Precolumn concentration of protein samples by displacement electrophoresis (isotachophoresis) followed by high-performance molecular sieve chromatography on a packed agarose column

The use of displacement electrophoresis for the concentration of dilute protein solutions and the construction of a column suitable for this purpose are described. The concentrated protein zone can be pumped directly from the electrophoresis column into a gel-filtration column, which greatly reduces losses of protein. Recoveries of 95% or better were obtained even for small amounts of protein. The electrophoretically concentrated samples gave virtually the same elution profiles as did samples injected in a small volume without the use of electrophoretic preconcentration.     

Predicting the elution behavior of proteins in affinity chromatography on non-porous particles.

Affinity chromatography on non-porous particles of microsize is particularly useful for the rapid analysis and micropreparative separation of proteins. The elution behavior of proteins in an affinity column packed with non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate was investigated both theoretically and experimentally, using the lysozyme-Cibacron Blue 3G-A affinity system. Equations used to predict the elution profiles, resulting from the elution by increasing the ionic strength (NaCl concentration) in the mobile phase, were obtained. The maximum adsorbate concentration, desorption rate constant and equilibrium constant under elution conditions were determined by matching experimental data with predicted elution profiles. Based on the parameters determined at a flow-rate of 0.5 ml/min and with 1 M NaCl in the elution buffer, the model equations could predict the elution profiles for other experimental runs, where different flow-rates and sodium chloride concentrations were used. Both the experimental and predicted results revealed that the affinity interaction kinetics are not significantly influenced by the flow-rate and, hence, the film mass transfer. To elute bound lysozyme from immobilized dye ligand, a higher value of the ionic strength leads to a faster elution and a sharper elution peak. The influence of elution conditions on the kinetic and thermodynamic parameters and, consequently, on the elution peak profiles was evaluated. The model equations can also predict the behavior of protein elution from an affinity column by changing the pH of the mobile phase, according to a previous study.     

Affinity chromatography without immobilized ligands; A new method for studying macromolecular interactions using high-performance liquid chromatography

A new method for studying macromolecular interactions was devised. The principle is based on affinity chromatography with a mobile zone of affinity ligand instead of a column with immobilized ligand. In this method, the difference in migration velocities between the moving zone of affinity ligand and a sample in a conventional gel-permeation column is utilized. The fast migrating zone (A zone), which is later injected into the column, and the slow migrating zone (B zone), which is injected beforehand, interfere with each other at the passing point in the column if A and B interact such as A + B ? AB. The zone interference deforms each elution profile of A and B, because the complex AB has a migration velocity different from the others. The elution profiles in zone-interference chromatography are calculated by computer simulation in the framework of the plate theory. The binding constant is calculated from the peak shift of elution volume of B in the zone-interference chromatogram. The interaction between single-strand DNA (A zone) and ribonuclease A (B zone) was studied.     

Systematic interpolation method predicts protein chromatographic elution from batch isotherm data without a detailed mechanistic isotherm model

Predicting protein elution for overloaded ion exchange columns requires models capable of describing protein binding over broad ranges of protein and salt concentrations. Although approximate mechanistic models are available, they do not always have the accuracy needed for precise predictions. The aim of this work is to develop a method to predict protein chromatographic behavior from batch isotherm data without relying on a mechanistic model. The method uses a systematic empirical interpolation (EI) scheme coupled with a lumped kinetic model with rate parameters determined from HETP measurements for non‐binding conditions, to numerically predict the column behavior. For two experimental systems considered in this work, predictions based on the EI scheme are in excellent agreement with experimental elution profiles under highly overloaded conditions without using any adjustable parameters. A qualitative study of the sensitivity of predicting protein elution profiles to the precision, granularity, and extent of the batch adsorption data shows that the EI scheme is relatively insensitive to the properties of the dataset used, requiring only that the experimental ranges of protein and salt concentrations overlap those under which the protein actually elutes from the column and possess a ±10% measurement precision.     

