Optimization of step gradient separations: Consideration of nonlinear adsorption

Nonlinear adsorption plays an important role in determining the chromatographic behavior of proteins in preparative ion-exchange chromatography. In this article, the steric mass action (SMA) isotherm is used in conjunction with a mass transport model to describe nonlinear cation-exchange chromatography. Excellent agreement is observed between simulated and experimental step gradient separations of the proteins alpha-chymotryp-sinogen A, cytochrome C, and lysozyme. A systematic method of selecting the optimum step gradient program for a given separation problem is presented and employed to study optimization of step gradient chromatography under conditions of high mass loading. This article includes consideration of the effects of the adsorption properties of the feed stream, the feed stream concentration, protein solubility, and otherconstraints on the optimum separation conditions.(c) John Wiley & Sons, Inc.     

Operating parameters for glutamic acid crystallization in displacement ion exchange chromatography

Glutamic acid can be crystallized inside cation exchange column when displacer NaOH concentration is high enough to concentrate displaced glutamic acid beyond its solubility limit. Resulting crystal layer of glutamic acid was moved with liquid phase through the column, and thus could be eluted from the column and recovered in fraction collector. For the purpose of enhancing crystal recovery, effects of operating parameters on the crystal formation were investigated. The increase in the degree of crosslinking of resin favored crystal recovery because of its low degree of swelling. Higher concentration of displacer NaOH was advantageous. If NaOH concentration is too high, however, crystal recovery was lowered due to the solubility-enhancing effects of high pH and ionic strength. The decrease of mobile phase flow rate enhanced crystal recovery because enough time to attain local equilibrium could be provided, but film diffusion would control the overall crystal formation with extremely low flow rate. Lower temperature reduced solubility of glutamic acid and thus favored crystal formation unless the rate of ion exchange was severely reduced. The ion exchange operated by displacement mode coupled with crystallization was advantageous in reducing the burden of further purification steps and in preventing purity-loss resulted from overlapping between adjacent bands.     

Comparison of maximum production rates and optimum operating/design parameters in overloaded elution and displacement chromatography

The results of a study of the optimization of the experimental conditions for maximum production rate in overloaded elution and displacement chromatography are discussed. This study is based on the use of the equilibrium-dispersive model of chromatography and the competitive Langmuir isotherms to calculate individual band profiles in the elution and displacement modes, and of a simplex algorithm to optimize the production rate. The operating parameters (sample size, mobile phase velocity, and the displacer concentration in the displacement model) and the column design parameters (column length and average particle diameter) are optimized simultaneously. Binary mixtures having relative concentrations 3:1 and 1:3, and separation factors of 1.2 to 1.8 are investigated. One of our major results is that, in both modes of chromatography, the maximum production rate is achieved at very low values of the retention factors, k', much lower than those used in current practice. In all cases, unless k' exceeds greatly that optimum value, the production rate is higher in overloaded elution than in displacement chromatography. This is particularly true for the extraction of a minor component, which is eluted second. (c) 1993 John Wiley & Sons, Inc.     

Changes in retention behavior of fluorescently labeled proteins during ion-exchange chromatography caused by different protein surface labeling positions

Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.     

Optimized operating parameters for the displacement separation of biomolecules in immobilized metal ion affinity chromatography

The ideal immobilized metal ion affinity chromatography (IMAC) model was employed to investigate the effect of operating parameters change on the displacement separation of biomolecules. By combining a lower initial mobile phase modifier (MPM) concentration and a higher final MPM concentration, the displacement chromatographic separation produced both higher concentration of feeds and better throughput in IMAC displacement separating systems.     

