Ion-exchange Displacement Chromatography Research Articles

Dynamic behavior of binary component ion-exchange displacement chromatography of proteins visualized by confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was introduced to visualize particle-scale binary component protein displacement behavior in Q Sepharose HP column. To this end, displacement chromatography of two intrinsic fluorescent proteins, enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP), were developed using sodium saccharin (NaSac) as a displacer. The results indicated that RFP as well as eGFP could be effectively displaced in the single-component experiments by 50mmol/L NaSac at 120 and 140mmol/L NaCl whereas a fully developed displacement train with eGFP and RFP was only observed at 120mmol/L NaCl in binary component displacement. At 140mmol/L NaCl, there was a serious overlapping of the zones of the two proteins, indicating the importance of induced-salt effect on the formation of an isotachic displacement train. CLSM provided particle-scale evidence that induced-salt effect occurred likewise in the interior of an adsorbent and was synchronous to the introduction of the displacer. CLSM results at 140mmol/L NaCl also demonstrated that both the proteins had the same fading rate at 50mmol/L NaSac in the initial stage, suggesting the same displacement ability of NaSac to both the proteins. In the final stage, the fading rate of RFP in the adsorbent became slow, particularly at lower displacer concentrations. In the binary component displacement, the two proteins exhibited distinct fading rates as compared to the single component displacement and the remarkable lagging of the fading rate was observed in protein displacements. It suggested that the co-adsorbed proteins had significant influence on the formation of an isotachic train and the displacement chromatography of the proteins. Therefore, this research provided particle-scale insight into the dynamic behavior and complexity in the displacement of proteins.

Isolation of monoclonal antibody charge variants by displacement chromatography.

This unit discusses the important parameters in designing and optimizing a separation of monoclonal antibody (mAb) charge variants from process streams by ion-exchange displacement chromatography, including sample preparation and selection of matrix, column, and appropriate buffer. A protocol is provided for determination of optimal column binding and displacement conditions, including cleaning and regeneration of the displacement columns.

Isotope effects of neodymium in different ligands exchange systems studied by ion exchange displacement chromatography

The isotope effects of neodymium in Nd-glycolate ligand exchange system were studied by using ion exchange chromatography. The separation coefficients of neodymium isotopes, ε’s, were calculated from the observed isotopic ratios at the front and rear boundaries of the neodymium adsorption band. The values of separation coefficients of neodymium isotopes, ε’s, for the Nd-glycolate ligand exchange system were compared with those of Nd-malate and Nd-citrate, which indicated that the isotope effects of neodymium as studied by the three ligands takes the following direction Malate>Citrate>Glycolate. This order agrees with the number of available sites for complexation of each ligand. The values of the plate height, HETP of Nd in Nd-ligand exchange systems were also calculated.

Displacement Chromatography of Proteins

This unit discusses the important parameters in designing and optimizing a separation by ion-exchange displacement chromatography, including preparing the sample and choosing a matrix, column, and buffer. Protocols are provided for testing a column, determining binding and elution conditions, displacing the sample, and cleaning, regenerating, and storing of displacement columns.

Displacer concentration effects in displacement chromatography: Implications for trace solute detection

In this paper, the utility of ion-exchange displacement chromatography for the concentration and enrichment of trace proteins is examined. Separations with varying displacer concentrations (1–25 mM neomycin sulfate) indicate that higher concentrations result in elevated protein concentrations, at the price of reduced yields. The results demonstrate that displacement chromatography carried out at relatively low displacer concentrations (2.5 mM) can produce significant concentration (8.5-fold) and enrichment (18-fold) of trace proteins present in the feed. Parametric simulations using the steric mass action model are carried out to investigate the concentration effects and enrichment factors observed over a wide range of feed, displacer and buffer counter-ion concentrations, and solute separation factors. The simulations confirm that trace components can be readily concentrated and enriched by displacement chromatography and that these effects will be more pronounced as the separation factor between trace and abundant components is increased. The results presented in this paper indicate the potential of displacement chromatography for improved separations where trace enrichment is critical such as proteomic applications.

