疏水膜色谱法对生物大分子的快速纯化

首次采用自制的分别级合了辛基、丁基、苯基及聚乙二醇-4000的4种常用疏水基团的纤维素疏水膜色谱柱,以键合了辛基、苯基的SepharoseCL－4B凝胶柱为对照,考察了疏水膜色谱柱对牛血清白蛋白（BSA）的动态吸附容量及流速对吸附容量的影响。疏水膜色谱柱对蛋白及酶具有较好的疏水吸附及纯化作用,但吸附容量比相应的琼脂糖凝胶柱低得多。增大流速及降低蛋白溶液质量浓度对疏水膜色谱柱的吸附容量影响较小,这些性能使膜色谱柱非常适合于大体积低质量浓度蛋白溶液（如基团工程培养液）的分离纯化。     

白蛋白在以染料为配基的醋酸纤维滤棒上的等温吸附行为(英文)

 以醋酸纤维滤棒为基质 ,染料CibacronBlueF3GA为配基 ,合成了一种新的染料亲和介质 ;分别以牛血清白蛋白 (BSA)和人血清白蛋白 (HSA)为对象 ,用静态法进行了吸附实验 ,得到了相应的亲和等温吸附曲线 ;对曲线按Langmuir模型和Freundlich模型分别进行拟合 ;结果表明 ,醋酸纤维滤棒染料亲和介质对BSA和HSA的等温吸附遵循Freundlich模型。采用该亲和介质装柱并分离实际样品人血浆 ,可得到纯化的人血清白蛋白。     

牛血清白蛋白在NVP/HEMA无规共聚物水凝胶上的吸附研究

合成了不同含水量的N-乙烯吡咯烷酮(NVP)／甲基丙烯酸-β-羟乙酯(HEMA)共聚物水凝胶．研究了温度、pH值，蛋白质初浓度及离子强度等因素对牛血清白蛋白吸附性能的影响．采用紫外分光光度法测定BSA的吸附量．结果表明，蛋白质初浓度越高．温度越高，离子强度越大；蛋白质内部更多的疏水性氧基酸残基暴露于外部．吸附量增加：在等电点pH=4．7附近蛋白质沉积量最大．     

聚苯乙烯—g—聚氧乙烯接枝共聚物表面阻抗蛋白质吸附的研究

利用放射性磺标记技术研究了血浆蛋白质－缓冲溶液体系在聚苯乙烯－ｇ－聚氧乙烯接枝共聚物表面的等温吸附和吸附动力学，材料表面蛋白质等温吸附量或平衡吸附量随表面ＰＥＯ含量增加而下降，吸附量最低值小于０．２５μｇ．ｃｍ＾－２；同时探讨了材料表面“梳状”结构对材料表面ＰＥＯ侧链阻抗蛋白质性能的影响。     

两种固定化金属螯合复合亲和膜色谱介质制备

以纤维素滤纸为基质,通过碱处理、环氧活化、偶联亚氨基二乙酸二钠、固定化Ｃｕ＾２＋后制得了大孔纤维素亲和膜。另外,在活化后的膜上通过共价交联覆盖上琼脂糖,制得了具有类似“三明治”结构且性能优于的复合亲和膜,装柱后分别制得固定化金属螯合亲和膜色谱柱。对两种亲和膜进行牛血清白蛋白等温吸附测定显示,两者的最大吸附量分别为１．１７ｍｇ／ｃｍ＾２和１．３０ｍｇ／ｃｍ＾２,与传统的琼脂糖凝胶类介质吸附量相当,表     

疏水性二氧化硅气凝胶吸附水中微量苯酚和甲醛的研究

气凝胶是一种新型纳米多孔非晶材料,其密度低、比表面积大、孔隙率高,可以作为吸附剂.本文研究了稻壳灰为硅源制成的合甲基的疏水性二氧化硅气凝胶对水中微量苯酚和甲醛的吸附性能.结果表明,疏水性气凝胶对水中微量苯酚和甲醛具有一定的吸附能力,实验中对苯酚的吸附容量是1.93mg/g,要大于对甲醛的吸附容量0.92mg/g;增大气凝胶的疏水化程度可以增加其对水中微量苯酚和甲醛的平衡吸附容量;苯酚的吸附平衡等温曲线符合Langmuir和Freundlich方程,甲醛的吸附平衡等温曲线符合Freundlich方程.     

