Conditions for purification of proteins by free-flow zone electrophoresis

The separation of proteins by free-flow zone electrophoresis is generally impaired by a number of secondary effects which cause spreading of the protein streams with resultant loss in resolution. A strategy is outlined, based on experimental observations and numerical modeling, which allows the operating conditions and separation chamber dimensions to be chosen so as to obtain complete separation of two proteins of known mobility. This approach takes into account some dispersive phenomena such as molecular diffusion, electroosmosis and residence time gradients. In some cases, the right operating conditions cannot be achieved on earth and electrophoresis separations in microgravity may then be justified.     

Use of full-column imaging capillary isoelectric focusing for the rapid determination of the operating conditions in the preparative-scale continuous free-flow isoelectric focusing separation of enantiomers

A rapid, simple method is proposed here for the identification of the experimental conditions that lead to satisfactory preparative-scale isoelectric focusing enantiomer separations in continuous free-flow electrophoretic units. The method first calls for the use of a commercially available, full-column imaging capillary electrophoretic system to find the background electrolyte composition that generates the largest pI difference between the bands of the enantiomers. The method then calls for the finding of the minimum residence time that permits full development of the pH gradient across the separation chamber of the continuous free-flow electrophoretic unit by measuring the pH in the sample-free carrier electrolyte fractions collected during these runs. Finally, the quality of the predicted preparative-scale separation is verified by analyzing the enantiomer-containing collected fractions by capillary electrophoresis using a 14-sulfated, single-isomer cyclodextrin as resolving agent. The pI difference values and production rate values observed in this work agree well with the literature values that were obtained by much more time-consuming methods.     

Simplified gradient generator for micro- and nano-liquid chromatography

We have developed a simple device to generate gradient elution profiles using split tubing array (STAR) units for micro- and nano-HPLC. This gradient device consists of a delivery pump, a splitter, tubes in parallel, and a relatively large-volume mixing chamber. In the mixing chamber, an initially filled water-rich solvent is adjusted to an organic-rich solvent by delivery of appropriate components through the split paths, in which the flow rate and residence time are controlled by the sizes of the tubes employed. A program was developed to describe the output gradient profiles, and the predicted gradient profiles were highly consistent with the observed ones. A linear gradient generated by this STAR system was successfully applied to micro-HPLC systems for separation of digested peptides derived from serum albumin with sufficient reproducibility. Further miniaturization of STAR systems for nano-HPLC coupled with tandem mass spectrometry was accomplished to separate digested peptides from serum albumin with a reproducibility of retention times of better than 0.2%, and the obtained spectra from the well-separated chromatographic peaks allowed protein identification with high confidence by protein database searching. We believe that this simple and reproducible gradient system using an isocratic pump will be especially advantageous for nanoLC-MS, instead of flow-controlled gradient pumps.     

Recycling and screen-segmented column isotachophoresis, two free-fluid approaches for fractionation of proteins.

Recycling and screen-segmented column isotachophoresis (ITP), two approaches for the milligrams to grams preparative-scale purification of proteins, are discussed and compared. Recycling ITP was performed in a recycling free-flow focusing apparatus. In this process, fluid flows rapidly through a narrow channel and the effluent from each channel is reinjected into the electrophoresis chamber through the corresponding input port. The residence time in the cell is of the order of 1 s per single pass, which does not allow complete separation, so recycling is essential to attain the steady state. Immobilization of the advancing zone structure is obtained via a controlled counterflow. Thirty fractions of about 4 ml each are obtained. Column ITP was executed in a Rotofor apparatus and in a similar column operated vertically and without rotation. These instruments feature a screen-segmented annular separation space with twenty subcompartments of about 2 ml each. With both approaches, the collected fractions were analysed separately for conductivity, pH and UV absorbance. Selected fractions were characterized by analytical electrophoretic methods. Examples presented include the cationic and anionic ITP behaviour of model proteins, including bovine serum albumin, ovalbumin and ribonuclease A, and the ITP removal of the major impurities from a commercial ovalbumin sample. These examples revealed that the screen-segmented column is suitable for ITP protein purification and operates optimally in a horizontal rotating mode and without internal cooling. The recycling experiments showed that counterflow improves separation and the steady-state patterns are dependent on the fluid layer thickness in the separation cell but, with a given gap, essentially independent of applied current and recycling pump rate.     