Multidimensional High-Throughput Screening of Displacers

A multidimensional, batch high-throughput screening (MD-HTS) protocol was developed to investigate the effects of various parameters on the selectivity of ion-exchange protein displacement systems. A variety of molecules were screened, and the results were employed to provided insights into the influence of displacer chemistry and concentration, resin chemistry, and mobile-phase salt counterion on the efficacy and selectivity of these nonlinear chromatographic systems. These results open up the possibility of tailoring the selectivity of displacement separations by choosing appropriate combinations of operating conditions using the MD-HTS technique. The screens were also employed for the identification of displacers and conditions for the separation of a challenging protein mixture by selective displacement chromatography. Column displacements were carried out with potential lead compounds identified from the MD-HTS screens, and the results confirmed that selective displacement could indeed be achieved for this model mixture. Furthermore, the results indicated that this approach is particularly useful when the order of elution is not changed, but the inherent selectivity is increased in the presence of the displacer. The results presented in this paper demonstrate the utility of the MD-HTS technique for rapid method development in protein ion-exchange displacement chromatography.

Anion-Exchange Displacement Centrifugal Partition Chromatography

Ion-exchange displacement chromatography has been adapted to centrifugal partition chromatography. The use of an ionic liquid, benzalkonium chloride, as a strong anion-exchanger has proven to be efficient for the preparative separation of phenolic acid regioisomers. Multigram quantities of a mixture of three hydroxycinnamic acid isomers were separated using iodide as a displacer. The displacement process was characterized by a trapezoidal profile of analyte concentration in the eluate with narrow transition zones. By taking advantage of the partition rules involved in support-free liquid-liquid chromatography, a numerical separation model is proposed as a tool for preliminary process validation and further optimization.

Isotope effects of copper in Cu(II) malate ligand exchange system studied by using ion exchange displacement chromatography

The ion exchange chromatography technique was used for studying copper isotope effects in Cu-malate ligand exchange system (LXS) in the temperature range 288–353K. A highly acidic cation exchange resin (TITECH-3, 37–88 μm) was used. It had been found that the heavier isotope, 65Cu, was enriched at the front boundary, while the lighter isotope, 63Cu, was enriched at the rear boundary of the copper band at all temperatures. Such finding means that the heavier isotope, 65Cu, is preferentially fractionated into the malic complex form in the solution phase. The values of the single stage separation coefficients, ϵ=S−1, of the 63Cu/65Cu isotopic pair have been calculated and found to decrease with the increase in temperature as 2.87×10−4, 2.39×10−4, and 2.13×10−4 at 288, 333, and 353K, respectively. The height equivalent to the theoretical plate (HETP), at every temperature, was also calculated.

The Effect of Temperature on Uranium Isotope Effects Studied by Cation Exchange Displacement Chromatography

The uranium isotope effect in the exchange system uranyl(VI)-malate ligand at 288-343 K has been studied by ion exchange displacement chromatography. At all temperatures 235U is enriched at the front of the uranium band. The single stage separation coefficient, (Ɛ = α - 1), increased from (0.9 ± 0.1) × 10-4 at 288 K to (2.9 ± 0.3) × 10-4 at 343 K. The equilibrium constant of the isotope exchange reaction equaled the separation factor at the current experimental conditions. The increase of the separation coefficient with temperature, which is in contrast to the uranium(IV)-ligand exchange systems, can be explained by the introduction of the field shift effect.

When non-linear effects help to overcome limiting mass transfer in the ion exchange displacement chromatography of amino acids

This work addresses the description of interrelated phenomena of adsorption and mass transfer, occurring when preparative ion exchange chromatography is used for the separation of amino acids (alanine and proline). We have considered the use of commercial resins, with large bead diameter (600 μm), inducing significant mass transfer limitations. The modelling was developed from recent works in the literature, and was able to properly describe non linear effects due to stationary phase saturation. We have validated this numerical tool with experiments of displacement chromatography (NaOH as the displacer). This modelling has enabled us to design a set of operating conditions, with high loading factors (40%) where non linear effects were shown to counterbalance mass transfer limitations and yield acceptable separations.

A theoretical approach to ion exchange displacement chromatography

In this paper, the Nernst–Planck equation, in which the strong coupling effects between the various species are considered by including electrical influence produced by ion transfer, is introduced to the study of ion exchange displacement chromatography and the solution of concentration distribution is obtained for the steady state. In addition, the limitations of the original differential equations are also disscussed for the case of ion exchange displacement chromatography. Copyright © 1999 John Wiley & Sons, Ltd.