大孔型疏水整体柱的制备及其表征

利用液体致孔剂正庚烷,将双甲基丙烯酸乙二醇酯(EDMA)和甲基丙烯酸缩水甘油酯(GMA)聚合成含有环氧基团的大孔型整体柱,用正丁胺修饰制备成疏水性整体柱。压汞法分析表明,疏水整体柱的孔隙率为60.2%,500nm以上的大孔占疏水整体柱孔隙率的65.7%。在2890cm/h的高流速下,疏水整体柱的背压只有10.9MPa。以牛血清白蛋白(BSA)为研究对象,动态吸附容量为14.4mg/g介质,在一定范围内,分离效果不受操作流速的影响。此外,在1445cm/h的流速下,3min内即可对细胞色素C、RNase A、溶菌酶和鸡卵清蛋白进行基线分离。结果表明,大孔型疏水整体柱可用于蛋白质的快速分离和分析。     

聚苯乙烯-g-聚氧乙烯接枝共聚物表面阻抗蛋白质吸附的研究

利用放射性碘标记技术研究了血浆蛋白质-缓冲溶液体系在聚苯乙烯-g-聚氧乙烯接枝共聚物表面的等温吸附和吸附动力学。材料表面蛋白质等温吸附量或平衡吸附量随表面PEO含量增加而下降;吸附量最低值小于0.25μg,cm^-2;同时探讨了材料表面”梳状“结构对材料表面PEO侧链阻抗蛋白质性能的影响。     

酚醛型吸附树脂对水体系中吡啶和N,N-二甲基苯胺的吸附性能研究

研究了酚醛型吸附树脂在水体系中对吡啶和N，N-二甲基苯胺的静态和动态吸附行为．结果表明，在水中树脂对吡啶和N，N-二甲基苯胺的吸附主要以疏水吸附机理进行；吸附吡啶和N．N-二甲基苯胺的初始阶段，即达到38．3～48．9％平衡吸附时，吸附速率数据和半经验速率方程很吻合：酚醛型吸附树脂等温吸附吡啶和N，N-二甲基苯胺的平衡吸附数据符合Langmuir方程，相关系数在0．99以上，酚醛型吸附树脂吸附吡啶和N，N-二甲基苯胺属单分子层吸附：用80％的乙醇溶液作洗脱剂来洗脱吸附吡啶已达饱和的JDW-2树脂，效果是很理想的．在3．6个床体积内洗脱率达91．52％，4．8个床体积内洗脱率达到94．85％。表明酚醛型吸附树脂具有优良的洗脱性能．     

多胺型螯合树脂对重金属离子的吸附研究

研究了含有三乙烯四胺功能基的酚醛树脂（FQ—34）对Cu2 、Ni2 ,Zn2 、Co2 的静态吸附动力学、等温吸附等过程。结果表明，液膜扩散是吸附的主控步骤，可用G．E．Boyd膜扩散方程来描述；等温吸附属单分子层吸附，可用Langmuir等温方程描述。同时，还对吸附机理作了初步探讨。     

Multiple-injection technique for isolating a target protein from multicomponent mixtures

An integrated chromatographic process comprising ion exchange (IEC) and hydrophobic interaction chromatography (HIC) for isolating a target protein form multicomponent mixtures has been analyzed. The model mixture contained immunoglobulin G that was the key product of the separation process, cytochrome C and ovalbumin. The adsorption characteristics and the mass transport kinetics of the model proteins have been determined along with their dependencies on the operating variables such as pH, temperature and the salt concentration for IEC as well as HIC media. Limitations of the process efficiency resulting from kinetic effects, solubility constraints and the necessity of the mobile phase exchange between chromatographic steps have been discussed. To improve the performance of the integrated process the multiple-injection technique has been suggested. This technique consisted in loading feed mixtures dissolved in a good solvent onto the column by several small-volume injections under conditions of strong protein adsorption. It allowed diminishing interactions between the sample-solvent and protein and elimination of undesired effects such as band splitting and band broadening. For the process design and optimization a dynamic model has been used accounting for thermodynamics and kinetics of the process. The optimization results indicated superiority of the multiple-injection technique over standard isocratic injections in terms of the process yield and productivity.     

Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography: Effect of ionic strength and protein characteristics

This work investigates the effects of ionic strength and protein characteristics on adsorption and transport of lysozyme, BSA, and IgG in agarose-based cation exchangers with short ligand chemistry and with charged dextran grafts. In all cases, the adsorption equilibrium capacity decreased with increasing salt. However, the adsorption kinetics was strongly influenced by the adsorbent structure and protein characteristics. For the smaller and positively charged lysozyme, the effective pore diffusivity was only weakly dependent on salt for the dextran-free media, but declined sharply with salt for the dextran-grafted materials. For this protein, the dextran grafts enhanced the adsorption kinetics at low salt, but the enhancement vanished at higher salt concentrations. For BSA, which was near its isoelectric point for the experimental conditions studied, the effective diffusivity was low for all materials and almost independent of salt. Finally, for the larger and positively charged IgG, the effective diffusivity varied with salt, reaching an apparent maximum at intermediate concentrations for both dextran-free and dextran-grafted media with the kinetics substantially enhanced by the dextran grafts for these conditions. Microscopic observations of the particles during protein adsorption at low ionic strengths showed transient patterns characterized by sharp adsorption fronts for all materials. A theory taking into account surface or adsorbed phase diffusion with electrostatic coupling of diffusion fluxes is introduced to explain the mechanism for the enhanced adsorption kinetics observed for the positively charged proteins.     

Hydrophobic interaction chromatography of proteins. III. Unfolding of proteins upon adsorption

Hydrophobic interaction chromatography (HIC) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. In contrast to reversed-phase chromatography, this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. Hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. When proteins are injected on HIC columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. The higher the salt concentration, the lower is the amount of eluted protein. The rest can be desorbed with a buffer of low salt concentration or water. It has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. This has been also corroborated by physicochemical measurements. The retention data of 5 different model proteins and 10 different stationary phases were evaluated. Partial unfolding of proteins upon adsorption on surfaces of HIC media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. Furthermore, we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain.     

Immobilized Stationary Phases for Hydrophobic Interaction Chromatography of Proteins

Immobilized stationary phases for hydrophobic interaction chromatography (HIC) of proteins are prepared by coating macroporous silica Daisogel of different porosity with hydrophobized cellulose derivatives. The polymer adsorbed on the silica surface afterwards was cross-linked with bifunctional compounds. A uniform polymer nanocoating was indicated using the nitrogen gas adsorption method BET and scanning electron microscopy. The absence of non-specific protein sorption of the synthesized adsorbents shows that the developed polymeric coating isolates silica surface from contact with the sorbate. The retention data of bovine serum albumin (BSA) in the HIC mode on different synthesized adsorbents were evaluated. It was shown that sorption capacity of such phases may vary over a wide range and depends mainly on the substitution degree of the immobilized polymer. The dynamic sorption capacity of BSA was up to 63 mg mL⁻¹. The results proved that the new stationary phases have significant promise for the separation and purification of proteins in the HIC mode.

     

一种疏水色谱填料的特性及应用的研究

 以交联壳聚糖为基质 ,正戊醛为配基 ,利用改进的方法制备了疏水作用色谱 (HIC)填料 ,并对该色谱填料的吸附行为和应用作了研究。结果表明 ,此类填料对蛋白质的吸附行为符合疏水相互作用理论 ,对α 淀粉酶的纯化活性回收率大于 80 %。     

Preparation of Functional Fluorescent Microspheres Used for HTS and Their Interaction with Biomolecules

There is considerable interest in protein adsorption onto microspheres because of its importance in a wide range of biomedical applications, such as artificial tissues and organs, drug delivery systems, biosensors, solid-phase immunoassays, immunomagnetic cell separation and immobilized enzymes or catalyst. It has been well known that the interaction between proteins and microspheres plays important roles in this process. Major interaction involved in the adsorption can be classified as electrostatic, hydrophobic and hydrogen-bonding. Indeed, adsorption of proteins onto microspheres is a complex process and often can involve many dynamic steps, from the initial attachment of the protein on the surface of microspheres to the equilibrium. Also the conformation of proteins probably occurs to a certain degree of deformation or structural change due to the large area of contact. Recently, much interest has been shown in sulfonated microspheres, since sulfonate-group itself is one of components in bio-bodies, as well as is sensitive to the change of pH or ionic strength. Indeed, so far, scanty investigations have been performed in the full range. Also few researches have involved the data on adsorption rate and the maximum amount of protein adsorbed, or the reversibility of the process and conformational change of protein adsorbed as well.In present study, BSA (bovine serum albumin) was chosen as the model protein and sulfonated PMMA [poly(methyl methacrylate)] microspheres as the matrix to investigate the adsorption process.The purpose is to show some information especially the intrinsic information involved by the adsorption process Adsorption of BSA onto sulfonated microspheres (MS) has been investigated as a function of time, protein concentration and pH. The adsorption appears to be a reversible process and the presence of sulfonate groups can play important roles in the adsorption process, so as to increase the amount of protein adsorbed and influences the interaction of BSA molecules. Fig. 1 also shows that the reciprocation between unadsorbed and adsorbed BSA or rearrangement of adsorbed BSA molecules does not produce visible change in the properties of the adsorbed protein. Close to the isoelectric point of BSA (pI 4.7), the amount of protein adsorbed exhibits a maximum. A higher or lower pH results in the significant decrease of the adsorption amount. This is related to the dependence of BSA conformations at different pH conditions.     

