pH, viscosity, initial concentration and the volume of adsorbents on the adsorption characteristics was investigated in detail. The results showed that the isotherms of lysozyme to CM Sepharose FF were well described by a Langmuir-type correlation. The two phase resistance model describing the dynamic adsorption process of lysozyme, papain, BSA to CM Sepharose FF was presented, and the pore diffusion coefficients were determined by using this model and the dynamic adsorption data.     

改进葡聚糖接枝技术制备高性能层析介质

利用改进的葡聚糖接枝技术,在以环氧氯丙烷为交联剂交联琼脂糖微球骨架的过程中加入葡聚糖溶液,在交联的同时接枝葡聚糖制得葡聚糖接枝型琼脂糖微球Rigose-Dex,再与盐酸2-氯三乙胺（DEAE）反应,获得葡聚糖接枝型高载量弱阴离子交换介质Rigose-Dex DEAE。以牛血清白蛋白（BSA）为模型蛋白,以商品化介质DEAE Sepharose 6FF为对照,系统研究了该葡聚糖接枝型Rigose-Dex DEAE的蛋白吸附性能,并进行了物理性能研究。结果表明,改进后葡聚糖接枝技术的最高接枝量为24.5mg/mL。自制Rigose-Dex DEAE可耐受700cm/h 的线性流速,对BSA的动态饱和载量为127.6mg/mL, 为商品介质DEAE Sepharose 6FF 载量的212%;具有在高流速下快速结合蛋白的能力,上样蛋白溶液在层析柱中停留2min即可基本达到饱和动态载量;重复使用性能好,经120 次在线清洗后,Rigose-Dex DEAE介质的动态载量为原始载量的90.4%。     

Protein adsorption dynamics in cation-exchange chromatography quantitatively studied by confocal laser scanning microscopy

A self-contained research system based on the technique of confocal laser scanning microscopy (CLSM) was put up to quantitatively analyze the dynamics of protein adsorption to porous cation exchanger by mathematical modeling. Bovine serum albumin adsorption to the cation exchanger SP Sepharose FF was performed by batch adsorption and micro-flow cell in which protein concentration in single absorbent was visualized by CLSM. The effects of ionic strength and the protein concentration in liquid phase (50 mmol/L acetate buffer, pH 5.0) on the adsorption dynamics were examined. The intraparticle concentration profile data experimentally obtained from CLSM were quantitatively analyzed by three diffusive mass transfer models (i.e., pore diffusion, surface diffusion and Maxwell-Stefan models (MSM)) in virtue of the attenuation equation for the CLSM visualization developed earlier. The nuance between the model simulations and experimental results of the developing protein distribution in a single adsorbent particle could thus be found out. Without salt addition to the buffer, the adsorption isotherm was strongly favorable, and the pore diffusion model (PorDM) and MSM gave similarly good simulations of the experiments, whereas the surface diffusion model was unreasonable in the model presumption. Moreover, it was observed that the experimentally obtained adsorption front was relative flatter as compared with the calculated results from the PorDM, which implied the possible existence of surface diffusion. With increasing salt concentration, the simulations became to deviate from the experiments. Especially, when the salt concentration approached 50 mmol/L, all the three mass transfer models could hardly give good simulation of the experiment. This was considered due to the difference in adsorption behavior between the fluorescence labeled and unlabeled proteins therein.     

Comparison of the adsorption characteristics of expanded bed adsorbent with conventional chromatographic adsorbent

The static and kinetic adsorption characteristics of Streamline DEAE and DEAE-Sepharose FF were studied under various operating conditions. The adsorption isotherms for the two types of adsorbents were obtained and found to fit well to a Langmuir-type expression. The adsorption kinetics of Streamline DEAE at different concentrations, temperatures, and viscosities were studied and a mathematical model including particle size distribution was developed to describe the adsorption performance of Streamline DEAE. Comparing the uptake curves of Streamline DEAE with DEAE-Sepharose FF, it could be concluded that Streamline DEAE achieves equilibrium faster to get equilibrium than DEAE-Sepharose FF, indicating that Streamline DEAE could be used in higher flow rate systems.     