A theoretical approach to ion exchange displacement chromatography

In this paper, the Nernst–Planck equation, in which the strong coupling effects between the various species are considered by including electrical influence produced by ion transfer, is introduced to the study of ion exchange displacement chromatography and the solution of concentration distribution is obtained for the steady state. In addition, the limitations of the original differential equations are also disscussed for the case of ion exchange displacement chromatography. Copyright © 1999 John Wiley & Sons, Ltd.

Isotope effects of europium in ligand exchange system and electron exchange system using ion-exchange displacement chromatography

The ion-exchange chromatography of europium has been carried out to study the europium isotope effects in a ligand exchange system (LXS) and an electron exchange system (EXS). In both cases highly acidic cation-exchange resin (AG-MP 50) with 100–200 mesh size particles was used. In the case of LXS, where EDTA was chosen as a ligand for Eu 3+ ions, the heavier isotope 153Eu has been enriched at the front boundary of the europium band. Such findings mean that the heavier isotope 153Eu is preferentially fractionated into the EDTA complex form in the solution phase. In the EXS, where Eu(II)–Eu(III) exchange reaction takes place, the heavier isotope 153Eu has been observed to be enriched in the front part of the europium band. The result shows that the heavier isotope is preferentially fractionated in the Eu(II) solution phase. The values of the single stage separation coefficients ( ε= S−1) of the 153Eu/ 151Eu isotopic pair have been calculated for LXS as 1.48·10 −5 at 80°C and for EXS as 2.13·10 −4 at 60°C.

Preliminary report on fractionation of fucans by ion-exchange displacement centrifugal partition chromatography

A new method combining ion-exchange displacement chromatography with centrifugal partition chromatography (CPC) was used for the fractionation of partially depolymerized fucans (polysulphated polysaccharides). The ion-exchanger was Amberlite LA2, a high-molecular-mass liquid secondary amine miscible with most common organic solvents and immiscible with aqueous solutions. Ion-exchange displacement centrifugal partition chromatography was performed with LA2 in methyl isobutyl ketone (MiBK) as the stationary phase, water as the mobile phase, Cl− as the carrier and OH− as the displacer. A complex mixture of partially depolymerized fucans was resolved into adjacent families characterized by their peak molecular mass and polydispersity. The Dubois test (sugar) and the azur A test (SO3−) confirmed the displacement mode of the process, and size-exclusion chromatographic controls confirmed its efficiency.

Isotope Effects of Copper in Ligand-Exchange System and Electron-Exchange System Observed by Ion-Exchange Displacement Chromatography

ABSTRACT Using cation-and anion-exchange resins, ion-exchange displacement chromatography of copper has been carried out to study the copper isotope effects in the ligand exchange system (LXS) and the electron exchange system (EXS), respectively. In the LXS, where malate was chosen as the ligand for Cu2+ ions, the heavier isotope 65Cu has been enriched at the front part of the copper chromatogram, which means that the heavier isotope 65Cu is preferentially fractionated into the malate complex in the solution phase. In the EXS, where the Cu(I)/Cu(II) exchange reaction takes place in the HCl-anion exchanger resin system, the lighter isotope 63Cu has been observed to be enriched at the Cu(I) chloride anion complex side. The result shows that the lighter isotope is preferentially fractionated into the anion exchange resin phase. The values of the single-stage separation coefficients (e = S — 1) of the 63Cu65Cu isotopic pair have been calculated for LXS as 2.8 X 10∼4 at 40°C and for EXS as 3.8 X 10∼4 at 60°C.

Productivity and operating regimes in protein chromatography using low-molecular-mass displacers

A theoretical and experimental study of ion-exchange displacement chromatography using the low-molecular-mass displacer neomycin sulfate, is carried out. A chromatography model utilizing the steric mass action (SMA) ion-exchange formalism is employed to predict the displacement behavior of proteins displaced by neomycin sulfate. An operating regime plot is developed from the SMA model to predict selective displacement chromatography using low-molecular-mass displacers. Numerical simulations are employed to examine the behavior of selective displacement systems and to investigate the productivity of displacement chromatography. In this study, it is seen that use of low-molecular-mass displacers allows “selective displacement” separations in which some impurities are removed by elution or by desorption within the displacer front. Further, low-molecular-mass displacers, like previously studied high-molecular-mass displacers, can provide high productivity chromatographic separations even for feed streams characterized by low separation factors.