Generalizing a two-conformation model for describing salt and temperature effects on protein retention and stability in hydrophobic interaction chromatography

A two-conformation adsorption model that includes the effects of salt concentration and temperature on both stability and adsorption has been developed to describe the effects of secondary protein unfolding on hydrophobic interaction chromatography (HIC). The model has been applied to a biotech protein and to beta-lactoglobulin on Phenyl Sepharose 6FF low sub HIC media. Thermodynamic property models for adsorption and protein stability with parameters obtained from experimental chromatographic data successfully describe observed chromatographic behavior over ranges of temperature and salt concentration, provide predictions of distribution among different conformers, and give a basis for calculating trends in retention strength and stability with changing conditions, that might prove useful in HIC process development.     

Expanded Bed Chromatography as a Tool for Nanoparticulate Separation: Kinetic Study and Adsorption of Protein Nanoparticles

Expanded bed adsorption was investigated together with its suitability for the practical recovery of nanoparticulate mimics of products such as plasmid DNA and viruses as putative gene therapy vectors. The study assessed the binding of protein nanoparticles fabricated from bovine serum albumin (BSA) with average size of 80 nm as a model system and viral size/charge mimic to the streamline DEAE adsorbent in the expanded bed column chromatography. The adsorption kinetics and adsorption mechanism for the BSA nanoparticles on the adsorbent were studied. In batch adsorption studies, the factors nanoparticle concentration, contact time and adsorbent amount, affecting adsorption isotherms were investigated. Subsequently the data were regressed against the Lagergren equation, which represents a first-order kinetics equation and also against a pseudo-second-order kinetics equation. The results demonstrated that the adsorption process followed a Langmuir isotherm equation. The kinetics of the adsorption process followed a pseudo-second-order kinetics model with a rate constant value of 0.025 g mg^?1 min^?1. The dynamic binding capacity of the BSA nanoparticles on an expanded bed was calculated. The recovery of the nanoparticles was more than 85%.     

Breakthrough Model of Recombinant Human-Like Collagen in Immobilized Metal Affinity Chromatography

The adsorption of recombinant human-like collagen by metal chelate media was investigated in a batch reactor and in a fixed-bed column. The adsorption equilibrium and kinetics had been studied by batch adsorption experiments. Equilibrium parameters and protein diffusivities were estimated by matching the models with the experimental data. Using the parameters of equilibrium and kinetics, various models, such as axial diffusion model, linear driving force model, and constant pattern model, were used to simulate the breakthrough curves on the columns. As a result, the most suitable isotherm was the Langmuir–Freundlich model, and the ionic strength had no effect on the adsorption capacity of chelate media. In addition, the pore diffusion model fitted very well to the kinetic data. The pore diffusivities decreased with increasing the initial protein concentration, however had little change with the ionic strength. The results also indicated that the models predict breakthrough curves reasonably well to the experimental data, especially at low initial protein concentration (0.3 mg ml⁻¹) and low flow rate (34 cm h⁻¹). By the results, we optimized the experimental conditions of a chromatographic process using immobilized metal affinity chromatography to purify recombinant human-like collagen.     

Fast adsorption and separation of bovine serum albumin and lysozyme using micrometer-sized macromesoporous silica spheres

Micrometer-sized silica spheres with bimodal macromesoporous structures are synthesized continuously by a modified gelation technology, and a novel coaxial microfluidic device is applied to control the size of the silica spheres. These spheres are used as a new protein adsorbent to realize fast adsorption and separation of protein mixture. BSA and lysozyme (LYS) are as model proteins with different sizes and pIs. High protein adsorption capacity and rapid adsorption rate have been achieved. Fast separation of a binary mixture of BSA and LYS through a short packed column (50 mm x 4.6 mm id) has also been successfully realized. LYS was selectively adsorbed while BSA flowed through the column in 12 s at a flow rate of up to 5.0 mL/min. As a result, an ultrafast adsorption and separation of BSA and LYS was obtained, showing great potential for applications in fast, large-scale protein separation processes of these monodispersed silica spheres.