Modeling the effects of column packing quality and residence time changes on protein monomer/aggregate separation

The packing quality of chromatography columns used for the purification of protein therapeutics is routinely monitored to ensure consistent and reproducible performance. In this work, we used established chromatography models to determine the effect of column packing quality and fluid residence time on the separation of protein therapeutic monomer and aggregate species using a hydrophobic interaction chromatography adsorbent (Phenyl Sepharose Fast Flow). The relationship between the number of theoretical plates, fluid residence time, and column separation performance was quantified using modeling simulations. The simulations showed the separation depended on both the fluid residence time and the number of theoretical plates. However, when the number of theoretical plates was increased to ≥150, the simulations predicted that the separation performance of the column was not significantly improved. The approach described here could be used as a method to quantify acceptable height equivalent of a theoretical plate values for columns, and serve as a tool to understand how column packing quality impacts a given chromatographic separation prior to column scale-up, as well as during the monitoring of column lifetime in the manufacturing of large scale protein therapeutics.     

Investigation of the effect of solution destabilization on flux enhancement in filtration

A simple model for the filtration of a destabilized colloidal suspension is proposed. It takes into account the effect of flocculation on the properties of the deposited layer. It is shown that the cake permeability is a function of the quantity ωt_R, where ω is the flocculation rate (collision frequency) and t_R is the residence time defined by the time from which the particle enters the membrane channel to the time that it attaches onto the membrane surface.     

Scale-up of free flow electrophoresis: I. Purification of alcohol dehydrogenase from a crude yeast extract by zone electrophoresis

The potential and limitations in scaling-up free-flow electrophoresis, with emphasis on zone electrophoresis, are demonstrated. Purification of alcohol dehydrogenase (ADH) from a crude yeast extract was chosen as a model for an industrial approach to enzyme purification. In zone electrophoresis the separation quality strongly depends on the pH and conductivity of the background electrolyte, its residence time and flow rate, as well as the applied voltage. Optimization of these parameters resulted in a purification factor of 5.3 and a yield of 96% ADH, using a Tris/HCl buffer, pH 8.0, and a conductivity of 1 mS/cm, with a residence time of 10 min at 500 V. The loading capacity of the method for a laboratory-sized free-flow electrophoresis apparatus was limited to a sample throughput of about 0.4 g/h. By increasing the chamber dimensions it was possible to purify the enzyme by a purification factor of 4.7 and a yield of 93% ADH, at a throughput of about 1 g total protein/h. By simultaneously applying the sample at 3 input positions the throughput could be increased to 2.75 g/h with a purification factor of 4.7 and an overall yield of 90%.     

The relative importance of transport phenomena in recycling isoelectric focusing.

The various transport phenomena involved in recycling isoelectric focusing are analyzed for their contributions to band spreading so as to find ways of improving the resolution of this liquid-phase protein purification method. A numerical model is proposed that takes into account diffusion, electroosmosis and electrophoretic migration as a function of pH. The electrohydrodynamic effects have so far been neglected in these calculations. The results of these calculations are compared with experimental measurements performed in different chamber geometries, with a variety of proteins and under different operating conditions, always chosen to avoid flow instabilities. This comparison shows that the resolution of this process is greatly impaired if the electroosmotic slip velocity at the wall is not suppressed.     

Radiotracer investigation of the pulp flow characteristics in the superphosphate production chamber

By means of radiotracer⁵¹Cr impregnated in the solid phase the residence time distribution of the pulp in the mixing chamber was obtained. The mathematical model constructed on the basis of the experimental distribution showed an unsatisfactory functioning of the reactor. Ways were proposed to correct this shortcoming, both in the existing chamber and in that which is being designed for a new plant.     

Hydrophobic interaction chromatography of proteins IV. Kinetics of protein spreading

Adsorption of proteins on surfaces of hydrophobic interaction chromatography media is at least a two-stage process. Application of pure protein pulses (bovine serum albumin and beta-lactoglobulin) to hydrophobic interaction chromatography media yielded two chromatographic peaks at low salt concentrations. At these salt concentrations, the adsorption process is affected by a second reaction, which can be interpreted as protein spreading or partial unfolding of the protein. The kinetic constants of the spreading reaction were derived from pulse response experiments at different residence times and varying concentrations by applying a modified adsorption model considering conformational changes. The obtained parameters were used to calculate uptake and breakthrough curves for spreading proteins. Although these parameters were determined at low saturation of the column, predictions of overloaded situations could match the experimental runs satisfactorily. Our findings suggest that proteins which are sensitive to conformational changes should be loaded at high salt concentrations in order to accelerate the adsorption reaction and to obtain steeper breakthrough curves.     