Protein adsorption onto diethylaminoethyl dextran modified anion exchanger: Effect of ionic strength and column behavior

Our previous studies on bovine serum albumin(BSA) adsorption to diethylaminoethyl dextran(DEAE dextran,DexD, grafting-ligand) and DEAE(D, surface-ligand) modified Sepharose FF resins found that all the grafted resins(FF-DexD and FF-D-DexD) exhibited extremely fast uptake rate(effective diffusivity, D_e, D_e/D_O 1.4),which was six times greater than the ungrafted resins(D_e/D_O 0.3). In this work, the influence of ionic strength(IS) on 6 typical DEAE dextran-grafted resins was investigated. Bath adsorption equilibria and kinetics, breakthrough, and linear gradient elution experiments were conducted. Commercial DEAE Sepharose FF was used for comparison. It is found that protein adsorption capacities on DEAE dextran-FF resins and the commercial resin decreased with increasing IS, but DEAE dextran-FF resins exhibited much higher capacity sensitivity to salt concentration. Besides, steeper decrease of adsorption capacities could be obtained at higher graftingligand or surface-ligand density. It is worth noting that the facilitating role of surface-ligand to the "chain delivery" effect was weakened after adding salt, leading to the less improvement in uptake rate by increasing surface-ligand density at higher IS. Although the uptake rates of the DEAE dextran-FF resins increased first and then decreased with increasing IS, they kept the extremely high level of De values(D_e/D_O 1.1) at the their working/binding IS range. Moreover, the DEAE dextran-FF resin displayed much higher adsorption capacities and De values than commercial ungrafted resin in their working condition. Furthermore, the column results of DEAE dextran-FF resins presented higher dynamic binding capacities than and similar elution ISs with DEAE Sepharose FF to achieve similar(or even higher) recoveries suggest the excellent chromatographic column performance of the DEAE dextran-FF resins. Finally, both high recovery and purity of BSA and γ-globulin could be easily achieved using the typical DEAE dextran-FF column, FF-D60-DexD160, to separate their binary mixtures,by step gradient elution. The research has provided new insights into the practical application of the series of DEAE-dextran grafted resins in protein chromatography and proved their superiority.     

盐种类和浓度对蛋白质疏水性吸附过程有效孔扩散系数的影响

研究了牛血清白蛋白在配基密度不同的两种疏水性吸附剂Phenyl Sepharose FF low sub和Phenyl Sepharose FF high sub上的吸附平衡和孔内传质动力学,重点考察了盐种类和浓度的影响.结果表明,Na2SO4溶液中盐浓度的增加导致蛋白质吸附容量的增大和解离常数的降低比(NH4)2SO4溶液更显著.利用孔扩散模型得到的有效扩散系数随盐浓度及配基密度的增大而提高,表明表面扩散作用对孔内传质的贡献随吸附容量提高而增大.     

离子强度和溶质浓度对蛋白质在Q Sepharose FF中吸附动力学的影响

采用孔扩散模型, 模拟不同盐浓度和不同蛋白质初始浓度条件下,吸附牛血清白蛋白(BSA)的动态吸附曲线并获得孔扩散系数;考察了离子强度和溶质浓度对蛋白质在阴离子交换剂Q Sepharose FF中吸附动力学的影响.结果表明,蛋白质的孔扩散系数随初始浓度的增大而下降;在氯化钠浓度小于0.10 mol•L^-1的范围内,蛋白质的孔扩散系数随着盐浓度的增加而增大,但当盐浓度增大到0.15 mol•L^-1时又有所降低,表明存在着一个最佳的离子强度,使蛋白质的孔扩散系数最大.     

柱层析法纯化基因工程HBsAg中试工艺的研究

由乙肝病毒S基因转化的哺乳动物细胞培养收液，经过Butyl-S-SepharoseFF层析，DEAESepharoseFF层析和Sepharose4FF层析，可获HBsAg纯品。产品检定结果表明，PAGE和SDS-PAGE电泳纯度均合格，小牛血清残余量、细胞DNA残余量和动物免疫效力也均符合规定标准．HBsAg终收率达40％左右．在此工艺中，疏水作用柱层析的纯化效率比较高，可去除绝大部分杂蛋白和细胞DNA。     

煤粉炉和循环流化床锅炉飞灰吸附汞动力学及其吸附机制

在固定床吸附反应器内对两台300MW等级燃煤发电机组循环流化床锅炉和煤粉锅炉飞灰样品进行气相零价汞吸附实验,通过改变实验工况研究温度、入口汞浓度和入口气体流量对飞灰汞吸附能力的影响。采用颗粒内扩散模型、准一阶和准二阶动力学模型、耶洛维奇（Elovich）模型对实验数据分别进行拟合,从动力学的角度探讨两种锅炉飞灰对气相零价汞吸附的影响机制以及两种锅炉飞灰与气相零价汞之间吸附动力学行为差异。结果表明：相同工况下循环流化床锅炉飞灰汞吸附过程的穿透时间和平衡吸附量远大于煤粉锅炉飞灰。吸附温度为150℃时,两种锅炉飞灰对气相零价汞的平衡吸附量最大。由于外扩散阻力随气体入口流量的增加而减小,入口汞浓度的增加可提高传质推动力,飞灰对汞的吸附得以增强。动力学分析表明飞灰的零价汞吸附由外扩散、内扩散和表面化学吸附共同控制,其中表面化学吸附是该吸附过程中的控制步骤;准二阶动力学模型和Elovich动力学模型更适合于描述该吸附过程。相同实验条件下,循环流化床锅炉飞灰吸附过程拟合所得的颗粒内扩散系数、准一阶动力学常数和初始吸附速率均大于煤粉锅炉飞灰。     