Prediction of the profiles of the elution bands of proteins in preparative liquid chromatography

A semiequilibrium model of chromatography has been developed to account for the elution profile of high concentration bands. The use of this model requires the prior determination of the equilibrium isotherms of the studied compounds in the chromatographic phase system. The model is applied here to a study of the isocratic elution bands of chicken albumin and conalbumin on a weak anion exchanger in aqueous buffered solutions. Satisfactory agreement is observed between the experimental elution profiles of large-sample-size bands of chicken albumin and conalbumin.     

Frontal gel chromatography of interacting systems: theoretical and experimental evaluation of the shapes of elution profiles for systems of the type A + B in equilibrium C

A theoretical and experimental study has been made of the advancing elution profile in frontal gel chromatography of interacting systems for which the elution volume of the complex is smaller than that of the larger reactant. First, Gilbert-Jenkins theory is used to delineate the form of the elution profile from the magnitudes of the elution volumes and concentrations of reacting species. This procedure resulted in the detection of a misinterpretation of certain patterns obtained in a gel chromatographic study of the interaction between myoglobin and ovalbumin. Second, a numerical computational procedure, which incorporates both axial dispersion and concentration-dependence of species elution volumes, is used to establish the influence of these two factors on boundary shapes for such systems. Third, frontal gel chromatography on Sephadex G-75 is used to compare experimental behavior with theoretical profiles predicted for the electrostatic interaction between cytochrome c and soybean trypsin inhibitor (pH 6.8, I 0.01). Results of these experiments serve as a guide for future conduct of experiments aimed at characterization of biologically important, reversible complex formation between proteins and/or other macromolecules.     

Ultra scale‐down approach to correct dispersive and retentive effects in small‐scale columns when predicting larger scale elution profiles

Ultra scale‐down approaches represent valuable methods for chromatography development work in the biopharmaceutical sector, but for them to be of value, scale‐down mimics must predict large‐scale process performance accurately. For example, one application of a scale‐down model involves using it to predict large‐scale elution profiles correctly with respect to the size of a product peak and its position in a chromatogram relative to contaminants. Predicting large‐scale profiles from data generated by small laboratory columns is complicated, however, by differences in dispersion and retention volumes between the two scales of operation. Correcting for these effects would improve the accuracy of the scale‐down models when predicting outputs such as eluate volumes at larger scale and thus enable the efficient design and operation of subsequent steps. This paper describes a novel ultra scale‐down approach which uses empirical correlations derived from conductivity changes during operation of laboratory and pilot columns to correct chromatographic profiles for the differences in dispersion and retention. The methodology was tested by using 1 mL column data to predict elution profiles of a chimeric monoclonal antibody obtained from Protein A chromatography columns at 3 mL laboratory‐ and 18.3 L pilot‐scale. The predictions were then verified experimentally. Results showed that the empirical corrections enabled accurate estimations of the characteristics of larger‐scale elution profiles. These data then provide the justification to adjust small‐scale conditions to achieve an eluate volume and product concentration which is consistent with that obtained at large‐scale and which can then be used for subsequent ultra scale‐down operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Numerical and experimental demonstrations of the need for caution in the use of zonal gel chromatography for characterizing ligand interactions with small acceptors

Quantitative expressions are presented for the evaluation of equilibrium constants for interactions of the type A + B in equilibrium C from experiments entailing the application of a small zone of acceptor-ligand mixture to a column of gel preequilibrated with ligand solution [J.P. Hummel and W.J. Dreyer (1962) Biochim. Biophys. Acta 63, 530-532]. Only in the event that identical elution volumes pertain to acceptor and complex does the steady-state binding constant (Kss) obtained by that method equal the thermodynamic equilibrium constant (K). Simulated elution profiles are then generated with parameters relevant to gel chromatography of the ATP-Mg2+ system on Sephadex G-10 in order to demonstrate the practical importance of the need for distinction between Kss and K in situations where acceptor and complex do not comigrate. A study of the interaction between soybean trypsin inhibitor and cytochrome c by gel chromatography on Sephadex G-75 is then used to illustrate the feasibility of combining information from Hummel-Dreyer experiments with the theoretical expressions to characterize systems under the more general conditions that the elution volumes of A and C differ. A finding of considerable theoretical interest in relation to the simulation of mass migration behavior is the demonstration that a truncation error is the source of zonal spreading in the theoretical-plate model of chromatography. This truncation error is shown to be the source of spreading generated whenever solution of an abbreviated (diffusion-free) continuity equation involves substituting first differences for first derivatives in the differential equation describing mass transport.     