Development of a simple ampholyte-free isoelectric focusing slab electrophoresis for protein fractionation

Sample preparation is often necessary to separate and concentrate various compounds prior to analysis of complex samples. In this regard, isoelectric focusing (IEF) is one of the best sample preparation methods. With this approach, however, carrier ampholytes have to be introduced into the samples, which may result in matrix interferences. In this paper, a simple ampholyte-free IEF free-flow electrophoresis design was developed for the separation of proteins. beta-Lactoglobulin, hemoglobin, myoglobin and cytochrome c were selected as model analytes. The experimental design took advantage of the electrolysis-driven production of H(+) and OH(-) ions that migrated from the anode and cathode, respectively, establishing a pH gradient spanning from 2.3 to 8.9. The separation chamber was filled with silanized glass beads as a support medium. Dialysis membranes were mounted at the two sides of the separation chamber (made of glass slides) and sealed with 2% agarose gel. The separated proteins drained from the outlets of the separation chamber and could be successfully collected into small glass tubes. The focusing process was visually observed and the separation was confirmed by capillary isoelectric focusing (cIEF) with pI markers.     

Particle-loaded hollow-fiber membrane adsorbers for lysozyme separation

The separation of lysozyme (LZ), a valuable enzyme naturally present in chicken egg white, was carried out using a new type of ion exchange hollow-fiber membranes. Functionalities were incorporated into the polymeric membranes by dispersing ion-exchange resins (IERs) in a microporous structure formed by phase inversion. The obtained hollow-fibers were composed of ion-exchange particles surrounded by a polymeric matrix and possessed both high static and dynamic adsorption capacities of more than 60 mg/ml membrane. The hollow-fiber membrane adsorbers were connected in series with different numbers of fibers thereby increasing the effective thickness and the protein residence time within the module. By choosing appropriate operation conditions, the membranes adsorbed solely LZ from fresh chicken egg white (eventually also the minor component avidin), whereas the adsorption of ovalbumin, ovotransferrin, and other low isoelectric point proteins was negligible. An average separation factor for LZ of about 150 was calculated by numerical integration of the protein concentrations in the elution curve during the filtration run. The effect of the filtration flow rate, protein concentration and ionic strength on the membrane's performance was investigated to determine the optimum operation parameters.     

电泳芯片结构和芯片电泳分离操作参数的模拟、优化和实验验证

选择了L-精氨酸和L-苯丙氨酸为分离样品体系,根据电泳实验提出样品基本参数,通过模拟计算考察了进样管道宽度和进样时间对进样方差的贡献;根据分离度与分离长度拟合曲线确定电泳芯片的有效分离长度;对化学发光柱后衍生管道施加的夹流电压进行了模拟优化,得出氨基酸体系分离分析的电泳芯片设计方案和操作参数为:进样管道宽度为分离管道宽度的1/2,简单进样充样时间应大于5 s,分离管道有效分离长度为30 mm,衍生夹流比1.0~1.6。根据模拟优化结果提出了电泳芯片设计方案,采用整体浇注法制作带有柱后衍生反应器的PDMS电泳芯片,按照模拟计算提出的电压操作参数实现了精氨酸和苯丙氨酸样品体系的准确进样、芯片电泳分离和柱后衍生化学发光检测。电泳过程模拟结果和实验结果相结合,考察了柱后衍生对样品谱带展宽的影响,简单进样过程样品泄露引起的谱峰拖尾现象,并讨论了夹流进样法对减小进样方差和抑制样品泄露的贡献。     