Application of chromatographic theory for process characterization towards validation of an ion-exchange operation

The behavior of ion-exchange chromatography is well understood with respect to changes in ionic strength, pH, resin ligand density, bed height, elution flow rate, and gradient slope. Their relative importance for any specific chromatographic situation varies. When a chromatographic operation utilized to purify a human therapeutic protein is prepared for validation before commercial production, numerous tests have to be performed to establish the relative importance of each operating parameter to define its future role and importance in the framework of in-process controls. This prioritization process is usually performed using a purely empirical approach. In this work, we demonstrate the application of a rational approach based on chromatographic theory to prioritize operating parameters. Both methodologies, empirical and rational, were performed to evaluate a specific ion-exchange chromatography operation for the preparative separation of closely related protein species. We show that the application of the rational approach has the potential to accelerate the evaluation and significantly reduce the amount of analytical testing needed.     

Influence of different metal flask systems on tooth displacement in complete upper dentures

doi: 10.1111/j.1741‐2358.2010.00392.x Influence of different metal flask systems on tooth displacement in complete upper dentures Objective: To verify the occurrence of tooth displacement in dentures processed by moist hot‐polymerisation using traditional and experimental metal flask systems. Materials and methods: Waxed complete dentures were randomly assigned to three groups (n = 10), including traditional (TF) and experimental flasks (DF and HHF). Metal pins were placed at the incisal edge of maxillary central incisors (I), buccal cusp of first premolars (P) and mesiobuccal cusp of second molars (M). Transversal (I–I, P–P and M–M) and anteroposterior (LI–LM and RI–RM) distances were measured before and after denture processing using an optical microscope. The dentures were processed by hot water curing cycle (9 h/74°C). Collected data were analysed by anova , Paired Student’s t‐test and Holm–Sidak method (p < 0.05). Results: All measured distances demonstrated contraction after polymerisation, except for LI–LM in TF and RI–RM in DF and HHF. Statistically significant differences were found between the distances P–P for DF and HHF, and M–M for all groups. Comparison among flask systems revealed statistically significant difference in the P–P distance for the TF and HHF. Conclusion: Different flask systems did not cause variation in tooth displacement during denture processing, with the exception of P–P distances for the TF and HHF. There is no evidence that different metal flask systems would promote tooth displacement of clinical significance.     

High-throughput screening of chromatographic separations: IV. Ion-exchange

Ion-exchange (IEX) chromatography steps are widely applied in protein purification processes because of their high capacity, selectivity, robust operation, and well-understood principles. Optimization of IEX steps typically involves resin screening and selection of the pH and counterion concentrations of the load, wash, and elution steps. Time and material constraints associated with operating laboratory columns often preclude evaluating more than 20-50 conditions during early stages of process development. To overcome this limitation, a high-throughput screening (HTS) system employing a robotic liquid handling system and 96-well filterplates was used to evaluate various operating conditions for IEX steps for monoclonal antibody (mAb) purification. A screening study for an adsorptive cation-exchange step evaluated eight different resins. Sodium chloride concentrations defining the operating boundaries of product binding and elution were established at four different pH levels for each resin. Adsorption isotherms were measured for 24 different pH and salt combinations for a single resin. An anion-exchange flowthrough step was then examined, generating data on mAb adsorption for 48 different combinations of pH and counterion concentration for three different resins. The mAb partition coefficients were calculated and used to estimate the characteristic charge of the resin-protein interaction. Host cell protein and residual Protein A impurity levels were also measured, providing information on selectivity within this operating window. The HTS system shows promise for accelerating process development of IEX steps, enabling rapid acquisition of large datasets addressing the performance of the chromatography step under many different operating conditions.     

Protected amino acids as novel low-molecular-weight displacers in cation-exchange displacement chromatography

Although the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state of the art of displacement chromatography has been the discovery that low-molecular-weight dendritic polymers can be successfully employed as displacers for protein purification in ion-exchange systems. In this article, protected amino acid esters (based on arginine and lysine) are shown to be useful displacers for protein purification in cation-exchange systems. A dynamic affinity plot is employed to evaluate the affinity of these low-molecular-weight compounds under dis-placement conditions. In contrast to large polyelectroyte displacers, the efficacy of these low-molecular-weight displacers was shown to be dependent on both the initial carrier salt concentration and the displacer concentration. In addition to the funcamental interest generated by low-molecular-weight displacers, it is likely that these displacers will have significant operatioal advantages as compared with large polyelectrolyte displacers. (c) 1995 John Wiley & Sons, Inc.     