蛋白质在离子交换介质中的动态吸附性能

研究了离子交换介质CM－Sepharose FF对几种不同的蛋白质－溶菌酶，木瓜蛋白酶，牛血清蛋白的动态吸附性能，考察了PH值，溶液粘度，初始蛋白浓度及吸附剂量等因素的影响，结果表明，这些参数会对吸附动态吸附性能产生不同程度的影响，而这些蛋白质也都显示了相似的规律，采用两相阻力模型描述了蛋白质在离子交换介质的动态吸附性能，计算得到了蛋白质在吸附剂颗粒中的扩散系数，有助于进一步研究蛋白质吸附过程的传质现象。     

蛋白质的膨胀床吸附过程穿透模型分析

引　言膨胀床吸附技术是近十几年来出现的一种新型生物分离模式 .该技术集细胞碎片清除、料液浓缩和蛋白质纯化等步骤于一身 ,大大提高了目标产物收率 ,降低了纯化时间和费用 .自 1993年Streamline系列膨胀床吸附介质和装置问世以来 ,国外已有不少利用膨胀床吸附技术提取蛋白质的报道[1,2 ] .已有研究表明 ,料液特性、吸附剂粒径等对膨胀床的流体力学特性和蛋白质在膨胀床内的吸附行为有着不同程度的影响[3～ 5] .考察这些影响因素有助于加深对膨胀床层析行为的理解 ,指导膨胀床层析的优化设计 .然而 ,由于膨胀床层析过程的复杂性 ,众多因素相互作用 ,某一参数的改变往往引起操作参数的系统性变化 ,因此实验条件下考察单一参数的改变对膨胀床层析过程的影响是不现实的[5,6 ] .　　　前期研究表明 ,对不同黏度的缓冲液 ,穿透模型能够较准确的预测蛋白质的穿透行为 .因此 ,本研究在前期研究的基础上[4 ,7] ,利用穿透模型分析了膨胀床吸附过程的质量传递和流体力学特1　穿透模型该模型假设[4 ,5] :①吸附剂颗粒为球形 ,有均一的尺寸和密度 ,离子交换基团均匀地分布在颗粒内部 ;②床内径向不存在浓度梯度 ;...     

长白白眉蝮蛇蛇毒类凝血酶的纯化及质量检测

长白白眉蝮蛇原毒经DEAE Sepharose FF ,CM Sepharose FF和G Sepharose CL 6B层析后 ,制得长白白眉蝮蛇蛇毒类凝血酶精品。该产品经质量检测完全符合卫生部颁发的质量标准 ,该工艺适合于大规模纯化长白白眉蝮蛇蛇毒类凝血酶。     

反离子对牛血清白蛋白在荷电葡聚糖接枝介质中色谱行为的影响

从前期开发的具有极高的吸附容量及传质速率的二乙氨乙基葡聚糖接枝离子交换介质中选取FF-D50-DexD100和FF-DexD100为典型代表,利用Cl^-、SCN^-、SO₄^2-、HPO₄^2-为模型反离子,以牛血清白蛋白（BSA）为模型蛋白,以商品化介质（Q Sepharose FF、Q Sepharose XL、DEAE Sepharose FF）为对照,在离子强度为0.06mol/L下,系统研究反离子对二乙氨乙基葡聚糖接枝介质的蛋白质吸附与洗脱行为的影响。结果表明,二乙氨乙基葡聚糖接枝介质对不同反离子的偏好性存在差异,且该偏好性差异与基团所处位置（接枝链配基或表面配基）无关。同时,介质偏好性弱的反离子会通过促进二乙氨乙基葡聚糖接枝介质的“链传递”效应加快蛋白质的传质速率,从而提高动态吸附容量。因此,在使用二乙氨乙基葡聚糖接枝介质进行蛋白质色谱柱分离过程中,可在吸附操作中使用HPO₄^2-,在洗脱操作中使用SCN^-来优化分离效果。     

一步离子交换层析从Cohn组分V上清液中分离人血清白蛋白

传统的血浆低温乙醇沉淀工艺中Cohn组分V上清液由于其乙醇浓度高(体积浓度40％),进一步回收残余蛋白困难而被作为废弃组分.本研究探索了采用一步层析从Cohn组分V上清中回收入血清白蛋白的方法.首先以牛血清白蛋白(BSA)为模型蛋白,比较了三种不同类型介质在不同乙醇-水溶液中的吸附容量.疏水介质在乙醇-水溶液中对BSA...     