Modelling the flow of liquid in continuous dialyzer

The paper deals with the modelling of the flow of liquid in a compartment of continuous dialyzer. Two simple models, the dispersion model and tanks-in-series model, were used. Their parameters Peclet number, the mean residence time of liquid in dialyzer compartment, the number of tanks and the mean residence time of liquid in each tank were determined from a nonideal step input of a tracer and its response. It has been found that in the range of the Reynolds number from 0.44 to 3.64, the Peclet number and the number of tanks are in the range from about 200 to 320 and from 100 to 310, respectively. Both these parameters and the mean residence time of liquid in the compartment of dialyzer and in each tank decrease with the increasing Reynolds number. Furthermore, it has been proved that the values of the mean residence time of liquid in the compartment of dialyzer calculated from the dispersion model agree well with those calculated from the tanks-in-series model. The obtained values of the Peclet number and the number of tanks indicate that the flow in the dialyzer does not significantly differ from the plug flow.     

Elucidating the relationship between the spreading coefficient, surface-mediated partial coalescence and the whipping time of artificial cream

We studied the whipping of artificial creams composed of a blend of sunflower oil and hydrogenated palm fat stabilized by protein or a mixture or protein and low molecular weight (lmw) surfactant. It was found that an increased whipping speed, decreased protein concentration, and the addition of lmw surfactant leads to shorter whipping times. Further, shorter whipping times were observed for WPI-stabilized cream compared to cream stabilized by sodium caseinate. In all cases, the decrease in whipping time was due to a decrease in the length of the second stage of whipping, the stage characterized by the adhesion of fat droplets to the air bubble surface. The decrease in whipping time could be accounted for by considering the influence of the experimental variables on the fraction of bubble surface area at which fat droplet spreading is possible. The same changes in parameters that promote droplet spreading at the air/water interface cause a decrease in the whipping time of our model creams. Correlating the whipping time of cream with the spreading behavior of fat droplets at the air/water interface represents a new insight into the mechanisms involved in the whipping of cream.     

Residence time distribution for electrokinetic flow through a microchannel comprising a bundle of cylinders

The electrokinetic flow of an electrolyte solution through a microchannel that comprises a bundle of cylinders is investigated for the case of constant surface potential. The system under consideration is simulated by a unit cell model, and analytical expressions for the flow field and the corresponding residence time distribution under various conditions are derived. These results are readily applicable to the assessment of the performance of a microreactor such as that which comprises a bundle of optical fibers. Numerical simulations are conducted to investigate the influences of the key parameters, including the thickness of the double layer, the strength of the applied electric field, the magnitude of the applied pressure gradient, and the characteristic sizes of a microchannel, on the residence time distribution. We show that the following could result in a shorter residence time: thin double layer, strong applied electric field, large applied pressure gradient, and small number of cylinders. Based on the thickness of the double layer, criteria are proposed for whether the flow field can be treated as a laminar flow or as a plug flow, two basic limiting cases in reactor design.     

Applicability of dynamic change of pH in the capillary zone electrophoresis of proteins

A method to extend the separation power of CZE is described. The method is based on the separation of sample components at two different pH values during one separation run, and involves dynamic buffering of the pH inside a separation capillary by controlling the flow of H+ ions from the anodic electrode chamber. By changing the anolyte in the chamber, a dynamic pH step is generated, which proceeds rapidly along the capillary and establishes the required new pH value. The use of the method has been demonstrated by the cationic separation of a model mixture of proteins.     

On the carrier effect mechanism in relation to mass transport processes in the arc discharge

The time of residence of impurity atoms in the arc discharge is calculated. Diffusion, ion motion in the electric field, and ambipolar diffusion are taken into account. It is shown for the first time that ambipolar diffusion contributes significantly to the total particle flow from the arc discharge zone. The effect of charge exchange on the speed of ion motion in the arc is estimated. The influence of a carrier on the residence time of atoms in the arc discharge zone is calculated. Attention is paid to the peculiarities of the mechanism of the carrier effect associated with halide compounds. An attempt is made to explain the influence of halide compounds on the residence time of atoms in the arc using the considered model of mass transport. The velocities of mass transport and the time of atoms in the discharge zone are calculated for the are with and without halide containing substances. The initial parameters of calculation (discharge temperature, electron density, degree of ionization, and coefficient of atom diffusion) are partly measured and partly taken from the literature. The results of the calculation are compared with experimental data published in the literature. The adopted mass transport model adequately accounts for the influence of a carrier on the residence time of atoms in the arc discharge zone.