肝素纯化工艺中离子交换树脂的选择与应用研究

研究比较了5种树脂对肝素的吸附能力,从中选出S5428阴离子交换树脂来纯化肝素。通过对各因素的研究,确定了树脂对肝素的静态、动态吸附以及解吸的最佳条件。结果表明:静态吸附的温度45℃,pH 8.0的条件下吸附2 h,肝素的吸附率为90.5%;层析柱的动态吸附温度45℃,肝素溶液进样浓度1.0 mg/mL,进样速度1.5 mL/min,树脂柱能处理1.5 BV肝素液而不发生泄露,吸附量为3.05 mg/mL,达到饱和吸附时可处理4BV的料液,吸附量为9.18 mg/mL;采用2.0 mol/L NaCl洗脱,洗脱流速1.5 mL/min,肝素解吸率可达95.8%,肝素效价可达150 U/mg,效价回收率98%。     

两种方法分离纤维蛋白原效果的比较

为了比较冷乙醇沉淀法和离子交换层析法在分离止血胶成分中的纤维蛋白原的效果,本实验将灭活病毒的步骤加在溶解Nitschmann组分I中进行,结果发现0．3％磷酸三丁酯／1％吐温-80不仅具有灭活病毒的作用,而且还能促进纤维蛋白原的溶解;分别用冷乙醇沉淀法和离子交换层析法分离纤维蛋白原,结果两种方法分离的纤维蛋白原均符合止血胶要求。冷乙醇沉淀法分离虽然凝血因子Ⅷ有一定损失,但纤维蛋白原纯度和回收率均高于离子交换层析法的分离效果,这提示冷乙醇沉淀法较离子交换层析法更适于分离止血胶用纤维蛋白原。     

Evaluation of Escherichia coli proteins that burden nonaffinity-based chromatography as a potential strategy for improved purification performance

Escherichia coli is a favored host for rapid, scalable expression of recombinant proteins for academic, commercial, or therapeutic use. To maximize its economic advantages, however, it must be coupled with robust downstream processes. Affinity chromatography methods are unrivaled in their selectivity, easily resolving target proteins from crude lysates, but they come with a significant cost. Reported in this study are preliminary efforts to integrate downstream separation with upstream host design by evaluating co-eluting host proteins that most severely burden two different nonaffinity-based column processes. Phosphoenolpyruvate carboxykinase and peptidase D were significant contaminants during serial purification of green fluorescent protein (GFP) by hydrophobic interaction and anion exchange chromatography. Ribosomal protein L25 dominated non-target binding of polyarginine-tagged GFP on cation exchange resin. Implications for genetic knockout or site-directed mutagenesis resulting in diminished column retention are discussed for these and other identified contaminants.     

Mechanistic studies of displacer–protein binding in chemically selective displacement systems using NMR and MD simulations