Applications of field-flow fractionation in proteomics: presence and future

Field-flow fractionation (FFF) represents a group of elution separation methods where external force fields act perpendicularly on analytes in a carrier liquid flows with nonuniform velocity profiles. It is an elution separation method that enables to separate analytes in relatively short times and collect fractions for further characterization or for investigation of their properties. Other advantages of FFF are small consumption of samples and gentle experimental conditions. These make FFF uniquely qualified for separation and purification of biological samples. The most promising are applications of different variants of flow FFF utilizing a cross flow through membrane channel walls to separate proteins. The separation is based on differences in protein diffusion coefficients, which allows calculating the size of macromolecules. Other FFF techniques (e.g., electrical, isoelectric, and sedimentation FFF) were also used for separation of biomolecules. FFF appears to be not only promising rapid technique for protein separation but it offers some other advantages in sample preparation, especially, focusing (hyperlayer) FFF techniques that enable preparation of homogeneous fractions of cells. Selected applications of FFF to protein analysis are described and future trends in application of FFF to proteomics are discussed.     

Physico-chemical model for DNA alkaline elution: New experimental evidence and differential role of DNA length,chain flexibility and superpacking

For a better understanding of data provided by DNA alkaline elution technique, a new analytical model has been developed which takes into consideration both the physicochemical properties of in situ DNA strand (length and flexibility/superpacking) and the geometric and hydrodynamic configuration of the elution apparatus (flow and filter conditions). Simulation by this model of experimental data previously obtained before and after carcinogens administration, has shown that for constant flow and filter conditions elution profiles are dependent, not only from DNA molecular weight, but also from a parameter critically related to modifications in chain flexibility/superpacking. This has been confirmed by several independent observations, including the time-dependent changes in non-denaturing lysing solution monitored by hydroxylapatite and alkaline elution techniques.     

Selection of monoclonal antibodies for immunoaffinity chromatography: model studies with antibodies against soy bean trypsin inhibitor

To facilitate selection of monoclonal antibodies for immunoaffinity chromatography, an ELISA screening procedure was developed. The assay is based on the avidin-biotin system and provides a profile of the monoclonal antibody which is based on the binding characteristics of the antigen binding site when exposed to different elution reagents. The elution profiles of 5 monoclonal antibodies to soy bean trypsin inhibitor (SBTI) were determined and for 2 of the antibodies the results obtained in the ELISA were verified using column experiments. The affinity constants were determined for the same 5 monoclonal antibodies and no correlation was seen with the ease of elution. The elution profiles presented here are easily obtained and the results indicate that a general screening procedure for suitable combinations of antibodies and elution conditions can be carried out using an elution ELISA assay when modified as described herein.     

The effect of pore-size distribution on the binding of proteins to porous resin beads

The effect of the distribution of pore radii in the resin beads on protein binding was taken into account to analyze the elution profiles of proteins from the polymer-packed column obtained by repetitive injection method. By assuming that the distribution of pore radii in the resin beads is logarithmic Gaussian, the theoretical curves obtained agreed well with the experimental data.     