Surface DEAE groups facilitate protein transport on polymer chains in DEAE‐modified‐and‐DEAE‐dextran‐grafted resins

Sepharose FF was modified with diethylaminoethyl‐dextran (DEAE‐dextran, DexD) and/or DEAE (D) to fabricate three types of ion exchangers FF‐DexD (grafting‐ligand resin), FF‐D (surface‐ligand resin), and FF‐D‐DexD (mixed‐ligand resin), for protein adsorption equilibria and kinetics study. It was found that both adsorption capacity and uptake rate (effective diffusivity, D_e) were significantly enhanced by grafting DEAE‐dextran. Notably, the D_e values on FF‐DexD and FF‐D‐DexD (D_e/D₀ > 1.4) were six times greater than those on FF‐D (D_e/D₀ < 0.3). More importantly, the increase of surface‐ligand density greatly enhanced uptake kinetics on FF‐D‐DexD. The results indicate that the surface ligands assisted the transport of bound proteins on polymer chains in the mixed‐ligand resins. That is, surface ligands worked as “transfer stations” between two neighboring chains, resulting in enhanced transport of bound proteins on chains. The research thus disclosed the unique role of surface ligands in facilitating protein uptake kinetics onto polymer‐grafted ion‐exchangers. © 2016 American Institute of Chemical Engineers AIChE J, 62: 3812–3819, 2016     

Streamline DEAE对蛋白酶复合物的吸附平衡研究

以人凝血酶原复合物(以下简称PCC)为目标产物,研究了扩张床用离子交换树脂Streamline DEAE对蛋白酶复合物的吸附平衡。测定了不同温度下的吸附等温线和吸附动力学曲线,并通过计算求得热力学和动力学参数。结果表明:Streamline DEAE对PCC的吸附符合Langmuir模型,吸附反应的焓热变为34.466 kJ/mol;吸附动力学曲线用拟二级速率方程拟和有着较好的吻合度。吸附过程中蛋白质分子在树脂颗粒内部的扩散为控制步骤,求得吸附反应的表观活化能为29.089 kJ/mol,表明此Streamline DEAE对PCC的吸附具有反应速率快,性能稳定等特点。     

Modelling of heavy metal adsorption into activated carbon from apricot stones in fluidized bed

The purpose of the present study is to investigate the behaviour of activated carbon from apricot stones (ACAS) in a closed circuit fluidized bed (CCFB) for removal of Pb, Cu, Cd and Zn ions from aqueous solution. The expansion characteristics of the bed are studied. A homogeneous solid phase diffusion model was used to describe the mass transfer inside the adsorbent particles. A piston-dispersion model was applied for the liquid phase in the bed and compared to the calculations, assuming perfect mixing conditions in the whole CCFB adsorber. The close correspondence between the experimentally determined adsorption in batch and fluidized bed operation modes support the observation that the CCFB can be described by a stirred batch adsorption model. The concentration profile at the bed exit is satisfactorily predicted by the model, thus confirming the equilibrium and kinetic parameters, determined from the laboratory batch adsorption runs.     

Expanded-Bed Adsorption of Proteins Using Ion-Exchangers

Abstract

The use of expanded beds of ion-exchange adsorbents for the adsorption of proteins from feedstocks containing whole cells is described. Such feedstocks can be applied to the bed without prior removal of particulate material by centrifugation or filtration, thus showing considerable potential for this approach in simplifying downstream processing flow-sheets. Stable, expanded beds can be obtained using simple equipment adapted from that used for conventional packed bed adsorption and chromatography processes. Ion-exchange adsorbents are likely to be chosen for such separations as a result of the robustness of the ion-exchange groups to cleaning and sanitization protocols. Frontal analysis involving the measurement of breakthrough curves indicates that the adsorption performance of an expanded bed is similar to that achieved when the same amount of adsorbent is used in a packed configuration at the same volumetric flow-rate. In addition, the adsorption performance of an expanded bed of a cation exchanger was not diminished when adsorption was carried out in the presence of high concentrations of cells. However, the performance of similar beds of anion exchangers was found to be reduced under some conditions depending on the type of cells, their concentration, and their age. The results from the expanded bed adsorption experiments were consistent with those from batch adsorption which showed that the adsorption characteristics of the anion exchangers were diminished in the presence of cells in contrast to results with a cation-exchanger system where little deterioration in performance was noted. The results suggest that cation exchangers might be expected to be of more use for expanded bed adsorption in the presence of cells.