A parallel batch screening technique was employed to identify chemically selective displacers which exhibited exclusive separation behavior for the protein pair α‐chymotrypsin/ribonuclease A on a strong cation exchange resin. Two selective displacers, 1‐(4‐chlorobenzyl)piperidin‐3‐aminesulfate and N′1′‐(4‐methyl‐quinolin‐2‐yl)‐ethane‐1,2‐diamine dinitrate, and one non‐selective displacer, spermidine, were selected as model systems to investigate the mechanism of chemically selective displacement chromatography. Saturation transfer difference (STD) NMR was used to directly evaluate displacer–protein binding. The results indicated that while binding occurred between the two chemically selective displacers and the more hydrophobic protein, α‐chymotrypsin, no binding was observed with ribonuclease A. Further, the non‐selective displacer, spermidine, was not observed to bind to either protein. Importantly, the binding event was observed to occur primarily on the aromatic portion of the selective displacers. Extensive molecular dynamic simulations of protein–displacer–water solution were also carried out. The MD results corroborated the NMR findings demonstrating that the binding of selective displacers occurred primarily on hydrophobic surface patches of α‐chymotrypsin, while no significant long term binding to ribonuclease A was observed. The non‐selective displacer did not show significant binding to either of the proteins. MD simulations also indicated that the charged amine group of the selective displacers in the bound state was primarily oriented towards the solvent, potentially facilitating their interaction with a resin surface. These results directly confirm that selective binding between a protein and displacer is the mechanism by which chemically selective displacement occurs. This opens up many possibilities for future molecular design of selective displacers for a range of applications. Biotechnol. Bioeng. 2009;102: 1428–1437. © 2008 Wiley Periodicals, Inc.     

Combination of size-exclusion chromatography and ion exchange adsorption for improving the proteomic analysis of plasma-derived extracellular vesicles

Extracellular vesicles (EVs) are lipid membrane vesicles released by live cells that carry a variety of biomolecules, including nucleic acids, lipids, and proteins. Recently, proteins in plasma-derived EVs have emerged as novel biomarkers with essential functions in the diagnosis and prognosis of human diseases. However, the current methods of isolating EVs from plasma often lead to coisolated impurities in biological fluids. Therefore, before performing any research protocol, the process of extracting EVs from plasma for proteomic analysis must be optimized. In this study, two EV isolation strategies, size exclusion chromatography (SEC) and SEC combined with ion exchange adsorption (SEC + IEA), were compared in terms of the purity and quantity of protein in EVs. Our results demonstrated that, compared to single-step SEC, SEC combined with IEA could produce plasma-derived EVs with a higher purity by decreasing the abundance of lipoprotein. Additionally, with MS analysis, we demonstrated that the combination approach maintained the stability and improved the purity of EVs in many plasma samples. Furthermore, by combining SEC with IEA, more cancer-associated proteins were detected in the plasma of various cancer samples.     

Scaled-up separation of cellobiohydrolase1 from a cellulase mixture by ion-exchange chromatography

Enzymatic hydrolysis of cellulose often involves cellulases produced by Trichoderma reesei, of which cellobiohydrolase1 (CBH1) is the most abundant (about 60% of total cellulases) and plays an important role in the hydrolysis of crystalline cellulose. A method for separating sufficient quantities from the bulk cellulase cocktail is highly desirable for many studies, such as those that aim to characterize binding and hydrolysis kinetics of CBH1. In this work, CBH1 was separated from other Spezyme CP cellulases by ion-exchange chromatography using an efficient modification of a smaller scale process. The ion-exchange column was connected to a vacuum manifold system to provide a steady flow through parallel columns and thus achieve scale-up for enzyme separation. With five 5-mL columns running in parallel, about 55 mg of CBH1 was separated from 145 mg of Spezyme CP in a single separation. Step elution was used to replace the continuous gradient used at smaller scale. The purified CBH1 was collected in the fraction eluted with a buffer containing 0.33 M salt and showed comparable purity and activity as the enzyme purified by a fast protein liquid chromatography system. The stability of separated CBH1 was studied for up to 2 days and good thermal stability was observed. Separated CBH1 also showed both high adsorption to bacterial microcrystalline cellulose with ~4 μmol/g maximum adsorption and a K(a) of 5.55 ± 2.34 μM(-1) , and good hydrolytic activity based on atomic force microscopy observations that show a reduction in fiber height.     