Kinetic analysis of the hydrodynamic transition accompanying protein folding using size exclusion chromatography. 1. Denaturant dependent baseline changes

Size exclusion profiles of proteins with persistent conformations exhibit broad asymmetric peaks whose shape and elution times are dependent on denaturant concentration. The collective elution profiles were precisely simulated by an apparent binding model that treats the denaturant dependence in terms of an apparent matrix binding. The model requires three experimentally measurable parameters: the elution time for the unbound protein, an apparent association equilibrium constant for binding, and an apparent exchange time for binding. The denaturant dependence for each of these parameters is related to the accessible surface area of the protein. © 1993 John Wiley & Sons, Inc.     

Ligand binding and self-association of proteins

Summary This review deals with the quantitative analysis of protein self-association and ligand binding when there is a significant mutual influence of the two processes. The particular points of interest are the evaluation of the pertinent equilibrium constants and the prediction and interpretation of concentration profiles in transport experiments, including gel elution and sedimentation velocity. The case of dimertetramer equilibrium with four binding sites for ligand is considered in detail. Three representative experimental studies are described which deal with hemoglobin, phosphorylase b, and tubulin.     

Pulse response in an immobilized-enzyme column: Elution profiles in reversible and consecutive reactions

A pulse of substrate solution was applied to an immobilized-enzyme column, in which the substrate was then converted by reversible or consecutive reactions. Immobilized glucose isomerase was used for the reversible reaction, and immobilized invertase and glucose oxidase for the consecutive reactions. The elution profiles of substrate and product were determined experimentally. These profiles were in good agreement with the ones predicted theoretically. The effect of some parameters on the elution profiles for reversible and consecutive reactions is discussed.     

Distinctive properties of fucosyl glycopeptides on human teratoma cells

Fucose-labeled glycopeptides from four human teratoma cell lines of independent origin show similar elution profiles on Sephadex G-50 column chromatography. The fucosyl glycopeptides elute in two major regions: one near the void volume, the other in fractions corresponding to a molecular weight of 2500-3000. These elution profiles are very different from those obtained with the other human cell lines examined which included 3 lymphomas, 2 colon carcinomas, and HeLa. The elution profiles of the human teratomas, however, show remarkable similarities to those obtained with murine embryonal carcinoma cell culture and early mouse embryos. These results suggest that the excluded G-50 fraction may well contain glycopeptides playing a role in mammalian embryogenesis.     

Cooperative elution of oligoadenylic acid in polyU immobilized chromatography.

Characterization of polyU immobilized chromatography was performed in order to use this technique as an analytical device. A method of analysis of the elution profile related to thermodynamic parameters was also developed. Sites of attachment of polyU to agarose gel activated by cyanogen bromide were studied using uridine diphosphate and adenine. Independent equilibrium dialysis of ApA for different states of polyU, in solution and immobilized in gel, were carried out. These results show that the immobilized polyU is attached to agarose only at the 5′-terminal phosphate groups and behaves as it does in solution. Column chromatography of ApA with the immobilized polyU was performed at several temperatures and concentrations. To analyze the elution profile, the theory of cooperative binding of oligonucleotides to polynucleotides was extended to the frame-work of plate theory. A computer simulation for the elution profiles was performed using thermodynamic parameters obtained by equilibrium dialysis. This simulation duplicated the experimental results. This fact shows that the peculiar leading form of elution profile is due to the cooperative binding. The thermodynamic parameters of the polyU–ApA system were obtained from the “peak trajectory.”     

凝胶过滤法纯化裂褶多糖检测方法的改进

凝胶过滤洗脱液中多糖含量一般采用硫酸-苯酚等化学显色法测定,然后根据洗脱曲线得到纯化的组分,但化学方法费时且消耗试剂.本研究采用350 nm非特征性吸收波长对2种不同纯度裂褶多糖样品PSG1和PSG2的洗脱液直接测定吸光度,与硫酸-苯酚法490 nm测定得到的洗脱曲线基本一致,即分别可得到4个和2个组分,同时采用HPLC对分离效果进行了检测.研究表明,采用350 nm波长直接检测可实现不同纯度裂褶多糖的分离.利用本法检测其他多糖的效果有待进一步研究.