Solute affinity in ion-exchange displacement chromatography

In this paper a new graphical method, derived from the Steric Mass Action model of non-linear ion-exchange chromatography, is presented for the determination of solute affinity in ion exchange displacement systems. The affinity of solutes in these systems is described by a dynamic affinity parameter which is a function of the linear steric mass action parameters of the solutes and the characteristic velocity of the displacer front. This method can be employed to determine the elution order of the feed solutes in the isotachic displacement train as well as the ability of a given molecule to act as a displacer for a given protein mixture. The ideal model of ion-exchange displacement chromatography and a transient model are employed to study selectivity reversals and resolving power in ion-exchange displacement systems. The work presented here provides a theoretical framework for studying dynamic affinity, resolving power, and displacer design for ion-exchange displacement chromatography.

Antibiotics as low-molecular-mass displacers in ion-exchange displacement chromatography

While 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 implementtion 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-mass dendritic polymers and protected amino acids can be successfully employed as displacers for protein purification in ion-exchange systems. In this paper, the efficacy of aminoglycosidic antibiotic displacers are investigated for protein purification in cation-exchange systems. The displacers neomycin B and streptomycin A are employed with model feed mixtures containing moderately to very strongly retained proteins. These experiments demonstrate that this new class of low-molecular-mass antibiotic displacers can indeed act as efficient protein displacers. In fact, the displacer neomycin B is the first low-molecular-mass displacer reported i the literature which can readily displace very strongly retained proteins such as lysozyme. In addition to the fundamental interest generaed by low-molecular-mass displacers, it is likely that these displacers will have significant operational advantages as compared to large polyelectrolyte displacers.

Production of rare-earth oxides of high purity

A novel industrial technology for producing rare earth oxides of high purity (>99.99%) by temperature pressurized ion-exchange displacement chromatography is reported. It was concluded from the technological parameters that the concentration of complex agent in the eluent can be several times higher than that used in the classical ion-exchange method if a certain amount of buffer is present; the height equivalent to a theoretical plate (HETP) decreases linearly with increase in the operating temperature, increases slightly with increase in the flow-rate of the eluent and depends on the particle size of the ion exchanger. Rare earth oxides of high purity were manufactured on an industrial scale using a solution of special composition as eluent, and enriched rare earth mixtures or single rare earth oxides with low grade as raw and processed material on a temperature pressurized ion-exchange multi-column operating system containing polytetrafluoroethylene as the inner liner.

Ion-exchange displacement chromatography of proteins Dendritic polymers as novel displacers

While the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of biopharmaceuticals, a major obstacle to the implementation of displacement chromatography has been the lack of appropriate displacer compounds. All protein displacement separations reported to date have employed relatively high-molecular-mass (> 2000) polyelectrolyte displacers. In this paper, results are presented on the discovery that low-molecular-mass dendritic polymers can be successfully employed as efficient displacers for protein purification in ion-exchange systems. Pentaerythritol-based dendritic polyelectrolytes ranging in molecular mass from 480 to 5100 were investigated as potential displacers for the purification of proteins in cation-exchange systems. The adsorption properties of these dendrimers were investigated using the steric mass action (SMA) model of non-linear ion-exchange chromatography. An analysis of the resulting SMA parameters using a dynamic affinity plot indicated that these dendrimers should have sufficient affinity to act as protein displacers. Displacement separations of protein mixtures in cation-exchange systems were carried out using zero-, first- and second-generation dendrimers. These experiments demonstrate that this new class of dendritic polyelectrolytes can indeed act as efficient protein displacers. The ability of a low-molecular-mass compound such as the “zero”-generation dendrimer ( M r 480) to displace proteins is very significant in that it represents a major departure from the conventional wisdom that large polyelectrolyte polymers are required to displace proteins in ion-exchange systems. In addition to the fundamental interest generated by low-molecular-mass displacers, it is likely that these displacers will have significant operational advantages as compared to large polyelectrolyte displacers.