蛋白质层析用离子交换和疏水作用层析介质的发展概况

层析是蛋白质纯化的关键技术之一 ,作为层析技术的核心———层析介质一直以来是层析技术研究的一个热点。近年来 ,越来越多的新型层析介质被开发出来 ,如粒度均匀的交联多糖、人工合成的大孔聚合物、触角型吸附剂、软胶包裹在硬胶表面等介质。主要介绍应用较为广泛的IEC和HIC介质的组成、特性及其在蛋白质纯化中的应用 ,还研究了与HIC技术相关的两种新技术 :亲硫层析和疏水电荷诱导层析 (HCIC) ,重点介绍了HCIC的介质及其应用 ,同时也讨论了在蛋白质纯化中应用的三相纯化策略 (富集、中间纯化和精制 )。结合我国的实际情况 ,就当前蛋白质纯化的离子交换和疏水层析介质面临的挑战和未来的发展进行讨论并提出了建议     

Competitive adsorption of labeled and native protein in confocal laser scanning microscopy

Confocal laser scanning microscopy has been previously applied to the study of protein uptake in porous chromatography resins. This method requires labeling the protein with a fluorescent probe. The labeled protein is then diluted with a large quantity of native protein so that the fluorescence intensity is a linear function of the labeled protein concentration. Ideally, the attachment of a fluorescent probe should not affect the affinity of the protein for the stationary phase; however, recent experimental work has shown that this assumption is difficult to satisfy. In the present study, we present a mathematical model of protein diffusion and adsorption in a single adsorbent particle. The differences in adsorption behavior of labeled and native protein are accounted for by treating the system as a two-component system (labeled and native protein) described by the steric mass action isotherm (SMA). SMA parameters are regressed from experimental linear gradient elution data for lysozyme and lysozyme-dye conjugates (for the fluorescent dyes Cy3, Cy5, Bodipy FL, and Atto635). When the regressed parameters are employed in the model, an overshoot in the labeled lysozyme concentration is predicted for Cy5- and Bodipy-labeled lysozyme, but not for Atto635-labeled lysozyme. The model predictions agree qualitatively well with recent work showing the dependence of the concentration overshoot on the identity of the attached dye and provide further evidence that the overshoot is likely caused by the change of binding characteristics due to the fluorescent label.     

火菇素提取新工艺及其生物活性测定

火菇素是一种具有抗癌活性的简单蛋白 ,从金针菇中分离纯化火菇素的改进工艺为 :金针菇子实体经乙酸液浸提、硫酸铵和乙醇分级沉淀后 ,通过DEAE SephadexA 5 0除去色素和部分杂蛋白 ,最后经CM SephadexC 5 0离子交换柱层析处理 ,得到了电泳均一的纯化蛋白 ,用微量透析法得到结晶 .理化性质研究结果表明火菇素为分子量 2 370 0 ,等电点pI 8 9,不含糖的简单蛋白 ,最大吸收波长λmax在 2 76～ 2 78nm ,最小吸收波长λmin在 2 5 0nm ,氨基酸组成不含蛋氨酸 .纯化产物对实验动物移植肿瘤的抑瘤作用实验的研究指出 ,火菇素对小鼠肉瘤S180 和艾氏腹水瘤有明显的抑制作用 ,且能延长小鼠的生存期 .毒性实验研究表明 ,未发现有明显的毒副作用     

木瓜凝乳蛋白酶的分离纯化及部分酶学性质研究

目的：从木瓜蛋白酶粗酶中分离木瓜凝乳蛋白酶,并对其部分酶学性质作初步研究。方法：采用离子交换层析分离木瓜凝乳蛋白酶,通过温度、Na^ 、Ca^2 及其它金属离子等来研究其对酶性质的影响。结果：经离子交换得到了在PAGE中呈单一区的木瓜凝乳蛋白酶,该酶在37℃保温24h后其活力只有原来的7．5％,低浓度的Na^ 、Ca^2 、K^ 、Cu^2 对其有激活作用。结论：木瓜凝乳蛋白酶的分离纯化及酶学性质都值得进一步研究。